RU2829874C1 - Versions based on adeno-associated virus, compositions and methods for pulmonary delivery - Google Patents

Abstract

FIELD: chemistry; biotechnology.

SUBSTANCE: invention relates to biochemistry. Described is a method of delivering a heterologous nucleic acid into a lung cell in a subject, involving administering to the subject a composition containing a recombinant adeno-associated virus (rAAV) vector. Said vector rAAV contains (i) a capsid containing a capsid protein containing an amino acid sequence presented as SEQ ID NO: 12, or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and containing Thr at amino acid 469 and Ala at amino acid 598, based on amino acid numbering presented in SEQ ID NO: 12, and (ii) a heterologous nucleic acid containing a nucleotide sequence coding a gene product. Introduction includes pulmonary, endobronchial, intranasal, intratracheal and/or intrabronchial introduction. Described is a corresponding composition containing said vector. What is also described is a method of treating cystic fibrosis in a subject, involving administering said composition to the subject. Use of said composition in preparing a medicinal agent for treating cystic fibrosis, wherein the medicinal agent is intended for pulmonary, endobronchial, intranasal, intratracheal or intrabronchial administration.

EFFECT: invention extends the range of products for treating pulmonary diseases.

30 cl, 35 dwg, 18 tbl, 7 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/016,246, filed April 27, 2020, and U.S. Provisional Patent Application No. 63/088,432, filed October 6, 2020, the entire disclosures of which are incorporated herein by reference.

Submitting a list of sequences via EFS-WEB

[0002] A machine-readable text file named "090400-5013-WO-Sequence-Listing" created on or about April 26, 2021, with a file size of approximately 186 KB, contains a sequence listing for the present application, which is incorporated herein by reference in its entirety.

State of the art

[0003] Adeno-associated virus (AAV)-based gene delivery vectors have shown promising results in both preclinical disease models and recent human clinical trials for the treatment of several target diseases. AAV-based vectors are essentially safe because wild-type AAV is non-pathogenic and has no etiologic association with any known diseases. In addition, AAV enables highly efficient gene delivery and robust transgene expression in many tissues, including liver, muscle, lung, retina, and brain.

[0004] AAV is a single-stranded DNA virus that contains two open reading frames, rep and cap. The former gene encodes four proteins required for genome replication (Rep78, Rep68, Rep52, and Rep40), and the latter expresses three structural proteins (VP1-3) that assemble to form the viral capsid. As its name suggests, active AAV replication depends on the presence of a helper virus such as adenovirus or herpesvirus. In the absence of a helper, it enters a latent state in which its genome is maintained episomally or integrated into the host chromosome. Numerous homologous primate AAV serotypes and numerous non-human primate serotypes have been identified. AAV2 has been best characterized as a gene delivery vehicle.

[0005] As of 2010, there are 75 ongoing clinical trials using AAV as a gene delivery vehicle. However, the high prevalence of anticapsid neutralizing antibodies resulting from the widespread exposure of multiple AAV variants and serotypes in the human population reduces the efficacy of AAV-based gene therapy. Such pre-existing immunity, as well as subsequent development of immunity due to vector administration, may hinder the broader adoption of AAV-based gene therapy. For example, to date, AAV has been most successful in clinical trials involving delivery to immune-privileged regions.

[0006] A recent analysis showed that the prevalence of IgG anti-AAV antibodies in humans was highest for AAV2 (72%) and AAV1 (67%), but antibodies to AAV9 (47%), AAV6 (46%), AAV5 (40%), and AAV8 (38%) were also present in a significant proportion of the population studied. Results from several studies have shown that humoral immunity to the AAV capsid during gene therapy can be prevented by reducing the amount of rAAV particles delivered. Unfortunately, administration of low doses of vector results in low transduction and, therefore, low expression of the therapeutic gene.

[0007] There is a need in the art to develop new AAV variants that are resistant to neutralization by anti-AAV antibodies.

The essence of the invention

[0008] In some embodiments, the present invention relates to a recombinant adeno-associated virus (rAAV) vector comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12, and (ii) a nucleic acid comprising, in 5' to 3' direction: (a) an AAV2 terminal repeat, (b) a promoter, (c) a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein that lacks amino acids 708-759 of the human CFTR protein sequence, (d) a polyadenylation sequence, and (e) an AAV2 terminal repeat.

[0009] In related embodiments, a nucleotide sequence encoding human CFTR or a biologically active fragment thereof encodes a native human CFTR protein and has the following sequence or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto:

ATGCAGAGGTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACCAATTTTGAGGAAAGGATACAGACAGCGCCTGGAATTGTCAGACATATACCAAATCCCTTCTGTTGATTCTGCTGACAATCTATCTGAAAAATTGGAAAGAGAATGGGATAGAGAGCTGGCTTCAAAGAAAAATCCTAAACTCATTAATGCCCTTCGGCGATGTTTTTTCTGGGAGATTTATG TTCTATGGAATCTTTTTATATTTAGGGGAAG TCACCAAAGCAGTACAGCCTCTCTTACTGGGAAGAATCATAGCTTCCTATGACCCGGATAACAAGGAGGAACGCTCTATCGCGATTTATCTAGGCATAGGCTTATGCCTTCTCTTTATTGTGAGGACACTGCTCCTACACCCAGCCATTTTTGGCCTTCATCACATTGGAATGCAGATGAGAATAGCTATGTTTAGTTTGATTTATAAGAAGACTTTAAAGCTGTCAAGCCGTGTTCTAGATAAAATAAGTATT GGACAACTTGTTAGTCTCCTTTCC AACAACCTGAACAAATTTGATGAAGGACTTGCATTGGCACATTTCGTGTGGATCGCTCCTTTGCAAGTGGCACTCCTCATGGGGCTAATCTGGGAGTTGTTACAGGCGTCTGCCTTCTGTGGACTTGGTTTCCTGATAGTCCTTGCCCTTTTTCAGGCTGGGCTAGGGAGAATGATGATGAAGTACAGAGATCAGAGCTGGGAAGATCAGTGAAAGACTTGTGATTACCTCAGAAATGAT TGAAAATATCCAATCTGTTAAGGCATACTGCTGGG AAGAAGCAATGGAAAAAATGATTGAAAACTTAAGACAAACAGAACTGAAACTGACTCGGAAGGCAGCCTATGTGAGATACTTCAATAGCTCAGCCTTCTTCTTCTCAGGGTTCTTTGTGGTGTTTTTATCTGTGCTTCCCTATGCACTAATCAAAGGAATCCTCCGGAAAATATTCACCACCATCTCATTCTGCATTGTTCTGCGCATGGCGGTCACTCGGCAATTTCCCTGGGCTGTACAAACATGG TATGACTCTCTTGGAGCAATAAACAAA ATACAGGATTTCTTACAAAAGCAAGAATATAAGACATTGGAATATAACTTAACGACTACAGAAGTAGTGATGGAGAATGTAACAGCCTTCTGGGAGGAGGGATTTGGGGAATTATTTGAGAAAGCAAAACAAAACAATAACAATAGAAAAACTTCTAATGGTGATGACAGCCTCTTCTTCAGTAATTTCTCACTTCTTGGTACTCCTGTCCTGAAAGATATTAATTTCAAGATAGAAAGAGGACAGTT GTTGGCGGTTGCTGGATCCACTGGAGCAG GCAAGACTTCACTTCTAATGGTGATTATGGGAGAACTGGAGCCTTCAGAGGGTAAAATTAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCCTGGATTATGCCTGGCACCATTAAAGAAAATTCATCTTTGGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAAAGCATGCCAACTAGAAGAGGACATCTCCAAAGTTTGCAGAGAAAGACAATATAGTTCTTGGAGAAGG TGGAATCACACTGAGTGGAGGTCAACGAGCAAGA ATTTCTTTAGCAAGAGCAGTATACAAAGATGCTGATTTGTATTTATTAGACTCTCCTTTTGGATACCTAGATGTTTTAACAGAAAAAGAAATATTTGAAAGCTGTGTCTGTAAACTGATGGCTAACAAAACTAGGATTTTGGTCACTTCTAAAATGGAACATTTAAAAGGCTGACAAAATATTAATTTTGCATGAAGGTAGCAGCTATTTTTATGGGACATTTTCAGAACTCCAAAATCTACAGCCAGACTTTAG CTCAAAACTCATGGGATGTGA TTCTTTCGACCAATTTAGTGCAGAAAGAAGAAATTCAATCCTAACTGAGACCTTACACCGTTTCTCATTAGAAGGAGATGCTCCTGTCTCCTGGACAGAAACAAAAAAACAATCTTTAAACAGACTGGAGAGTTTGGGGAAAAAAGGAAGAATTCTATTCTCAATCCAATCAACTCTATACGAAAATTTTCCATTGTGCAAAAGACTCCCTTACAAATGAATGGCATCGAAGAGGATTCTGATGAGCCTTTA GAGAGAAGGCTGTCCTTAGTACCAG ATTCTGAGCAGGGAGAGGCGATACTGCCTCGCATCAGCGTGATCAGCACTGGCCCCACGCTTCAGGCACGAAGGAGGCAGTCTGTCCTGAACCTGATGACACACTCAGTTAACCAAGGTCAGAACATTCACCGAAAGACAACAGCATCCACACGAAAAGTGTCACTGGCCCCTCAGGCAAACTTGACTGAACTGGATATATATTCAAGAAGGTTATCTCAAGAAACTGGCTTGGAAATAAGTGAAGAAATTAACGAA GAAGACTTAAAGGAGTGCTT TTTTGATGATATGGAGAGCATACCAGCAGTGACTACATGGAACACATACCTTCGATATATTACTGTCCACAAGAGCTTAATTTTTGTGCTAATTTGGTGCTTAGTAATTTTTCTGGCAGAGG TGGCTGCTTCTTTGGTTGTGCTGTGGCTCCTTGGAAACACTCCTCTTCAAGACAAAGGGAATAGTACTCATAGTAGAAATAACAGCTATGCAGTGATTATCACCAGCACCAGTTCGTATTAT GTGTTTTACATTTACGTGGGAGTAGCCGACACTT TGCTTGCTATGGGATTCTTCAGAGGTCTACCACTGGTGCATACTCTAATCACAGTGTCGAAAATTTTACACCACAAAATGTTACATTCTGTTCTTCAAGCACCTATGTCAACCCTCAACACGTTGAAAGCAGGTGGGATTCTTAATAGATTCTCCAAAGATATAGCAATTTTGGATGACCTTCTGCCTCTTACCATATTTGACTTCATCCAGTTGTTATTAATTGTGATTGGAGCTATAGCAGTTGTC GCAGTTTTTACAACCCTACATCTTTGTTGCA ACAGTGCCAGTGATAGTGGCTTTTATTATGTTGAGAGCATATTTCCTCCAAACCTCACAGCAACTCAAACAACTGGAATCTGAAGGCAGGAGTCCAATTTTCACTCATCTTGTTACAAGCTTAAAAGGACTATGGACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACTCTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCCAAATGAGAATA GAAATGATTTTTTGTCATCTTCTT CATTGCTGTTACCTTCATTTCCATTTTAACAACAGGAGAAGGAGAAGGAAGAGTTGGTATTATCCTGACTTTAGCCATGAATATCATGAGTACATTGCAGTGGGCTGTAAACTCCAGCATAGATGTGGATAGCTTGATGCGATCTGTGAGCCGAGTCTTTAAGTTCATTGACATGCCAACAGAAGGTAAACCTACCAAGTCAACCAAACCATACAAGAATGGCCAACTCTCGAAAGTTATGATT ATTGAGAATTCACACGTGAAGAAAGATGACATC TGGCCCTCAGGGGGCCAAATGACTGTCAAAGATCTCACAGCAAAATACACAGAAGGTGGAAATGCCATATTAGAGAACATTTCCTTCTCAATAAGTCCTGGCCAGAGGGTGGGCCTCTTGGGAAGAACTGGATCAGGGAAGAGTACTTTGTTATCAGCTTTTTTGAGACTACTGAACACTGAAGGAGAAATCCAGATCGATGGTGTGTCTTGGGATTCAATAACTTTGCAACAGTGGAGGAAAGCCTTT GGAGTGATACCACAGAAAGTATTTATTTT TTCTGGAACATTTAGAAAAAACTTGGATCCCTATGAACAGTGGAGTGATCAAGAAATATGGAAAGTTGCAGATGAGGTTGGGCTCAGATCTGTGATAGAACAGTTTCCTGGGAAGCTTGACTTTGTCCTTGTGGATGGGGGCTGTGTCCTAAGCCATGGCCACAAGCAGTTGATGTGCTTGGCTAGATCTGTTCTCAGTAAGGCGAAGATCTTGCTGCTTGATGAACCCAGTGCTCATTTGGAT CCAGTAACATACCAAATAATTAGAAGAACTCTAA AACAAGCATTTGCTGATTGCACAGTAATTCTCTGTGAACACAGGATAGAAGCAATGCTGGAATGCCAAATTTTTGGTCATAGAAGAGAACAAAGTGCGGCAGTACGATTCCATCCAGAAACTGCTGAACGAGAGGAGCCTCTTCCGGCAAGCCATCAGCCCCTCCGACAGGGTGAAGCTCTTTCCCCACCGGAACTCAAGCAAGTGCAAGTCTAAGCCCCAGATTGCTGCTCTGAAAGAGGA GACAGAAGAAGGGTGCAAGATACAAGGCTTTAG (SEQ ID NO:42).

[0010] In preferred embodiments, the nucleotide sequence encoding human CFTR or a biologically active truncated CFTR protein comprises the following nucleotide sequence or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto:

ATGCAGCGCAGCCCACTGGAGAAGGCAAGCGTGGTGTCCAAGCTGTTCTTTTCCTGGACCAGGCCTATCCTGAGGAAGGGATACAGGCAGCGGCTGGAGCTGAGCGACATCTATCAGATCCCTTCTGTGGACAGCGCCGATAATCTGTCCGAGAAGCTGGAGAGAGAGTGGGATAGGGAGCTGGCCTCTAAGAAGAACCCAAAGCTGATCAATGCCCTGCGGAGATGCTTCTTTTGGCGGTT CATGTTCTACGGCATCTTCCTGTATCTGGGCGAGGTGACCAAGGCCGTGCAGCCACTGCTGCTGGGCAGAATCATCGCCTCTTACGACCCCGATAACAAGGAGGAGAGGAGCATCGCCATCTATCTGGGCATCGGCCTGTGCCTGCTGTTTATCGTGAGGACACTGCTGCTGCACCCAGCCATCTTCGGCC TGCACCACATCGGCATGCAGATGAGAATCGCCATGTTCAGCCTGATCTACAAGAAGACCCTGAAGCTGAGCTCCAGGGTGCTGGACAAGATCTCCATCGGCCAGCTGGTGTCCCTGCTGTCTAACAATCTGAACAAGTTTGATGAGGGACTGGCCCTGGCACACTTCGTGTGGATCGCACCACTGCAGGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCAAGCGCCTT TTGCGGACTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGCAGGACTGGGACGCATGATGATGAAGTACAGAGACCAGAGGGCCGGCAAGATCTCTGAGCGGCTGGTCATCACCAGCGAGATGATCGAGAACATCCAGTCCGTGAAGGCCTATTGTTGGGAGGAGGCCATGGAGAAGATGATCGAGAATCTGCG CCAGACAGAGCTGAAGCTGACCAGAAAGGCCGCCTACGTGAGGTACTTCAACTCTAGCGCCTTCTTTTTCTCTGGCTTTTTCGTGGTGTTCCTGAGCGTGCTGCCATACGCCCTGATCAAGGGCATCATCCTGCGGAAGATCTTTACCACAATCTCCTTCTGCATCGTGCTGAGAATGGCCGTGACAAGGCAGTTTCCCTGGGCCGTGCAGACCTGGTATGACTCTCTGGGCGCCATCAATAAGAT CCAGGATTTCCTGCAGAAGCAGGAGTACAAGACACTGGAGTATAACCTGACCACAACCGAGGTGGTCATGGAGAATGTGACCGCCTTCTGGGAGGAGGGCTTTGGCGAGCTGTTCGAGAAGGCCAAGCAGAACAATAACAATCGCAAGACATCTAACGGCGACGATAGCCTGTTTTTCAGCAATTTT TCCCTGCTGGGCACCCCCGTGCTGAAGGACATCAACTTCAAGATCGAGAGGGGACAGCTGCTGGCAGTGGCAGGCTCCACAGGCGCCGGCAAGACCTCTCTGCTGATGATGATCATGGGCGAGCTGGAGCCAAGCGAGGGCAAGATCAAGCACTCCGGCCGGATCTCTTTTTGCAGCCAGTTCTCCTGGATCATGCCCGGCACCATCAAGGAGAATATCATCTTTGGCGTGTCCTACGATGAGTACAG ATATAGGTCTGTGATCAAGGCCTGTCAGCTGGAGGAGGACATCAGCAAGTTCGCCGAGAAGGATAACATCGTGCTGGGCGAGGGCGGCATCACACTGAGCGGAGGACAGAGGGCAAGGATCTCCCTGGCCAGAGCCGTGTACAAGGACGCCGATCTGTATCTGCTGGACAGCCCCTTTGGCTATC TGGATGTGCTGACCGAGAAGGAGATCTTCGAGTCCTGCGTGTGCAAGCTGATGGCCAATAAGACAAGGATCCTGGTGACCTCTAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGATCCTGCACGAGGGCTCCTCTTACTTTTATGGCACATTCAGCGAGCTGCAGAATCTGCAGCCTGACTTCAGCTCCAAGCTGATGGGCTGTGACTCCTTTTGATCAGTTCTCTGCCGAGAGGCG CAACTCCATCCTGACAGAGACCCTGCACAGATTCTCTCTGGAGGGCGACGCACCCGTGAGCTGGACAGAGACCAAGAAGCAGTCCTTTAAGCAGACCGGCGAGTTCGGCGAGAAGAGGAAGAATTCTATCCTGAACCCTATCAATAGCACACTGCAGGCCCGGAGAAGGCAGTCTGTGCTGAACCTGATGAC CCACAGCGTGAACCAGGGCCAGAATATCCACAGAAAGACAACCGCCAGCACAAGGAAGGTGTCCCTGGCACCTCAGGCAAACCTGACCGAGCTGGACATCTACTCCCGCCGGCTGTCTCAGGAGACCGGACTGGAGATCTCTGAGGAGATCAATGAGGAGGATCTGAAGGAGTGCTTTTTTCGACGATATGGAGAGCATCCCAGCCGTGACAACCTGGAACACATACCTGCGCTATATCACCGTGCA CAAGTCCCTGATCTTTGTGCTGATCTGGTGTCTGGTCATCTTCCTGGCAGAGGTGGCAGCATCTCTGGTGGTGCTGTGGCTGCTGGGCAACACACCCCTGCAGGACAAGGGCAATTCTACCCACAGCCGCAACAATTCCTACGCCGTGATCATCACATCTACCTCTAGCTACTACGTGTTCTACATC TATGTGGGCGTGGCCGATACACTGCTGGCCATGGGCTTTTTCCGGGGCCTGCCCCTGGTGCACACACTGATCACCGTGAGCAAGATCCTGCACCACAAGATGCTGCACAGCGTGCTGCAGGCCCCTATGTCCACACTGAACACCCTGAAGGCCGGCGGCATCCTGAATCGGTTTTTCCAAGGACATCGCCATCCTGGACGATCTGCTGCCTCTGACCATCTTTGATTTCATCCAGCTGCTGCTGA TCGTGATCGGAGCAATCGCAGTGGTGGCCGTGCTGCAGCCTTACATCTTCGTGGCCACAGTGCCAGTGATCGTGGCCTTTATCATGCTGCGCGCCTATTTCCTGCAGACCAGCCAGCAGCTGAAGCAGCTGGAGAGCGAGGCCGGTCCCCTATCTTTACACACCTGGTGACCTCCCTGAAGGGACTGT GGACACTGAGGGCCTTCGGCCGGCAGCCATACTTTGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACAGCCAATTGGTTTCTGTATCTGAGCACCCTGCGCTGGTTTCAGATGCGGATCGAGATGATCTTCGTGATCTTTTTCATCGCCGTGACCTTCATCTCCATCCTGACAACCGGAGAGGGAGAGGGAAGAGTGGGAATCATCCTGACACTGGCCATGAACATCATGTCTACCCTGC AGTGGGCCGTGAATTCCTCTATCGACGTGGATAGCCTGATGAGATCTGTGAGCAGGGTGTTTAAGTTCATCGACATGCCCACAGAGGGCAAGCCTACAAAGAGCACCAAGCCATACAAGAACGGCCAGCTGTCCAAAGTGATGATCATCGAGAATTCTCACGTGAAGAAGGACGATATCTGGCCATCCGG (SEQ ID NO:43)

[0011] SEQ ID NO:43 is a nucleotide sequence that has been codon-optimized for expression in humans and encodes a biologically active truncated human CFTR protein lacking amino acids 708-759. In some embodiments, the invention provides an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:43 or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto, wherein optionally the nucleotide sequence is operably linked to an expression control regulatory sequence. The present invention also relates to plasmids and vectors comprising the nucleic acid sequence of SEQ ID NO:43 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical thereto, and host cells comprising such plasmids and vectors. The present invention also relates to the use of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:43 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical thereto, wherein optionally the nucleotide sequence is operably linked to an expression control regulatory sequence, for the treatment of cystic fibrosis or a lung disease associated therewith, as described in the present invention, or to use in the preparation of a medicament for the treatment of cystic fibrosis or a lung disease associated therewith.

[0012] In some aspects, the promoter is a constitutive promoter, optionally a truncated cytomegalovirus immediate/early enhancer/promoter (CMVie), and operably linked to a nucleotide sequence encoding human CFTR or a biologically active fragment thereof.

[0013] In yet other aspects, the promoter is a tissue-specific promoter, wherein the promoter preferably directs the preferential expression of a nucleic acid in a lung cell and is operably linked to a nucleotide sequence encoding human CFTR or a biologically active fragment thereof.

[0014] In preferred embodiments, the promoter is a truncated CMVie promoter and is operably linked to a nucleotide sequence encoding human CFTR or a biologically active fragment thereof. In a particularly preferred embodiment, the CMVie promoter is CMV173 having the following sequence or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto:

ACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGT (SEQ ID NO:44)

[0015] In a particularly preferred embodiment, the rAAV vector comprises (i) a capsid comprising the capsid protein of SEQ ID NO:12, and (ii) a nucleic acid comprising, in 5' to 3' direction: (a) an AAV2 terminal repeat (b) a promoter (c) a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence, (d) a polyadenylation sequence, and (e) an AAV2 terminal repeat, wherein the nucleic acid comprises, in 5' to 3' direction, the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to it:

TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATA ACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAG CAGAGCTCGTTTAGTGAACCGTCAGAATTCTCGAGTGATCGAAAGAGCCTGCTAAAGCAAAAAAGAAGTCACCATGCAGCGCAGCCCACTGGAGAAGGCAAGCGTGGTGTCCAAGCTGTTCTTTTCCTGGACCAGGCCTATCCTGAGGAAGGGATACAGGCAGCGGCTGGAGCTGAGCGACATCTATCAGATCCCTTCTGTGGACAGCGCCGATAATCTGTCCGAGAAGCTGGAGAGAGAGTGGG ATAGGGAGCTGGCCTCTAAGAAGAACCCAAAGCTGATCAATGCCCTGCGGAGATGCTTCT TTTGGCGGTTCATGTTCTACGGCATCTTCCTGTATCTGGGCGAGGTGACCAAGGCCGTGCAGCCACTGCTGCTGGGCAGAATCATCGCCTCTTACGACCCCGATAACAAGGAGGAGAGGAGCATCGCCATCTATCTGGGCATCGGCCTGTGCCTGCTGTTTATCGTGAGGACACTGCTGCTGCACCCAGCCATCTTCGGCCTGCACCACATCGGCATGCAGATGAGAATCGCCATGTT CAGCCTGATCTACAAGAAGACCCTGAAGCTGAGCTCCAGGGTGCTGGACAAGATCTCCATCGGCCA GCTGGTGTCCCTGCTGTCTAACAATCTGAACAAGTTTGATGAGGGACTGGCCCTGGCACACTTCGTGTGGATCGCACCACTGCAGGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCAAGCGCCTTTTGCGGACTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGGCAGGACTGGGACGCATGATGATGAAGTACAGAGACCAGAGGGCCGGCAAGATCTCTGAGCGGCT GGTCATCACCAGCGAGATGATCGAGAACATCCAGTCCGTGAAGGCCTATTGTTGGGAGGAGGCCA TGGAGAAGATGATCGAGAATCTGCGCCAGACAGAGCTGAAGCTGACCAGAAAGGCCGCCTACGTGAGGTACTTCAACTCTAGCGCCTTCTTTTTCTCTGGCTTTTTCGTGGTGTTCCTGAGCGTGCTGCCATACGCCCTGATCAAGGGCATCCTGCGGAAGATCTTTACCACAATCTCCTTCTGCATCGTGCTGAGAATGGCCGTGACAAGGCAGTTTCCCTGGGCCGTGCAGACC TGGTATGACTCTCTGGGCGCCATCAATAAGATCCAGGATTTCCTGCAGAAGCAGGAGTACAAGAC ACTGGAGTATAACCTGACCACAACCGAGGTGGTCATGGAGAATGTGACCGCCTTCTGGGAGGAGGGCTTTGGCGAGCTGTTCGAGAAGGCCAAGCAGAACAATAACAATCGCAAGACATCTAACGGCGACGATAGCCTGTTTTTCAGCAATTTTTCCCTGCTGGGCACCCCCGTGCTGAAGGACATCAACTTCAAGATCGAGAGGGGACAGCTGCTGGCAGTGGCAGGCTCCACAGGCGCCGGCA AGACCTCTCTGCTGATGATGATCATGGGCGAGCTGGAGCCAAGCGAGGGCAAGATCAAGC ACTCCGGCCGGATCTCTTTTTGCAGCCAGTTCTCCTGGATCATGCCCGGGCACCATCAAGGAGAATATCATCTTTGGCGTGTCCTACGATGAGTACAGATATAGGTCTGTGATCAAGGCCTGTCAGCTGGAGGAGGACATCAGCAAGTTCGCCGAGAAGGATAACATCGTGCTGGGCGAGGGCGGCATCACACTGAGCGGAGGACAGAGGGCAAGGATCTCCCTGGCCAGAGCCGTGTACAAGGA CGCCGATCTGTATCTGCTGGACAGCCCCTTTGGCTATCTGGATGTGCTGACCGAGAAGGAG ATCTTCGAGTCCTGCGTGTGCAAGCTGATGGCCAATAAGACAAGGATCCTGGTGACCTCTAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGATCCTGCACGAGGGCTCCTCTTACTTTTATGGCACATTCAGCGAGCTGCAGAATCTGCAGCCTGACTTCAGCTCCAAGCTGATGGGCTGTGACTCCTTTGATCAGTTCTCTGCCGAGAGGCGCAACTCCATCCTGACAGAGACCCT GCACAGATTCTCTCTGGAGGGCGACGCACCCGTGAGCTGGACAGAGACCAAGAAGCAGTCCTT TAAGCAGACCGGCGAGTTCGGCGAGAAGAGGAAGAATTCTATCCTGAACCCTATCAATAGCACACTGCAGGCCCGGAGAAGGCAGTCTGTGCTGAACCTGATGACCCACAGCGTGAACCAGGGCCAGAATATCCACAGAAAGACAACCGCCAGCACAAGGAAGGTGTCCCTGGCACCTCAGGCAAACCTGACCGAGCTGGACATCTACTCCCGCCGGCTGTCTCAGGAGACCGGACTGGAGAT CTCTGAGGAGATCAATGAGGAGGATCTGAAGGAGTGCTTTTTTCGACGATATGGAGAGCATC CCAGCCGTGACAACCTGGAACACATACCTGCGCTATATCACCGTGCACAAGTCCCTGATCTTTGTGCTGATCTGGTGTCTGGTCATCTTCCTGGCAGAGGTGGCAGCATCTCTGGTGGTGCTGTGGCTGCTGGGCAACACACCCCTGCAGGACAAGGGCAATTCTACCCACAGCCGCAACAATTCCTACGCCGTGATCATCACATCTACCTCTAGCTACTACGTGTTCTACATCTATGTGGG CGTGGCCGATACACTGCTGGCCATGGGCTTTTTCCGGGGCCTGCCCCTGGTGCACACACTGAT CACCGTGAGCAAGATCCTGCACCACAAGATGCTGCACAGCGTGCTGCAGGCCCTATGTCCACACTGAACACCCTGAAGGCCGGCGGCATCCTGAATCGGTTTTTCCAAGGACATCGCCATCCTGGACGATCTGCTGCCTCTGACCATCTTTGATTTCATCCAGCTGCTGCTGATCGTGATCGGAGCAATCGCAGTGGTGGCCGTGCTGCAGCCTTACATCTTCGTGGCCACAGTGCCAGTG ATCGTGGCCTTTATCATGCTGCGCGCCTATTTCCTGCAGACCAGCCAGCAGCTGAAGCAGCTG GAGAGCGAGGGCCGGTCCCCTATCTTTACACACCTGGTGACCTCCCTGAAGGGACTGTGGACACTGAGGGCCTTCGGCCGGCAGCCATACTTTGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACAGCCAATTGGTTTCTGTATCTGAGCACCCTGCGCTGGTTTCAGATGCGGATCGAGATGATCTTCGTGATCTTTTTCATCGCCGTGACCTTCATCTTCCATCCTGACAACCGGAGAGG GAGAGGGAAGAGTGGGAATCATCCTGACACTGGCCATGAACATCATGTCTACCCTGCAGTG GGCCGTGAATTCCTCTATCGACGTGGATAGCCTGATGAGATCTGTGAGCAGGGTGTTTAAGTTCATCGACATGCCCACAGAGGGCAAGCCTACAAAGAGCACCAAGCCATACAAGAACGGCCAGCTGTCCAAAGTGATGATCATCGAGAATTCTCACGTGAAGAAGGACGATATCTGGCCATCCGGAGGACAGATGACCGTGAAGGATCTGACAGCCAAGTATACCGAGGGCGGCAACGCCATCC TGGAGAATATCTCCTTTTCTATCAGCCCTGGACAGAGGGTGGGACTGCTGGGACGGACA GGCTCCGGCAAGTCTACCCTGCTGAGCGCCTTCCTGAGGCTGCTGAATACAGAGGGCGAGATCCAGATCGACGGCGTGAGCTGGGATTCCATCACCCTGCAGCAGTGGAGAAAGGCCTTTGGCGTGATCCCTCAGAAGGTGTTTATCTTCTCCGGCACCTTCAGGAAGAACCTGGACCCATACGAGCAGTGGTCTGATCAGGAGATCTGGAAGGTGGCCGACGAAGTGGGCCTGAGA TCTGTGATCGAGCAGTTTCCAGGCAAGCTGGACTTCGTGCTGGTGGATGGAGGATGCGTGCTGAGCCA CGGACACAAGCAGCTGATGTGCCTGGCCAGGTCTGTGCTGAGCAAGGCCAAGATCCTGCTGCTGGACGAGCCAAGCGCCCACCTGGATCCCGTGACATACCAGATCATCAGAAGGACCCTGAAGCAGGCCTTTGCCGATTGCACCGTGATCCTGTGCGAGCACCGCATCGAGGCCATGCTGGAGTGCCAGCAGTTCCCTGGTCATCGAGGAGAACAAGGTGCGGCAGTATGACAGCATCCAGAA GCTGCTGAATGAGCGGAGCCTGTTTCGGCAGGCCATCTCCCCCTCTGATCGCGTGAAGCTGT TCCCTCACCGGAACAGCTCCAAGTGTAAGTCCAAGCCCCAGATCGCCGCCCTGAAGGAGGAGACAGAGGAGGAGGTGCAGGACACCAGACTGTGAAATAAAACATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAACAACGGCCGGCCGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGC GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO: 45).

[0016] A nucleic acid having the nucleotide sequence of SEQ ID NO:45 comprises, in 5' to 3' direction: (a) an AAV2 terminal repeat, (b) a CMV173 promoter of SEQ ID NO:44, (c) a codon-optimized nucleotide sequence encoding a biologically active truncated human CFTR protein lacking amino acids 708-759 of SEQ ID NO:43, (d) a polyadenylation sequence, and (e) an AAV2 terminal repeat.

[0017] The present invention also relates to methods for treating cystic fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an infectious rAAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12, and (ii) a nucleic acid comprising a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence, operably linked to a promoter. In preferred embodiments, the nucleotide sequence encoding CFTR has the sequence of SEQ ID NO: 43, and/or the promoter has the sequence of SEQ ID NO: 44, and/or the nucleic acid comprises the sequence of SEQ ID NO: 45. In some aspects, the subject is administered an amount of rAAV effective to reduce one or more features of cystic fibrosis, non-limiting examples of which include upper and lower airway inflammation, aberrant epithelial cytokine signaling, and elevated IgE levels.

[0018] In yet other aspects, the present invention relates to methods of treating a cystic fibrosis-associated lung disease, including, but not limited to, an upper respiratory tract disease, a lower respiratory tract disease, a nasopharyngeal disease, sinusitis, and/or a salivary gland disease associated with cystic fibrosis, comprising administering to a subject a therapeutically effective amount of an infectious rAAV comprising (i) a capsid comprising the capsid protein of SEQ ID NO:12, and (ii) a nucleic acid comprising a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence, operably linked to a promoter. In preferred embodiments, the nucleotide sequence encoding CFTR has the sequence of SEQ ID NO: 43, and/or the promoter has the sequence of SEQ ID NO: 44, and/or the nucleic acid comprises the sequence of SEQ ID NO: 45.

[0019] The rAAV gene therapy vectors of the present invention, comprising a capsid comprising a capsid protein of SEQ ID NO:12 and nucleic acid sequences encoding CFTR or a biologically active fragment thereof (e.g., comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO:43, optionally linked to a promoter of SEQ ID NO:44 and/or comprising the nucleotide sequence of SEQ ID NO:45), can be administered to a patient in a variety of ways to achieve and maintain a therapeutically effective level of CFTR or a fragment thereof, for the treatment of cystic fibrosis or a lung disease associated therewith.

[0020] In some aspects, the infectious rAAV is administered to a subject with cystic fibrosis in one or more doses, each dose comprising from about 1×10 ¹³ to about 1×10 ¹⁵ vector genomes (vg), from about 1×10 ¹³ to about 1×10 ¹⁴ vg, from about 1×10 ¹⁴ to about 1×10 ¹⁵ vg, or from about 1×10 ¹⁵ to about 5×10 ¹⁵ vg. In some preferred aspects, each dose comprises about 1×10 ¹⁴ vg or about 1×10 ¹⁵ vg of rAAV.

[0021] In some aspects, the treatment comprises administering to the subject no more than a single dose and is effective to achieve a prolonged and sustained therapeutic concentration of CFTR or a biologically active fragment thereof. In related aspects, the treatment comprises administering no more than a single dose by inhalation of about 1×10 ¹³ to about 1×10 ¹⁵ plaque forming units (pfu), viral particles (vp), or viral genomes (vg) of rAAV comprising the capsid protein of SEQ ID NO:12 and the nucleic acid of SEQ ID NO:45 to a human with cystic fibrosis. In other aspects, the dosage treatment may be a multiple administration regimen.

[0022] Methods relating to the administration of AAV vectors to humans have been previously described in Kay et al. (2000, Nat. Genet., 24:257-261), the entire contents of which are incorporated herein by reference. In some preferred embodiments, infectious rAAV is administered to a subject by pulmonary, endobronchial, intranasal, intratracheal, and/or intrabronchial administration. In some preferred embodiments, infectious rAAV is administered by nebulizer.

[0023] In related aspects, the present invention provides an infectious rAAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12 and (ii) a nucleic acid comprising a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence operably linked to a promoter, for use in the treatment of cystic fibrosis or for use in the preparation of a medicament for the treatment of cystic fibrosis. In some preferred embodiments, the nucleotide sequence encoding the human CFTR protein comprises or consists of the sequence of SEQ ID NO: 43 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto, operably linked to a promoter comprising the sequence of SEQ ID NO: 44 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto. In particularly preferred embodiments, the rAAV comprises a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO: 45.

[0024] In yet other embodiments, the present invention relates to a pharmaceutical composition suitable for inhalation, comprising (i) an rAAV, an infectious rAAV comprising (a) a capsid comprising the capsid protein of SEQ ID NO:12, and (b) a nucleic acid comprising a nucleotide sequence encoding one or more gene products in (ii) a buffer comprising from about 10 mM to about 50 mM citrate, from about 70 mM to about 150 mM NaCl, and optionally a surfactant, preferably a nonionic surfactant such as Pluronic F-68, more preferably about 0.005% Pluronic F68, and having a pH of from 5 to 7, preferably having a pH of about 6.0. In some preferred aspects, the pharmaceutical composition comprises from about 20 mM to about 50 mM citrate, from about 85 mM to about 125 mM NaCl, and about 0.005% Pluronic F68, and has a pH of about 6.0. In some particularly preferred aspects, the pharmaceutical composition comprises about 20 mM citrate, about 125 mM NaCl, and about 0.005% Pluronic F68, and has a pH of about 6.0. In preferred embodiments, the pharmaceutical composition comprises a rAAV comprising (a) a capsid comprising the capsid protein of SEQ ID NO:12, and (b) a nucleic acid comprising a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence operably linked to a promoter. In related embodiments, the rAAV comprises a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:45.

[0025] In some aspects, the pharmaceutical composition comprises from 10 ¹¹ to 10 ¹⁴ vector genomes (vg) per ml. In some preferred embodiments, the pharmaceutical composition comprises from about 1×10 ¹³ to about 9×10 ¹³ vg/ml, preferably from about 2×10 ¹³ to 6×10 ¹³ vg/ml. In other preferred embodiments, the pharmaceutical preparation comprises about 1×10 ¹³ wg/mL, about 2×10 ¹³ wg/mL, about 3×10 ¹³ wg/mL, about 4×10 ¹³ wg/mL, about 5×10 ¹³ wg/mL, about 6×10 ¹³ wg/mL, about 7×10 ¹³ wg/mL, about 8×10 ¹³ wg/mL, or about 9×10 ¹³ wg/mL. In a particularly preferred embodiment, the pharmaceutical composition comprises from about 2×10 ¹³ wg/mL to about 5×10 ¹³ wg/mL.

[0026] In some embodiments, the pharmaceutical composition is formulated as a liquid/suspension suitable for aerosol delivery. In related embodiments, the pharmaceutical composition is formulated as an aerosol and/or is an inhalation dosage form.

[0027] The present invention also provides methods of delivering a heterologous nucleic acid to a lung cell, comprising contacting the lung cell with an rAAV virion comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12, and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. In some embodiments, the heterologous nucleic acid encodes a protein and/or small interfering RNA. In some preferred embodiments, the lung cell is any cell of the lung or trachea. In other preferred embodiments, the lung cell is an airway epithelial cell, including, but not limited to, an alveolar epithelial cell, a bronchial (primary, secondary, or tertiary) epithelial cell, or a tracheal epithelial cell. In some preferred aspects, the lung cell is a ciliated airway epithelial cell. In some preferred aspects, the lung cell is a lung alveolar epithelial cell type 1 (AECI) or type 2 (AECII). In other embodiments, the lung cell is a smooth muscle or endothelial cell. In yet other embodiments, the lung cell is a basal cell, a goblet cell, or an oocyte. In particularly preferred embodiments, the rAAV comprises a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:45.

[0028] The present invention also provides methods of delivering a heterologous nucleic acid to the lungs of a subject (e.g., a human), comprising administering to the subject an rAAV virion comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12, and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. In some embodiments, the heterologous nucleic acid encodes a protein and/or small interfering RNA. In related embodiments, methods of delivering a heterologous nucleic acid to the upper respiratory tract, nasopharynx, sinuses, oral/buccal region, and/or salivary glands of a subject (e.g., a human) comprise administering to the subject an rAAV virion comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12, and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. In related aspects, the rAAV or a pharmaceutical composition comprising it is administered to a subject by pulmonary, endobronchial, intranasal, intratracheal and/or intrabronchial administration. In particularly preferred embodiments, the rAAV comprises a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:45.

[0029] The present invention also provides methods for treating a lung disease comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV) comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12, and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products, wherein the one or more gene products are operably linked to a promoter. In some aspects, the heterologous nucleic acid comprises a nucleotide sequence encoding multiple gene products, in which case expression of the multiple gene products (e.g., 2) can be mediated by multiple (e.g., 2) independent promoters or can be mediated by a single promoter, wherein the multiple transgenes are separated by an internal ribosome entry site (IRES) or a 2A peptide sequence. In preferred embodiments, the heterologous nucleic acid encodes a therapeutic protein and/or a therapeutic small interfering RNA. In related aspects, the gene product(s) delivered by the rAAV reduces the level of an interfering gene product and/or introduces or supplements the level of a supporting gene product. In particularly preferred embodiments, the rAAV comprises a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:45. In other preferred embodiments, the rAAV comprises a nucleic acid comprising a nucleotide sequence encoding alpha-1-antitrypsin.

[0030] In some aspects, the lung disease is selected from pulmonary arterial hypertension, pulmonary hypertension, lung cancer (primary, secondary, and metastatic), surfactant deficiency, viral and/or bacterial infection, cystic fibrosis, acute bronchitis, pneumonia (including viral, bacterial, and fungal pneumonia), respiratory tract infection (including pharyngitis, croup, aspergillosis, coccidioidomycosis, hantavirus pulmonary syndrome, and histoplasmosis), chemical and hypersensitivity pneumonitis, tuberculosis and other mycobacterial infections (including, but not limited to, Mycobacterium avium ), sarcoidosis, respiratory syncytial virus infection, pulmonary edema, acute respiratory distress syndrome (ARDS), pneumoconiosis (including black lung disease, asbestosis, and silicosis), interstitial lung disease (including sarcoidosis and autoimmune disease), pulmonary embolism, pleural effusion, pleurisy, mesothelioma, pneumothorax, acute bronchitis, bronchiolitis (including bronchiolitis obliterans), sudden infant death syndrome, sleep apnea, bronchiectasis, bronchopulmonary dysplasia, cryptogenic organizing pneumonia, e-cigarette or vaping product use-associated lung injury (EVALI), Middle East respiratory syndrome (MERS), primary ciliary dyskinesia, severe acute respiratory syndrome (SARS), alpha-1 antitrypsin deficiency, asthma, interstitial lung disease, and COVID-19 (coronavirus disease 2019). In other aspects, the lung disease is chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF). In related aspects, there is provided a method of treating COVID-19 comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV) comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12 and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products operably linked to one or more promoters, or a pharmaceutical composition comprising the rAAV, wherein the gene product(s) knocks down, modifies, and/or overexpresses a viral or host cell gene product to reduce or eliminate viral pathogenicity or replication in either the lungs or the nasopharynx, and/or expresses a neutralizing antibody against an epitope on the virus.

[0031] In some aspects, genes that can be targeted to treat IPF include, but are not limited to, SFTPA1 (surfactant A1) and caveolin-1. Genes that may be targeted for treatment of COPD include, but are not limited to, alpha-1-antitrypsin, alpha-1-antichymotrypsin, alpha-1-macroglobulin, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 12 (MMP12), microsomal epoxide hydrolyase, CYP1A1, glutathione S-transferase, heme oxygenase-1, TGF-beta-1, TNF-alpha, IL-1 complex, IL-8, IL-13, human leukocyte antigen (HLA-B7 and Bw16), vitamin D-binding protein, and beta-2-adrenergic receptor.

[0032] In related aspects, the rAAV or pharmaceutical composition is administered by pulmonary, endobronchial, intranasal, intratracheal, and/or intrabronchial administration to treat a pulmonary disease in a subject in need thereof. In some preferred embodiments, the infectious rAAV is administered using a nebulizer.

[0033] In yet other aspects, at least one dose of about 10 ¹² to 10 ¹⁴ vector genomes (vg)/kg rAAV is administered to a subject for treating a pulmonary disease. In related aspects, the subject is administered from about 1×10 ¹¹ to about 1×10 ¹⁴ wg/kg, from about 1×10 ¹² to about 9×10 ¹³ wg/kg, from about 1×10 ¹² wg/kg to about 9×10 ¹² wg/kg, preferably from about 2×10 ¹² wg/kg to about 3×10 ¹² wg/kg, more preferably about 2.6×10 ¹² wg/kg, about 2.7×10 ¹² wg/kg, about 2.8×10 ¹² wg/kg, about 2.9×10 ¹² wg/kg, about 3.0×10 ¹² wg/kg, or about 3.1×10 ¹² wg/kg. In preferred embodiments, the subject is administered one or more doses in one or more dosages, each dose comprising from about 1×10 ¹³ to about 1×10 ¹⁵ vector genomes (vg), from about 1×10 ¹³ to about 1×10 ¹⁴ vg, from about 1×10 ¹⁴ and about 1×10 ¹⁵ vg, or about 1×10 ¹⁵ and about 5×10 ¹⁵ vg rAAV. In some preferred aspects, each dose comprises about 1×10 ¹⁴ vg or about 1×10 ¹⁵ vg rAAV.

[0034] The present invention further relates to infectious recombinant adeno-associated virus (rAAV) virions that comprise a variant capsid protein and a heterologous nucleic acid. The invention also relates to variant adeno-associated virus (AAV) capsid proteins (and/or nucleic acid encoding variant AAV capsid proteins) that confer on the infectious rAAV virion increased resistance to human neutralizing anti-AAV antibodies. The present invention further relates to host cells comprising an infectious rAAV virion and/or a nucleic acid encoding a variant AAV capsid protein of the present invention. The present disclosure further provides libraries of the above virions, capsid proteins, nucleic acids, and/or host cells; wherein the variant AAV capsid protein of at least one library member comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33.

[0035] The present invention also relates to methods of delivering a heterologous nucleic acid to a target cell, wherein the target cell is contacted with a claimed infectious rAAV virion. The present invention further relates to methods of delivering a gene product to a subject, wherein the methods typically comprise administering an effective amount of a claimed rAAV virion to a subject in need thereof. The present invention also relates to compositions and kits for use in the claimed methods.

[0036] Features of the present invention include an infectious recombinant adeno-associated virus (rAAV) virion comprising (a) a variant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33; and (b) a heterologous nucleic acid. In some cases, the variant AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity to an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the variant AAV capsid protein comprises an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33.

[0037] Features of the present invention include an infectious recombinant adeno-associated virus (rAAV) virion comprising (a) a variant adeno-associated virus (AAV) capsid protein that comprises an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 10 and comprises the amino acid substitutions N312K, N449D, D472N, N551S, 1698V, and L735Q relative to SEQ ID NO: 2; and (b) a heterologous nucleic acid. In some cases, the variant AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 10. In some cases, the rAAV exhibits increased resistance to human neutralizing anti-AAV antibodies compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). In some cases, rAAV exhibits at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater resistance to human neutralizing anti-AAV antibodies than the resistance exhibited by AAV2. In some cases, rAAV exhibits enhanced transduction of mammalian cells in the presence of human neutralizing anti-AAV antibodies compared to the transduction of mammalian cells exhibited by wild-type AAV serotype 2 (AAV2). In some cases, the mammalian cells are liver cells, pancreatic cells, skeletal muscle cells, cardiac muscle cells, fibroblasts, retinal cells, joint synovial cells, lung cells, T cells, neurons, glial cells, stem cells (e.g., hematopoietic stem cells, hematopoietic progenitor cells, neural stem cells, neural progenitor cells, neural crest stem cells, embryonic stem cells, induced pluripotent stem cells (iPS), mesenchymal stem cells, mesodermal stem cells, liver stem cells, pancreatic stem cells, pancreatic progenitor cells, muscle stem cells, retinal stem cells, etc.), endothelial cells, or tumor cells. In some cases, the heterologous nucleic acid comprises an RNAi agent. In some cases, the heterologous nucleic acid contains a nucleotide sequence encoding a polypeptide.

[0038] Features of the present invention include an isolated nucleic acid comprising a nucleotide sequence that encodes a variant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the encoded variant AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity to an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the encoded variant AAV capsid protein comprises an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33.

[0039] Features of the present invention include an isolated nucleic acid comprising a nucleotide sequence that encodes a variant adeno-associated virus (AAV) capsid protein that comprises an amino acid sequence that has at least about 95% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 10 and includes the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q relative to SEQ ID NO: 2.

[0040] In some cases, the encoded variant AAV capsid protein (encoded by the isolated nucleic acid) confers on the infectious recombinant adeno-associated virus (rAAV) virion increased resistance to human neutralizing anti-AAV antibodies compared to the resistance exhibited by AAV2 (wild type AAV serotype 2). In some cases, the increased resistance is at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater than the resistance exhibited by AAV2. In some cases, the encoded variant AAV capsid protein (encoded by isolated nucleic acid) confers the infectious recombinant adeno-associated virus (rAAV) virion enhanced transduction of mammalian cells in the presence of neutralizing human anti-AAV antibodies compared to transduction exhibited by AAV2.

[0041] Features of the present invention include an isolated host cell comprising the claimed nucleic acid as described above. In some cases, the host cell is stably transfected with the nucleic acid. In some cases, the host cell further comprises a nucleic acid comprising a nucleotide sequence encoding an AAV rep protein. In some cases, the host cell further comprises a recombinant AAV vector.

[0042] Features of the present invention include a method of delivering a heterologous nucleic acid to a target cell, comprising contacting the target cell with the claimed virion (described above). In some cases, the target cell is a liver cell, a pancreatic cell, a skeletal muscle cell, a cardiac muscle cell, a fibroblast, a retinal cell, a joint synovial cell, a lung cell, a T cell, a neuron, a glial cell, a stem cell (e.g., a hematopoietic stem cell, a hematopoietic progenitor cell, a neural stem cell, a neural progenitor cell, a neural crest stem cell, an embryonic stem cell, an induced pluripotent stem cell (iPS), a mesenchymal stem cell, a mesodermal stem cell, a liver stem cell, a pancreatic stem cell, a pancreatic progenitor cell, a muscle stem cell, a retinal stem cell, etc.), an endothelial cell, or a tumor cell. In some cases, the target cell is in vitro. In some cases, the target cell is in vivo.

[0043] Features of the present invention include a method of delivering a gene product to a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the claimed infectious recombinant adeno-associated virus (rAAV) virion (described above). In some cases, the heterologous nucleic acid of the rAAV virion comprises an RNA interfering agent. In some cases, the heterologous nucleic acid of the rAAV virion comprises a nucleotide sequence encoding a polypeptide. In some cases, the step of administering comprises indirect delivery of the infectious rAAV virion. In some cases, the step of administering comprises direct delivery of the infectious rAAV virion.

[0044] Features of the present invention include a variant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least about 90% identity to an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the AAV capsid protein comprises an amino acid sequence having at least about 95% amino acid sequence identity to an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33. In some cases, the AAV capsid protein comprises an amino acid sequence shown in one of SEQ ID NOs: 11-13 and 26-33.

[0045] Features of the present invention include a variant adeno-associated virus (AAV) capsid protein that comprises an amino acid sequence that has at least about 95% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 10 and comprises the amino acid substitutions N312K, N449D, D472N, N551S, 1698V, and L735Q relative to SEQ ID NO: 2. In some cases, the variant AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 10. In some cases, the variant AAV capsid protein confers on the infectious recombinant adeno-associated virus (rAAV) virion increased resistance to human neutralizing anti-AAV antibodies compared to the resistance exhibited by AAV2. In some cases, the increased resistance is at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater than the resistance exhibited by AAV2. In some cases, the variant AAV capsid protein confers an infectious recombinant adeno-associated virus (rAAV) virion increased transduction of mammalian cells in the presence of neutralizing human anti-AAV antibodies compared to transduction exhibited by AAV2.

[0046] Features of the present invention include a library comprising at least one of: (i) two or more infectious rAAV virions, each comprising a variant adeno-associated virus (AAV) capsid protein and a heterologous nucleic acid; (ii) two or more isolated nucleic acids, each comprising a nucleotide sequence encoding a variant AAV capsid protein; (iii) two or more host cells, each comprising a nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein; and (iv) two or more variant AAV capsid proteins; wherein the variant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33.

[0047] Features of the present invention include a method of producing and identifying a modified infectious rAAV virion that exhibits an altered infectivity property compared to a parent virion comprising a parent capsid protein, the method comprising: (a) producing variant adeno-associated virus (AAV) capsid proteins from a parent capsid protein, wherein the parent capsid protein comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33, and wherein each variant AAV capsid protein comprises at least one amino acid substitution relative to the parent capsid protein; (b) producing variant AAV virions, each comprising a variant AAV capsid protein obtained in step (a); and (c) analyzing the variant AAV virions obtained in step (b) for the altered infectivity property to identify the modified infectious rAAV virion. In some cases, generating a library of variant AAV capsid proteins comprises a mutagenesis method selected from the group consisting of: polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, loop exchange mutagenesis, fragment shuffling mutagenesis, and a combination thereof. In some cases, the altered infectivity property is increased resistance to human anti-AAV neutralizing antibodies compared to the resistance exhibited by the parental virion. In some cases, the altered infectivity property is increased transduction of mammalian cells in the presence of human anti-AAV neutralizing antibodies compared to the transduction exhibited by the parental virion. In some cases, the modified rAAV infectious virion comprises a modified AAV capsid protein comprising an amino acid sequence having at least about 90% amino acid sequence identity to the parental capsid protein.

[0048] Features of the present invention include a method for producing a variant AAV capsid protein from a parent capsid protein, wherein the method comprises: subjecting a nucleic acid comprising a nucleotide sequence encoding the parent capsid protein to a type of mutagenesis selected from the group consisting of: polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, loop exchange mutagenesis, fragment shuffling mutagenesis, and a combination thereof; wherein the parent capsid protein comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33.

Brief description of the figures

[0049] Fig. 1A-B shows directed evolution of AAV to enhance antibody evasion.

[0050] Figs. 2A-2B show neutralization profiles of antibody evading variants using human IVIG.

[0051] Figs. 3A-3C show neutralization profiles of antibody evasion variants using human serum obtained from subjects who were excluded from clinical trials with hemophilia B due to the presence of high titers of neutralizing anti-AAV antibodies.

[0052] Fig. 4A-4B show the amino acid sequences of the loop replacement/shuffling and saturation mutagenesis clones.

[0053] Fig. 5 shows the tropism of AAV variants in vitro.

[0054] Fig. 6A-6B show localization and neutralization of novel AAV variants in vivo.

[0055] Fig. 7A-7D show the number of antibody evasion variants.

[0056] Fig. 8A-8I shows the Shuffle 100-1 capsid protein sequence (SEQ ID NO: 11) aligned with the wild-type AAV1-9 capsid protein sequences (SEQ ID NOs: 1-9).

[0057] Fig. 9A-9I shows the Shuffle 100-3 capsid protein sequence (SEQ ID NO: 12) aligned with the wild-type AAV1-9 capsid protein sequences (SEQ ID NOs: 1-9).

[0058] Fig. 10A-I shows the Shuffle 100-7 capsid protein sequence (SEQ ID NO: 13) aligned with the wild-type AAV1-9 capsid protein sequences (SEQ ID NOs: 1-9).

[0059] Fig. 11 shows the neutralizing antibody titers of library clones and parental serotypes in the serum of immunized mice.

[0060] Fig. 12 shows the directed evolution process used to identify the capsid variant "A101" (containing the capsid protein of SEQ ID NO:12) with enhanced gene delivery to the lungs in the presence of human neutralizing antibodies.

[0061] Fig. 13A-13B: Fig. 13A shows the estimated genetic diversity of the capsid libraries used for the directed evolution process. The total diversity of the libraries is greater than 1 billion genetic variants. Fig. 13B shows the productivity of the capsid libraries. All capsid libraries were generated at a level sufficient to provide material for study in the in vivo therapeutic vector evolution program. The viral genomes (vg) delivered represent the target dose, excluding losses associated with the delivery device and route of administration.

[0062] Fig. 14A-14B: Fig. 14A shows external PCR amplification of viral genomes from isolated AT II cells after a) AeroProbe® administration or b) nebulizer administration in the first round of selection. Bands in blue boxes represent successful amplification of viral genomes. The temperature gradient represents the annealing temperatures used during PCR corresponding to each lane in the gel. Fig. 14B shows internal PCR amplification of viral genomes from isolated AT II cells after a) AeroProbe® administration or b) nebulizer administration in the first round of selection. Bands in blue boxes represent successful amplification of viral genomes.

[0063] Fig. 15A-15B: Fig. 15A shows the frequency of the chimeric motif in the sequencing analysis for the study. The sequencing analysis is based on the overall frequency in the sequenced population for both the AeroProbe and Nebulizer delivery devices. Fig. 15B shows the frequency of the A101 variant in the chimeric motif for the study. The sequencing analysis is based on the overall frequency in the sequenced population for the AeroProbe and Nebulizer delivery devices.

[0064] Fig. 16 shows a schematic of the sampling of lungs (Examples 3 and 7). Schematic representation of the sampling of the trachea and lungs. The circles in the right lung represent adjacent samples obtained for DNA and protein extraction. Samples oriented along the long and short axes for tissue sectioning are represented by squares.

[0065] Figure 17 shows the variant capsid (containing the capsid protein of SEQ ID NO:12). Transduction with NHP serum samples at a 1:10 serum dilution. Serum samples from NHPs selected for inclusion in the study were assayed for neutralizing anti-AAV antibodies. Transduction in the presence of the 1:10 serum dilution (compared to transduction in the absence of serum) occurred in all NHPs. NHPs selected for inclusion in the study are indicated by yellow bars. Error bars = standard deviation, n=3 (internal replicates).

[0066] Figure 18 shows variant capsid-mediated genome biodistribution. Quantification of viral genomes in the lung and additional systemic organs using qPCR with primers and probe against the EGFP transgene. Viral genomes were detected in all 48 samples (n=16 samples per NHP; n=3 NHP). All skeletal muscle (triceps brachii, vastus lateralis), diaphragm, kidney, spleen, brain, and spinal cord samples tested were below the limit of quantification. Mean ± SEM; n=3 NHPs (n=16 lung biopsy sites per NHP, n=10 liver biopsy sites per NHP, n=15 heart biopsy sites per NHP, n=9 skeletal muscle biopsy sites per NHP, n=2 kidney samples per NHP, n=1 spleen sample per NHP, n=8 brain biopsy sites per NHP, n=3 spinal cord biopsy sites per NHP).

[0067] Figure 19 shows variant capsid-mediated protein expression in the lung. Quantification of EGFP protein expression in the lung using an ELISA against EGFP protein. EGFP expression was observed in all 48 lung samples (n=16 samples per NHP; n=3 NHP). EGFP expression was observed in 10 liver samples positive for viral genomes (n=10 samples per NHP; n=3 NHP). Mean ± SEM.

[0068] Figure 20 shows variant capsid-mediated protein localization in the lung. Representative images of EGFP expression in the trachea (a-b), bronchi (c, e, g), and alveoli (d, f, h) of NHP V002969. Sections marked with white boxes in the trachea (b), alveoli (d), and bronchi (e) are shown as enlarged images in i, j, and k, respectively. Approximate image locations are indicated by magenta boxes in the diagram. EGFP expression is detected with anti-GFP antibody (red) in all images. Nuclei were counterstained with DAPI (blue).

[0069] Figures 21A-21D show characterization of non-human primate type II alveolar epithelial cells. NHP AECII cells were greater than 90% positive for LysoTracker staining as demonstrated by fluorescence microscopy (Figure 17A) and quantified by flow cytometry (Figure 21B). Surfactant protein C, a mature marker of AECII cells, was clearly visible at days 1 and 5 after plating (Figure 21C). AECII cells decreased in proliferation rate in culture over time as demonstrated by EdU incorporation (Figure 21D). EdU=5-ethynyl-2'-deoxyuridine, error bars=standard deviation, n=3 internal replicates.

[0070] Figures 22A-22B show characterization of the non-human primate type II alveolar epithelial cell vector. The rAAV capsid containing the capsid protein of SEQ ID NO:12 (4D-A101) showed a higher transduction rate than the AAV5 capsid, both of which carry CAG-eGFP, in ALI AECII NHP cell cultures. Quantification of eGFP-positive cells by flow cytometry (Figure 22A). Representative ICC images of eGFP positive cells (Figure 22B). Time post infection was 3 days, total of 5 days in culture. Error bars = standard deviation, n=3 internal replicates. Student's t-test, p<0.05 versus AAV5.

[0071] Figures 23A-23D show characterization of human alveolar epithelial type II cells. Human AECII cells were approximately 80% positive for LysoTracker staining until day 11 in culture, when they declined to 50% as assessed by fluorescence microscopy (Figure 23A) and quantified by flow cytometry (Figure 23B). Surfactant protein C, a mature marker of AECII cells, was clearly visible at days 5 and 11 after plating (Figure 23C). AECII cells decreased in proliferation rate over time in culture as assessed by EdU incorporation (Figure 23D). EdU=5-ethynyl-2'-deoxyuridine, error bars=standard deviation, n=3 internal replicates.

[0072] Figure 24 shows the characterization of vectors for human alveolar epithelial type II cells. The capsid containing the capsid protein of SEQ ID NO:12 (4D-A101) showed a higher transduction rate than the AAV5 capsid, both of which carry CAG-eGFP, in human ALI AECII cell cultures. Representative images of ICC eGFP-positive cells. Time after infection is 6 and 10 days, total of 7 and 11 days in culture.

[0073] Figure 25 shows the in vitro neutralization profiles of wild-type AAV1, AAV2, AAV5, AAV8, AAV9, and rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 (4D-A101). rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 demonstrated superior ability to evade neutralizing anti-AAV antibodies in human IVIG compared to wild-type AAV. AAV.CAG.Luciferase vectors were incubated with dilutions of IVIG prior to infection of 2V6.11 cells at an MOI of 1000. Vectors capable of evading antibodies were transduced and luciferase activity was measured 48 h post infection. IVIG=intravenous immunoglobulin, error bars=standard deviation, n=3, internal replicates. *p < 0.05 for 4D-A101 versus AAV1, AAV2, AAV8 and AAV9, †p < 0.05 for 4D-A101 versus AAV5.

[0074] Figure 26 shows a plot of net charge versus pH for the A101 capsid proteins VP1 and VP3.

[0075] Figure 27 shows a pH solubility graph of A101-GFP after 1 day of storage at room temperature.

[0076] Figures 28A-C show that transduction results in robust protein expression and membrane localization in HEK2v6.11 cells. HEK2v6.11 were transduced with 4D-710 and probed by Western blotting (Figure 28A) with anti-CFTR antibody (Figure 28A). Representative images (Figure 28B) show cells analyzed by immunocytochemistry, anti-CFTR (red), F-actin (green), DAPI, nuclear (blue). Scale bars represent 100 μM (Figure 28B) and 25 μM (Figure 28C).

[0077] Figures 29A-B show transduction of 16HBE14o-G542X cells with 4D-710. Droplet digital PCR (ddPCR) with reverse transcription-ddPCR (RT-ddPCR) was performed on RNA isolated from HBE cultures transduced with 4D-710 at increasing MOIs (Figure 29A). Exogenous CFTRΔR transcript levels were detected and quantified as copies/μL above a set threshold and plotted on a linear scale. BLQ, below the limit of quantification. NT, untransduced. Immunocytochemistry of HBE cultures transduced at MOIs of 35,000 and 50,000 (Figure 29B). Blue represents DAPI and red represents CFTR protein. Scale bar, 100 μm.

[0078] Figure 30 shows ex vivo transduction of healthy ALI lung cultures with 4D-710. ddPCR was performed on cDNA prepared from RNA extracted from 4D-710 transduced cultures. Two primer/probe sets were prepared to specifically differentiate the codon-optimized human CFTRΔR transgene from the endogenous human CFTR gene. Quantitative analysis analyzed the number of droplets above a specified threshold containing the transcript of the primer/probe set of interest. BLQ, below limit of quantification. NT, untransduced.

[0079] Figure 31 shows transduction of NHP serum samples with 4D-A101 at a 1:10 serum dilution. Serum samples from NHPs eligible for inclusion in the study were assayed for neutralizing anti-AAV antibodies to the 4D-710 capsid (4D-A101 containing the capsid protein of SEQ ID NO:12). Transduction in the presence of the 1:10 serum dilution (compared to transduction in the absence of serum) occurred in all NHPs. Error bars = standard deviation, n = 3 (internal replicates).

[0080] Figures 32A-32C show quantification of viral genomes using qPCR with primers and probe against the CFTRΔR transgene. In Figure 32A, viral genomes were consistently detected in lung samples distributed throughout the right lung. In Figure 32B, individual animal lung samples are labeled with the approximate region and lobe of the lung: alveoli (green), primary/secondary bronchi (blue), tertiary/inferior bronchi (red), superior lobe (circle), middle lobe (square), inferior lobe (triangle), accessory lobe (diamond). In Figure 32C showed that viral genomes were quantified in animals dosed at 3 x 10 ¹³ vg in all heart, liver, brain, skeletal muscle (triceps brachii, vastus lateralis, diaphragm), spinal cord, pancreas, kidney, and testis samples tested and were below the limit of quantification. All three animals had detectable viral genomes in the tracheobronchial (TB) lymph node and one animal had detectable viral genomes in the spleen. Mean ± SD.

[0081] Figures 33A-33B show expression of the 4D-710 transgene transcript in the lung. Quantification of CFTRΔR transcript was performed using RT-qPCR with primers and probe against the 4D-710 transgene. Figure 33A shows that transcripts were detected in right lung samples distributed by lobe in animals treated with 3 x 10 ¹³ vg and in all vehicle-treated animals, transcript levels were below BLQ. Figure 33B shows individual lung samples from animals dosed with 3× ¹⁰¹³ vg, labeled with approximate lung area and lobe: alveoli (green), primary/secondary bronchi (blue), tertiary/inferior bronchi (red), superior lobe (circle), middle lobe (square), inferior lobe (triangle), accessory lobe (diamond). Mean ± standard deviation.

[0082] Fig. 34A-B show 4D-710 protein expression in the lungs. CFTR protein expression in the lungs is shown using immunohistochemical staining. Fig. 34A shows CFTR expression in tracheal epithelium, bronchial epithelium, and alveolar sections in each treatment group, representative images. Fig. 34B shows CFTR protein expression in tracheal epithelium, bronchial epithelium, and alveolar sections of 3 x 10 ¹³ g treated animals (individual animals shown), representative images.

[0083] Fig. 35 shows a graph of the solubility of A101-Luc versus pH.

Detailed description of the invention

[0084] Definitions

[0085] Adeno-associated virus is a nonpathogenic parvovirus consisting of a 4.7-kb single-stranded DNA genome within a nonenveloped icosahedral capsid. "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that function as an origin of viral replication and a packaging signal. The rep ORF encodes four nonstructural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP1-3) that assemble to form the 60-mer viral capsid. Finally, the ORF present as an alternative reading frame in the cap gene produces assembly activating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and is involved in the capsid assembly process.

[0086] There are several naturally occurring serotypes and over 100 variants of AAV, each of which differs in amino acid sequence, particularly in the hypervariable regions of the capsid proteins, and thus in its gene delivery properties. No association has been demonstrated between any AAV and any human disease, making recombinant AAV attractive for clinical applications.

[0087] As used herein, the term "AAV" encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 (AAV-3 or AAV3), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAVs that infect primates, "non-primate AAV" refers to AAVs that infect non-primate mammals, "bovine AAV" refers to AAVs that infect mammals belonging to the bovine subfamily, etc.

[0088] As used herein, the term “4D-A101” or “A101” refers to an AAV capsid comprising a capsid protein of the sequence SEQ ID NO:12.

[0089] As used herein, the term "4D-710" refers to a recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO:12, and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45.

[0090] The genomic sequences of the various AAV serotypes, as well as the sequences of the native terminal repeats (TR), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in publicly available databases such as GenBank. See, for example, GenBank accession numbers NC-002077.1 (AAV-1), AF063497.1 (AAV-1), NC-001401.2 (AAV-2), AF043303.1 (AAV-2), J01901.1 (AAV-2), U48704.1 (AAV-3), NC-001729.1 (AAV-3), NC-001829.1 (AAV-4), U89790.1 (AAV-4), NC-006152.1 (AAV-5), AF085716.1 (AAV-5), AF028704.1 (AAV-6), NC-006260.1 (AAV-7), AF513851.1 (AAV-7), AF513852.1 (AAV-8) NC-006261.1 (AAV-8), and AY530579.1 (AAV-9); the disclosure of which is incorporated herein by reference for the disclosure of AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Publication Nos. WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Patent No. 6,156,303.

[0091] Sequences of natural capsid proteins associated with AAV serotypes are known in the art and include: AAV1 (SEQ ID NO: 1), AAV2 (SEQ ID NO: 2), AAV3 (SEQ ID NO: 3), AAV4 (SEQ ID NO: 4), AAV5 (SEQ ID NO: 5), AAV6 (SEQ ID NO: 6), AAV7 (SEQ ID NO: 7), AAV8 (SEQ ID NO: 8), and AAV9 (SEQ ID NO: 9). The term "variant AAV capsid protein" is an AAV capsid protein comprising an amino acid sequence that includes at least one substitution (including a deletion, insertion, etc.) relative to one of the naturally occurring AAV capsid protein sequences shown in SEQ ID NOs: 1-9.

[0092] "AAV virion" or "AAV viral particle" refers to a viral particle consisting of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[0093] "Recombinant" when applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps and other procedures that result in a construct different from the polynucleotide found in nature. A recombinant virus is a virus particle containing a recombinant polynucleotide. The terms accordingly include replicates of the original polynucleotide construct and progeny of the original viral construct.

[0094] If an AAV virion contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a target cell, an RNAi agent or a CRISPR agent to be delivered to a target cell, etc.), it is typically referred to as a "recombinant AAV virion (rAAV)" or "rAAV viral particle." Typically, the heterologous polynucleotide is flanked by at least one, and typically two, AAV inverted terminal repeat (ITR) sequences.

[0095] The term "rAAV vector" encompasses rAAV virions (i.e., rAAV viral particles) (e.g., an infectious rAAV virion), which by definition include an rAAV polynucleotide; and also includes polynucleotides encoding rAAV (e.g., a single-stranded polynucleotide encoding rAAV (ss-rAAV); a double-stranded polynucleotide encoding rAAV (ds-rAAV), e.g., plasmids encoding rAAV, etc.).

[0096] The term "packaging" refers to a series of intracellular events that lead to the assembly and encapsulation of the AAV particle.

[0097] The AAV rep and cap genes refer to polynucleotide sequences encoding adeno-associated virus replication and encapsidation proteins. The AAV rep and cap are referred to herein as the AAV "packaging genes."

[0098] An "AAV helper virus" refers to a virus that allows an AAV (e.g., wild-type AAV) to replicate and be packaged by a mammalian cell. A number of such AAV helper viruses are known in the art, including adenoviruses, herpesviruses, and proxy viruses such as vaccinia virus. Adenoviruses encompass many different subgroups, although adenovirus type 5, subgroup C, is commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and are available from repositories such as the ATCC. Herpes family viruses include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from the ATCC repositories.

[0099] "Helper virus function(s)" refers to the function(s) encoded in the genome of the helper virus that enables replication and packaging of the AAV (in combination with other replication and packaging requirements described herein). As described herein, "helper virus function" can be achieved in a number of ways, including by providing a helper virus or by providing, for example, polynucleotide sequences encoding the desired function(s) to the producer cell in trans. For example, a plasmid or other expression vector containing nucleotide sequences encoding one or more adenoviral proteins is transfected into the producer cell along with the rAAV vector.

[00100] The term "infectious" virus or virus particle is a virus that contains a properly assembled viral capsid and is capable of delivering a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any replicative capacity of the virus. Methods for enumerating infectious virus particles are described elsewhere in this specification and in documents in the art. Viral infectivity can be expressed as the ratio of infectious virus particles to all virus particles. Methods for determining the ratio of infectious virus particles to all virus particles are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11:S337. See also the examples.

[00101] As used herein, the term "tropism" refers to the preferential targeting of a virus (e.g., AAV) to certain host species or certain cell types within a host species. For example, a virus that can infect heart, lung, liver, and muscle cells has a broader (i.e., increased) tropism than a virus that can only infect lung and muscle cells. Tropism can also include a dependence of the virus on certain types of host cell surface molecules. For example, some viruses can only infect cells with surface glycosaminoglycans, while other viruses can only infect cells with sialic acid (such dependencies can be tested using various cell lines deficient in certain classes of molecules as potential host cells for viral infection). In some cases, the tropism of a virus describes the relative preferences of the virus. For example, the first virus may be able to infect all cell types, but infects cells with surface glycosaminoglycans at a much higher level. A second virus may be considered to have the same (or identical) tropism as a first virus if the second virus also prefers the same characteristics (e.g., the second virus also infects cells with surface glycosaminoglycans at a higher level), even if the absolute transduction efficiency is not the same. For example, a second virus may be more efficient than the first virus at infecting any given cell type tested, but if the relative preferences are similar (or identical), the second virus may be considered to have a similar (or identical) tropism as the first virus. In some embodiments, the tropism of a virion comprising a subject variant AAV capsid protein of the present invention is unchanged compared to a naturally occurring virion. In some embodiments, the tropism of a virion comprising a subject variant AAV capsid protein of the present invention is increased (i.e., broadened) compared to a naturally occurring virion. In some embodiments, the tropism of a virion comprising said variant AAV capsid protein is reduced compared to a naturally occurring virion.

[00102] A "replication-competent" virus (e.g., a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and is also capable of replication in an infected cell (i.e., in the presence of a helper virus or helper virus function). In the case of AAV, the ability to replicate typically requires the presence of functional AAV packaging genes. Typically, the rAAV vectors described herein are replication-incompetent in mammalian cells (especially human cells) by lacking one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility that replication-competent AAVs will be generated by recombination between the AAV packaging genes and the incoming rAAV vector. In many embodiments, the rAAV vector preparations described herein contain little or no replication-competent AAV (rcAAV, also referred to as RCA) (e.g., below about 1 rcAAV per ¹⁰² rAAV particles, below about 1 rcAAV per ¹⁰⁴ rAAV particles, below about 1 rcAAV per ¹⁰⁸ rAAV particles, below about 1 rcAAV per ¹⁰¹² rAAV particles, or no rcAAV).

[00103] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form.

[00104] A polynucleotide or polypeptide has a certain percentage "sequence identity" with another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids is the same when the two sequences are compared. Sequence similarity can be determined using a number of different methods. Sequences can be aligned using methods and computer programs to determine identity, including BLAST, available from the World Wide Web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available from the Genetics Computing Group (GCG) package of Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other alignment methods are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow gaps in the sequence. The Smith-Waterman algorithm is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70:173-187 (1997). Also, the program GAP, which uses the Needleman-Wunsch alignment method, can be used for sequence alignment. See J. Mol. Biol., 48:443-453 (1970).

[00105] "Gene" refers to a polynucleotide that performs a function in a cell. For example, a gene may contain an open reading frame capable of encoding a specific protein after its transcription and translation. On the other hand, a gene may encode an untranslated product of a functional RNA (e.g., an aptamer, interfering RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), etc.).

[00106] A "gene expression product" or "gene product" is a molecule obtained as a result of the expression of a particular gene, as defined above. Gene expression products include, for example, a polypeptide, an aptamer, an interfering RNA, a messenger RNA (mRNA), rRNA, tRNA, a non-coding RNA (ncRNA), and the like.

[00107] An "RNA interfering agent" or "RNAi agent" includes any agent (or a polynucleotide encoding such an agent) that can be used to alter the expression of a gene (as defined above). Examples of RNAi agents known to those skilled in the art include, but are not limited to, (i) siRNA agents; (ii) antisense RNA; (iii) CRISPR agents; (iv) zinc finger nuclease agents; and (v) transcription effector-like activator nuclease (TALEN)-based agents.

[00108] (i) The siRNA agent ("small interfering" or "short interfering RNA" (or siRNA)) is an RNA duplex of nucleotides that targets a gene of interest ("target gene"). "RNA duplex" refers to a structure formed by the complementary pairing of two sections of an RNA molecule to form a double-stranded RNA (dsRNA) region. The siRNA is "targeted" to a gene when the nucleotide sequence of the duplex region of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the siRNA duplex is less than 30 nucleotides long. In some embodiments, the duplex may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides long. In some embodiments, the duplex length is 19-25 nucleotides. The duplex region of the RNA in the miRNA may be part of a hairpin structure. MiRNA agents that contain a hairpin may also be referred to as "shRNA (short hairpin RNA) agents." In addition to the duplex portion, the hairpin structure may contain a loop region located between the two sequences that form the duplex. The loop may be of varying lengths. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides long. The hairpin structure may also include 3' or 5' overhangs. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length. Typically, the level of an expression product (e.g., mRNA, polypeptide, etc.) of a target gene is reduced by an siRNA agent (e.g., miRNA, shRNA, etc.) that contains specific double-stranded nucleotide sequences complementary to each other to at least a 19-25 nucleotide segment (e.g., 20-21 nucleotide sequences) of a target gene transcript, including the 5'-untranslated (UT) region, ORF, or 3'-UT region. In some embodiments, the small interfering RNAs are approximately 19-25 nucleotides in length. See, for example, PCT applications WO0/44895, WO99/32619, WO01/75164, WO01/92513, WO01/29058, WO01/89304, WO02/16620, and WO02/29858; and U.S. Patent Publication No. 20040023390 for descriptions of siRNA technology. The siRNA and/or shRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may also include a promoter. The nucleic acid sequence may also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal.

[00109] (ii) antisense RNA is RNA that is complementary to a gene expression product. For example, antisense RNA that targets a specific mRNA is an RNA-based agent (or may be a modified RNA) that is complementary to the mRNA, where hybridization of the antisense RNA to the mRNA alters the expression of the mRNA (e.g., by altering the stability of the RNA, altering the translation of the RNA, etc.). The term "antisense RNA" also includes nucleic acids encoding the antisense RNA.

[00110] (iii) CRISPR agents. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea by using CRISPR RNA (crRNA) to silence foreign nucleic acids. The Cas 9 protein (or its functional equivalent and/or variant, i.e., Cas9-like protein) naturally possesses DNA endonuclease activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNA). In some cases, two molecules are covalently linked to form a single molecule (also called a single guide RNA ("sgRNA")). In this way, Cas9 or a Cas9-like protein binds to a DNA-targeting RNA (this term covers both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates Cas9 or a Cas9-like protein and directs the protein to a target nucleic acid sequence. If Cas9 or a Cas9-like protein retains its natural enzymatic function, it will cleave the target DNA, creating a double-strand break that can result in a genome alteration (i.e., editing: deletion, insertion (if a donor polynucleotide is present), substitution, etc.), thereby altering gene expression. Some Cas9 variants (which are covered by the term Cas9-like) have been altered such that they have reduced cleavage activity DNA (in some cases they cleave one strand instead of both strands of the target DNA, while in other cases their DNA cleavage activity is greatly reduced or absent). Cas9-like proteins with reduced DNA cleavage activity (even without DNA cleavage activity) can still be targeted to the target DNA and can block RNA polymerase activity. Thus, enzymatically inactive Cas9-like proteins can be targeted to a specific location in the target DNA by DNA-targeting RNA to block transcription of the target DNA. Detailed information on CRISPR agents can be found in, for example, (a) Jinek et al., Science, 2012 Aug. 17; 337(6096):816-21: "A programmed dual-RNA-guided DNA endonuclease in adaptive bacterial immunity"; (b) Qi et al., Cell, 2013 Feb. 28; 152(5):1173-83: "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression" and (c) U.S. Patent Application Ser. No. 13/842,859 and PCT Application PCT/US13/32589; all of which are incorporated herein by reference in their entireties. Thus, as used herein, the term "CRISPR agent" encompasses any agent (or nucleic acid encoding such an agent) comprising naturally occurring and/or synthetic sequences that can be used in a Cas9-based system (e.g., Cas9 or a Cas9-like protein; any component of a DNA-targeting RNA, e.g., crRNA-like RNA, tracrRNA-like RNA, single guide RNA, etc.; a donor polynucleotide, etc.).

[00111] (iv) Zinc finger nuclease (ZFN) agents. Zinc finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences, and this allows zinc finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double-strand breaks. For more information on the use of ZFNs, see, for example, Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Ochiai et al. Genes Cells. August 2010; 15(8):875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10):759-65; Ekker et al, Zebrafish 2008 Summer; 5(2):121-3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91; Geurts et al, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371/journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA. 2011 Jul. 19; 108(29):12013-7; and Yu et al, Cell Res. 2011 November; 21(11):1638-40; which are all incorporated herein by reference for their teachings related to ZFNs. The term “ZFN agent” includes a zinc finger nuclease and/or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[00112] (v) Transcriptional activator-like nuclease (TALEN) agents. Transcriptional activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing the TAL (transcription activator-like) effector DNA-binding domain to a DNA cleavage domain. TALENs can be rapidly engineered to bind virtually any desired DNA sequence, and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cellular genome) by inducing double-strand breaks. For further information on the use of TALENs, see, e.g., Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8):695-6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; which are all incorporated by reference herein for their teachings related to TALENs. The term “TALEN agent” includes TALENs and/or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

[00113] The term "regulatory element" or "regulatory sequence" is a nucleotide sequence involved in the interaction of molecules that facilitates the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, rate, or specificity of a process and may be up- or down-regulatory. Regulatory elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a region of DNA capable, under certain conditions, of binding RNA polymerase and initiating transcription of a coding region, typically located downstream (3') of the promoter.

[00114] "Operably linked" or "functionally linked" refers to a juxtaposition of genetic elements where the elements are in a relationship that allows them to act in the expected manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region, so long as this functional relationship is maintained.

[00115] An "expression vector" is a vector containing a region that encodes a polypeptide of interest and is used to effect expression of the protein in a proposed target cell. The expression vector also contains regulatory elements operatively linked to the coding region to facilitate expression of the protein in the target. The combination of regulatory elements and the gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[00116] "Heterologous" means derived from a molecule that is genotypically distinct from the rest of the molecule to which it is being compared. For example, a polynucleotide that is genetically engineered into a plasmid or vector derived from another species is a heterologous polynucleotide. A promoter that is removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid that is not normally found in naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by naturally occurring wild-type AAV.

[00117] "Peptide 2A" refers to "self-cleaving" peptides of approximately 20 amino acids that produce equimolar levels of multiple genes from the same mRNA and can be used in place of IRES elements in multicistronic vectors. Non-limiting examples include T2A, P2A, E2A, and F2A peptide sequences.

[00118] The terms "genetic alteration" and "genetic modification" (and grammatical variations thereof) are used interchangeably herein to refer to a process in which a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell or may be an additional copy or an improved version of an element already in the cell. The genetic alteration can be accomplished, for example, by transfecting the cell with a recombinant plasmid or another polynucleotide by any method known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. The genetic alteration can also be accomplished, for example, by transduction or infection with a DNA or RNA virus or viral vector. Typically, the genetic element is introduced into a chromosome or minichromosome in the cell; but any change that alters the phenotype and/or genotype of the cell and its progeny is included in this term.

[00119] A cell has been "genetically modified" or "transformed" or "transfected" with exogenous DNA (e.g., by a recombinant virus) when such DNA is introduced into the cell. The presence of exogenous DNA results in a permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the cell's genome. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell capable of stable growth in vitro for many generations.

[00120] A cell is considered to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during prolonged culture of the cell in vitro and/or over an extended period of time in vivo. Typically, such a cell is "hereditarily" altered (genetically modified) in the sense that a genetic change is introduced into it that is also inherited by the progeny of the altered cell.

[00121] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. Polypeptides, such as antiangiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivery of a gene product to a mammalian subject and composition thereof, refer to the corresponding intact polypeptide or any fragment or engineered derivative thereof that retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding antiangiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivering a gene product to a mammalian subject (which may be referred to as "transgenes" to be delivered to the mammalian subject by a recipient cell) include polynucleotides encoding the intact polypeptide or any fragment or engineered derivative having the desired biochemical function.

[00122] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, protein, or other substance refers to a preparation of the substance, at least without certain other components that may also be present where the substance or similar substance originates or was originally obtained. Thus, for example, an isolated substance may be obtained by using a purification method to enrich it from an original mixture. The enrichment may be measured in absolute terms, such as by weight per volume of solution, or it may be measured relative to a second, potentially interfering substance present in the original mixture. Increasing the enrichment of embodiments of the present disclosure becomes increasingly isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is, in some embodiments, purified, such as to about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure or higher.

[00123] As used herein, the terms "treatment", "therapy" and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or a symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse effect associated with the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the development of a disease (and/or symptoms caused by the disease) in a subject who may be predisposed to the disease or is at risk for the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (and/or symptoms caused by the disease), i.e. stopping its progression; and (c) alleviating the disease (and/or symptoms caused by the disease), i.e. ensuring regression of the disease (and/or symptoms caused by the disease).

[00124] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans; non-human primates, including monkeys; sport mammals (e.g., horses); farm mammals (e.g., sheep, goats, etc.); domestic mammals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[00125] In some embodiments, the subject is a human who has previously been naturally exposed to AAV and, as a result, contains anti-AAV antibodies (i.e., neutralizing antibodies to AAV). In some embodiments, the subject is a human who has previously been administered an AAV vector (and, as a result, may contain antibodies against AAV) and who requires re-administration of the vector to treat another disease state or to further treat the same condition. Based on the positive results of clinical trials involving delivery of an AAV gene to, for example, the liver, muscle, and retina - all tissues affected by neutralizing antibodies against this carrier - there are a variety of such therapeutic uses/targets for treating diseases.

[00126] As used herein, the term "effective amount" is an amount sufficient to achieve beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of a compound (e.g., an infectious rAAV virion) is an amount sufficient to alleviate, reduce, stabilize, reverse, prevent, slow, or delay the progression of (and/or symptoms associated with) a particular disease state (e.g., cancer). Accordingly, an effective amount of an infectious rAAV virion is an amount of infectious rAAV virion that is capable of evading the neutralizing activity of individual anti-AAV antibodies, thereby effectively delivering a heterologous nucleic acid to a target cell (or cells) of a subject.

[00127] Before further describing the present invention, it should be understood that the invention is not limited to the particular embodiments described, as they may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[00128] Where a range of values is specified, it is intended that each intermediate value, to within a tenth of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range and any other specified or intermediate value within the specified range is encompassed by the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed by the invention taking into account any specifically excluded limit within the specified range. Where a specified range includes one or both limits, ranges excluding one or both of those included limits are also included in the invention.

[00129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications referenced herein are hereby incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[00130] It should be noted that, as used herein and in the appended claims, the singular forms include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an infectious virion of a recombinant adeno-associated virus (rAAV)" includes a plurality of such virions, and reference to "an infectious virion of a recombinant adeno-associated virus (rAAV)" includes reference to one or more such virions and equivalents thereof known to those skilled in the art, and so forth. It should further be noted that the claims may be drafted so as to exclude any optional element. As such, this statement is intended to serve as a precursor to the use of such exclusive terminology as "solely," "only," and the like, in connection with the recitation of elements of the claims or the use of a "negative" limitation.

[00131] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable combination. All combinations of embodiments related to the invention are encompassed by the present invention and are disclosed herein in the same way as if each combination were individually and expressly disclosed. In addition, all subcombinations of various embodiments and elements thereof are also specifically encompassed by the present invention and are disclosed herein in the same way as if each such subcombination were individually and expressly disclosed herein.

[00132] The publications cited herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to precede such publication by virtue of prior invention. Furthermore, the publication dates cited may differ from the actual publication dates, which may require independent verification.

[00133] The present invention relates to infectious recombinant adeno-associated virus (rAAV) virions that comprise a variant capsid protein and a heterologous nucleic acid. The invention also relates to variant adeno-associated virus (AAV) capsid proteins (and/or nucleic acid encoding variant AAV capsid proteins) that confer on the infectious rAAV virion increased resistance to human neutralizing anti-AAV antibodies. The present disclosure further relates to host cells comprising an infectious rAAV virion and/or a nucleic acid encoding a claimed variant AAV capsid protein. The present disclosure further relates to libraries of the above virions, capsid proteins, nucleic acids, and/or host cells; wherein the variant AAV capsid protein of at least one library member comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33.

[00134] The present invention also relates to methods of delivering a heterologous nucleic acid to a target cell, wherein the target cell is contacted with an infectious rAAV virion of a subject. The present invention also relates to methods of delivering a gene product to a subject, wherein the methods typically comprise administering an effective amount of the subject's rAAV virion to an individual in need thereof. The present invention also relates to compositions and kits for use in the disclosed methods. In many embodiments, a disclosed infectious rAAV virion, a disclosed nucleic acid, a disclosed variant AAV capsid protein, a disclosed host cell, etc. are isolated.

[00135] AAV variant capsid polypeptides

[00136] A variant AAV capsid polypeptide of the present invention (or a variant AAV capsid protein encoded by a nucleic acid of the present invention) confers on an infectious rAAV virion comprising the variant AAV capsid polypeptide increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV comprising a wild-type capsid protein. In some embodiments, the increased stability is at least about 1.5 times (e.g., at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times), at least about 7.5 times, at least about 10 times, at least about 12 times, at least about 15 times, at least about 17 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, etc.) higher than the stability, expressed by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV containing a wild-type capsid protein.

[00137] It can be said that the variant AAV capsid protein of the present invention (or the variant AAV capsid protein encoded by the nucleic acid of the present invention) confers on the infectious rAAV virion increased transduction of mammalian cells in the presence of human neutralizing antibodies to AAV, compared to the transduction exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV containing a wild-type capsid protein. In some embodiments, the increased transduction is at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) higher than transduction exerted by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV containing a wild-type capsid protein.

[00138] In some embodiments, a variant AAV capsid protein (or a variant AAV capsid protein encoded by a nucleic acid of the present invention) exhibits reduced binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, a variant AAV capsid protein can exhibit at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) lower binding (e.g., reduced affinity) to a neutralizing antibody that binds wild-type AAV capsid protein compared to the binding affinity of the antibody to wild-type AAV capsid protein.

[00139] In some embodiments, a neutralizing anti-AAV antibody binds to a variant AAV capsid protein of the present invention (or a variant AAV capsid protein encoded by a nucleic acid of the present invention) with an affinity of less than about 10 ^-7 M, less than about 5 x 10 ^-6 M, less than about 10 ^-6 M, less than about 5 x 10 ^-5 M, less than about 10 ^-5 M, less than about 10 ^-4 M, or less.

[00140] The term "variant capsid protein" does not encompass wild-type AAV capsid proteins. A "variant AAV capsid protein" does not comprise an amino acid sequence present in a natural AAV capsid protein. For example, a variant capsid protein does not comprise an amino acid sequence that is 100% identical to any of the sequences shown in SEQ ID NOs: 1-9. In other words, said variant capsid protein does not comprise an amino acid sequence shown in any of SEQ ID NOs: 1-9. A variant capsid protein may differ in amino acid sequence from a "parent" or "original" AAV capsid protein, wherein the parent AAV capsid protein may be a wild-type AAV capsid protein or a non-wild-type AAV capsid protein.

[00141] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 90% (such as at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33.

[00142] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by said nucleic acid) comprises an amino acid sequence having at least about 90% (such as at least about 92%, at least about 95%%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33.

[00143] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by said nucleic acid) comprises an amino acid sequence that has at least about 95% (such as at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence shown in SEQ ID NO:10, and includes the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q relative to the AAV capsid protein AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00144] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence that has at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:10 and includes the amino acid substitutions N312K, N449D, D472N, N551S, I698V, and L735Q relative to the AAV capsid protein AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00145] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence that has at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence shown in SEQ ID NO:31, and includes the amino acid substitutions N312K, N449D, N551S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00146] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence that has at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:31 and includes the amino acid substitutions N312K, N449D, N551S, and I698V relative to the AAV capsid protein AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00147] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence that has at least about 95% (such as at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence shown in SEQ ID NO:32 and includes the amino acid substitutions D180N, N312K, Q385R, N449D, N551S, I698V, and S721T relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00148] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence that has at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:32 and includes the amino acid substitutions D180N, N312K, Q385R, N449D, N551S, I698V, and S721T relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00149] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence that has at least about 95% (such as at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence shown in SEQ ID NO:33, and includes the amino acid substitutions N312K, N449D, T450A, N551S, and I698V relative to the AAV capsid protein AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00150] In some embodiments, a subject variant AAV capsid protein (or a variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence that has at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:33 and includes the amino acid substitutions N312K, N449D, T450A, N551S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2) or the corresponding positions in another parental AAV serotype.

[00151] Exemplary variant AAV capsid proteins include, but are not limited to (see Fig. 8-10 for selected sequence alignment examples):

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLK YNHADAEFQQRLQGDTSFGGNLGRAVFQAKKRVLEPLGLVEQAGETAPGKKRPLIESPQQPDSSTGIGKKGKQPAKKRLNFGQTGDSESVPD PQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSDYQLPYVLGSAHEGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKD LLFSRGSPTGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASN TALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL;

[00154] Shuffle 100-3 (nucleotide sequence) (SEQ ID NO: 24):

atggctgctgatggttatcttccagattggctcgaggacactctctctgaaggaataagacagtggtggaagctcaaacctggcccaccaccaccaaagcccgcagagcggcataaggacgacagcaggggtcttgtgcttcctgggtacaagtacctcggacccttcaacggactcgacaagggagagccggtcaacgaggcagacgcagcggccct cgagcacgacaaggcctacgaccagcagctcaaggccggtgacaacccctacctcaag tacaaccacgccgacgcggagttccagcagcggcttcagggcgacacatcgtttgggggcaacctcggcagagcagtcttccaggccaaaaagagggttcttgaacctctctggtctggttgagcaagcgggtgagacggctcctggaaagaagagaccgttgattgaatccccccagcagcccgactcctccacggggtat cggcaaaaaaggcaagcagccggctaaaaagagactcaattttggtcagactggcgactcagagtcagtccccgacc cacaacctctcggagaacctccagcaacccccgctgctgtgggacctactacaatggcttcaggtggtggcgcaccaatggcagacaataacgaaggcgccgacggagtgggtaatgcctcaggaaattggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgcacctgggccttgcccacctacaataaccacctctaca agcaaatctccagtgcttcaacggggggccagcaacgacaaccactacttcggctaca gcaccccctgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcagcgactcatcaacaacaattggggattccggcccaagagactcaacttcaaactcttcaacatccaagtcaaggaggtcacgacgaatgatggcgtcacaaccatcgctaataaccttaccagcacggttcaagtcttctcggactcagactatcag ctcccgtacgtgctcgggtcggctcacgagggctgcctcccgccgttcccagcaga cgtcttcatggtgccacagtatggatacctcaccctgaacaacgggagtcaggcagtaggacgctcttcattttactgcctggagtactttccttctcagatgctgcgtaccggaaacaactttaccttcagctacacttttgaggacgttcctttccacagcagctacgctcacagccagagtctggaccgtctcatgaat cctctcatcgaccagtacctgtattacctgaacagaactcagaatcagtccggaagtgcccaaaacaaggactt gctgtttagccgggggtctccaactggcatgtctgttcagcccaaaaactggctacctggaccctgttatcggcagcagcgcgtttctaaaacaaaaacagacaacaacaacagcaactttacctggactggtgcttcaaaatataaccttaatgggcgtgaatctataatcaaccctggcactgctatggcctcacacaaagac gacaaagacaagttctttcccatgagcggtgtcatgatttttggaaaggagagcgccggagcttcaaacact gcattggacaatgtcatgatcacagacgaagaggaaatcaaagcactaaccccgtggccactgaaagatttgggactgtggcagtcaatctccagagcagcagcacagaccctgcgaccggagatgtgcatgccatgggagccttacctggaatggtgtggcaagacagacgtatacctgcagggtcctatttgggccaaaattcct cacacggatggacactttcacccgtctcctctcatgggcggctttggactcaagaacccgcctcct cagatcctcatcaaaaacacgcctgttcctgcgaatcctccggcggagttttcagctacaaagtttgcttcattcatcacccagtattccacaggacaagtgagcgtggagattgaatgggagctcagaaagaaaacagcaaacgctggaatcccgaagtgcagtatacatctaactatgcaaaatctgccaacgtt gatttcactgtggacaacaatggactttatactgagcctcgccccattggcacccgttacctcacccgtcccctgtaa.

[00155] Shuffle 100-7 (amino acid sequence) (SEQ ID NO: 13); Shuffle 100-7 (nucleotide sequence) (SEQ ID NO: 25); Shuffle 10-2 (amino acid sequence) (SEQ ID NO: 26); Shuffle 10-2 (nucleotide sequence) (SEQ ID NO: 34); Shuffle 10-6 (amino acid sequence) (SEQ ID NO: 27); Shuffle 10-6 (nucleotide sequence) (SEQ ID NO: 35); Shuffle 10-8 (amino acid sequence) (SEQ ID NO: 28); Shuffle 10-8 (nucleotide sequence) (SEQ ID NO: 36); Shuffle 100-2 (amino acid sequence) (SEQ ID NO: 29); Shuffle 100-2 (nucleotide sequence) (SEQ ID NO: 37); SM 10-1 (amino acid sequence) (SEQ ID NO: 30); SM 10-1 (nucleotide sequence) (SEQ ID NO: 38); SM 10-8 (amino acid sequence) (SEQ ID NO: 31); SM 10-8 (nucleotide sequence) (SEQ ID NO: 39); SM 100-3 (amino acid sequence) (SEQ ID NO: 32); SM 100-3 (nucleotide sequence) (SEQ ID NO: 40); SM 100-10 (amino acid sequence) (SEQ ID NO: 33); and SM 100-10 (nucleotide sequence) (SEQ ID NO: 41).

[00156] Nucleic Acids and Host Cells

[00157] The present invention relates to nucleic acids comprising nucleotide sequences encoding a variant AAV capsid protein (as described above), as well as to host cells comprising a nucleic acid of the present invention. The nucleic acids and host cells are useful for producing rAAV virions (as described below).

[00158] The present invention provides host cells, such as isolated host cells, comprising a nucleic acid of the present invention. A host cell of the present invention may be referred to as a "genetically modified host cell" and is typically an isolated cell, such as a cell in vitro culture. A host cell of the present invention is useful for producing an rAAV virion of the present invention, as described below. When said host cell is used to produce said rAAV virion, it is referred to as a "packaging cell". In some embodiments, said host cell is stably genetically modified (i.e., stably transfected) with a nucleic acid of the present invention. In other embodiments, said host cell is transiently genetically modified (i.e., transiently transfected) with a nucleic acid of the present invention.

[00159] The nucleic acid of the present invention is stably or transiently introduced into a host cell using well-known methods, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. For stable transformation, the nucleic acid of the present invention typically further comprises a selectable marker, such as any of several well-known selectable markers, such as neomycin resistance, and the like.

[00160] The host cell of the present invention is produced by introducing the claimed nucleic acid into any of a variety of cells, such as mammalian cells, including, for example, mouse cells and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, wherein suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, mouse 3T3 fibroblasts, C3H10T1/2 fibroblasts, CHO cells, and the like.

[00161] In some embodiments, a host cell of the present invention comprises, in addition to a nucleic acid comprising a nucleotide sequence encoding a mutant capsid protein, a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, the subject host cell further comprises an rAAV vector, as described below. As described in more detail below, the rAAV virion is generated using a host cell of the present invention.

[00162] Infectious rAAV virions

[00163] An infectious rAAV virion of a subject comprises a variant AAV capsid protein and a heterologous nucleic acid (described in more detail below) and exhibits increased resistance to human neutralizing anti-AAV antibodies compared to the resistance exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV containing a wild-type capsid protein. By "increased resistance" is meant that the infectious rAAV virion of the subject exhibits increased infectivity in the presence of human anti-AAV antibodies. As described above, viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Thus, increased infectivity means an increased ratio of infectious viral particles to total viral particles. To determine the resistance of AAV to human anti-AAV antibodies, AAV infectivity is measured in the presence of varying concentrations of human anti-AAV antibodies to obtain the antibody concentration (e.g., serum concentration, IVIG concentration, etc.) (mg/mL) required to reduce the gene delivery efficiency (i.e., infectivity) to 50% of that in the absence of human anti-AAV antibodies. A virus that requires a higher antibody concentration to reduce gene delivery efficiency to 50% compared to the absence of human anti-AAV antibodies is considered to have increased resistance to antibody neutralization. Thus, a two-fold increase in resistance means a two-fold increase in the antibody concentration required to reduce gene delivery efficiency to 50% compared to the absence of human anti-AAV antibodies. In some embodiments, the infectious rAAV virion of the subject exhibits resistance to human neutralizing anti-AAV antibodies by at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least approximately 300-fold, etc.) than the resistance exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or wild-type capsid protein-containing AAV.

[00164] It can be said that the infectious rAAV virion of the present invention exhibits increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies. In some embodiments, an infectious rAAV virion of the present invention exhibits at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, (at least approximately 300-fold, etc.) in the presence of antibodies that neutralize human AAV than transduction exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV containing the wild-type capsid protein.

[00165] In some embodiments, the subject's infectious rAAV virion exhibits reduced binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, an infectious rAAV virion of a subject can have reduced binding (e.g., reduced affinity) by at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least approximately 300-fold, etc.) with a neutralizing antibody that binds wild-type AAV capsid protein, compared to the binding affinity of the antibody to wild-type AAV capsid protein.

[00166] In some embodiments, the neutralizing anti-AAV antibody binds to an infectious rAAV virion of a subject with an affinity of below about 10 ^-7 M, below about 5 x 10 ^-6 M, below about 10 ^-6 M, below about 5 x 10 ^-5 M, below about 10 ^-5 M, below about 10 -4 M, or below.

[00167] In some embodiments, an infectious rAAV virion of the present invention exhibits an increased in vivo residence time compared to a wild-type AAV. For example, an infectious rAAV virion of the present invention exhibits a residence time that is at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more longer than the residence time of a wild-type AAV.

[00168] The extent to which a subject's claimed infectious rAAV virion exhibits reduced binding to a neutralizing antibody and/or increased resistance to a neutralizing antibody can be determined using any suitable assay known to one of skill in the art.

[00169] In some embodiments, an infectious rAAV virion of the present invention comprises wild-type Rep78, Rep68, Rep52, and Rep40 proteins. In other embodiments, an infectious rAAV virion of the present invention comprises, in addition to one or more variant capsid proteins, one or more mutations in one or more Rep78, Rep68, Rep52, and Rep40 proteins.

[00170] Heterologous nucleic acids

[00171] A suitable heterologous DNA molecule (also referred to herein as a "heterologous nucleic acid") for use in a subject rAAV vector (e.g., a subject rAAV infectious virion) can be any heterologous nucleic acid. In some embodiments, the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide (e.g., a protein that confers certain desired characteristics on a target cell, such as a fluorescent protein that allows cells to be analyzed, an enzyme that provides an activity that is absent or altered in the target cell, etc.). In some embodiments, the heterologous nucleic acid comprises an RNA interference agent (as defined above).

[00172] A claimed heterologous nucleic acid will typically be below about 5 kb in size and will include, for example, a gene (nucleotide sequence) that encodes a protein that is defective or absent in the recipient or target cell; a gene that encodes a protein that has a desired biological or therapeutic effect (e.g., antibacterial, antiviral, or antitumor/anti-cancer activity); a nucleotide sequence that encodes RNA that inhibits or reduces the production of a harmful or otherwise undesirable protein (e.g., a nucleotide sequence that encodes an RNA interference agent, as defined above); and/or a nucleotide sequence that encodes an antigenic protein.

[00173] Suitable heterologous nucleic acids include, but are not limited to, those encoding proteins useful for the treatment of endocrine, metabolic, hematological, cardiovascular, neurological, musculoskeletal, urological, pulmonary and immune diseases, including diseases such as inflammatory diseases, autoimmune chronic and infectious diseases such as acquired immunodeficiency syndrome (AIDS), cancer, hypercholesterolemia, lysosomal storage diseases such as activator deficiency/GM2 gangliosidosis, alpha-mannosidosis, aspargal glucosaminuria, cholesteryl ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease, GM1 gangliosidosis, I-cell disease/mucolipidosis II, infantile free sialic acid storage disease/ISSD, juvenile hexosaminidase A deficiency, Krabbe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, mucopolysaccharidoses (including Hurler pseudopolydystrophy/mucolipidosis IIIA, MPS I Hurler syndrome, MPS I Scheie syndrome, MPS I Hurler-Scheie syndrome, MPS I II Hunter syndrome, Sanfilippo syndrome type A/MPS III A, Sanfilippo syndrome type B/MPS III B, Sanfilippo syndrome type C/MPS III C, Sanfilippo syndrome type D/MPS III D, Morquio syndrome type A/MPS IVA, Morquio syndrome type B/MPS IVB, MPS IX/hyaluronidase deficiency, MPS VI Marco-Lamy syndrome, MPS VII Sly syndrome, mucolipidosis I/sialidosis, mucolipidosis type IIIC and mucolipidosis type IV), multiple sulfatase deficiency, Niemann-Pick disease, neuronal ceroid lipofuscinoses, Pompe disease/glycogen storage disease type II, pycnodysostosis, adult-onset Sandhoff disease/GM2 gangliosidosis, childhood-onset Sandhoff disease/GM2 gangliosidosis, juvenile-onset Sandhoff disease/GM2 gangliosidosis, Schindler disease, Salla disease/sialic acid storage disease, Tay-Sachs disease/GM2 gangliosidosis and Wolman disease, insulin disorders such as diabetes, growth disorders, various blood disorders including various anemias, thalassemia and hemophilia; genetic defects such as cystic fibrosis, Gaucher disease, Hurler syndrome, adenosine deaminase (ADA) deficiency, emphysema, etc.

[00174] Suitable heterologous nucleic acids include, but are not limited to, those that encode any of a variety of proteins including, but not limited to: interferon (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ); insulin (e.g., novolin, humulin, humalog, lantus, ultralente, etc.); erythropoietin ("EPO"; e.g., Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp® (darbepoetin-α); NeoRecormon®, Epogin® (epoetin-α), etc.); antibody (e.g. monoclonal antibody) (e.g. Rituxan® (rituximab); Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab); Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab), Erbitux™ (cetuximab), Avastin® (bevacizumab), etc.), including antigen-binding fragment of a monoclonal antibody (e.g. Lucentis® (ranibizumab)); blood factor (e.g. Activase® (alteplase) tissue plasminogen activator; NovoSeven® (recombinant human factor VIIa); factor VIIa; factor VIII (e.g. Kogenate®); factor IX; β-globin; hemoglobin, etc.); colony stimulating factor (e.g. Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastim); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocytic colony stimulating factor, etc.); growth hormone (e.g. somatotropin, e.g. Genotropin®, Nutropin®, Norditropin®, Saizen®, Serostim®, Humatrope®, etc.; human growth hormone, etc.); interleukin (e.g. IL-1; IL-2, including e.g. Proleukin®; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.); growth factor (e.g. Regranex® (beclappermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim; stem cell factor); keratinocyte growth factor; acidic fibroblast growth factor, stem cell factor, basic fibroblast growth factor, hepatocyte growth factor, etc.); soluble receptor (e.g. soluble TNF-α binding receptor such as Enbrel® (etanercept); soluble VEGF receptor; soluble interleukin receptor; soluble T-cell γ/σ receptor, etc.); enzyme (e.g. α-glucosidase; Cerazyme® (imiglucarase; β-glucocerebrosidase, Ceredase® (alglucerase); enzyme activator (e.g. tissue plasminogen activator); chemokine (e.g. IP-10; Mig; Groα/ IL-8, RANTES; MIP-1α; MIP-1β; MCP-1; PF-4, etc.); angiogenic agent (e.g. vascular endothelial growth factor (VEGF); antiangiogenic agent (e.g. soluble VEGF receptor); protein vaccine; neuroactive peptide such as bradykinin, cholecystokinin, hastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, delta sleep inducing peptide and the like; other proteins such as thrombolytic agent, atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human alpha-1-antitrypsin, leukemia inhibitory factor, transforming growth factor, insulin-like growth factor, luteinizing hormone, macrophage activating factor, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, tissue inhibitor of metalloproteinases; vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; leukemia inhibitory factor; IL-1 receptor antagonist (e.g., Kineret® (anakinra)); ion channel, such as cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin, a tumor suppressor; lysosomal enzyme acid α-glucosidase (GAA) analog, and the like. Suitable nucleic acids also include those encoding a functional fragment of any of the foregoing proteins; and nucleic acids encoding functional variants of any of the foregoing proteins.

[00175] Suitable heterologous nucleic acids also include those that encode antigenic proteins. The subject rAAV vector comprising a heterologous nucleic acid encoding an antigenic protein is suitable for stimulating an immune response to the antigenic protein in a mammalian host. The antigenic protein is derived from a self-antigen, an allergen, a tumor/cancer-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects the mammalian host. As used herein, the term "nucleic acid encoding an antigenic protein derived from" includes nucleic acids encoding wild-type antigenic proteins, such as a nucleic acid isolated from a pathogenic virus that encodes a viral protein; synthetic nucleic acids produced in a laboratory that encode antigenic proteins whose amino acid sequence is identical to a naturally occurring antigenic protein; Synthetic nucleic acids produced in a laboratory that encode antigenic proteins that differ in amino acid sequence (e.g., from one amino acid to about 15 amino acids) from the natural antigenic protein but that nevertheless induce an immune response to the corresponding naturally occurring antigenic protein; Synthetic nucleic acids produced in a laboratory that encode fragments of antigenic proteins (e.g., fragments of from about 5 amino acids to about 50 amino acids, such fragments containing one or more antigenic epitopes), such fragments induce an immune response to the corresponding naturally occurring antigenic protein; and the like.

[00176] Similarly, an antigenic protein that is "derived from" a self-antigen, an allergen, a tumor/cancer-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian host includes proteins that are identical in amino acid sequence to a naturally occurring antigenic protein and proteins that differ in amino acid sequence (e.g., from one amino acid to about 15 amino acids) from a naturally occurring antigenic protein but that nevertheless induce an immune response to the corresponding naturally occurring antigenic protein; and fragments of antigenic proteins (e.g., fragments of from about 5 to about 100 amino acids, such as from about 5 to about 50 amino acids, wherein the fragments contain one or more antigenic epitopes), such fragments induce an immune response to the corresponding naturally occurring antigenic protein.

[000177] In some embodiments, an immune response to an antigenic protein encoded by a subject rAAV vector will induce a protective immune response to a pathogenic organism that displays the antigenic protein or antigenic epitope (or a protein or epitope that is cross-reactive with the rAAV-encoded antigenic protein or antigenic epitopes) in a mammalian host. In some embodiments, a cytotoxic T lymphocyte (CTL) response to an antigenic protein encoded by an rAAV is induced in the mammalian host. In yet other embodiments, a humoral response to an rAAV-encoded antigenic protein is induced in the mammalian host, resulting in the production of antibodies specific for the antigenic protein. In many embodiments, a TH1 immune response to an antigenic protein encoded by an rAAV is induced in the mammalian host. Suitable antigenic proteins include tumor/cancer-associated antigens, viral antigens, bacterial antigens, and protozoan antigens; and antigenic fragments thereof. In some embodiments, the antigenic protein is derived from an intracellular pathogen. In other embodiments, the antigenic protein is an autoantigen. In yet other embodiments, the antigenic protein is an allergen.

[00178] Tumor/cancer-specific antigens include, but are not limited to, any of the various MAGE (melanoma-associated antigen E), including MAGE 1 (e.g., GenBank Accession Number M77481), MAGE 2 (e.g., GenBank Accession Number U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank Accession Numbers X54156 and AA494311); and melanoma antigen p97 (e.g., GenBank Accession Number M12154). Other tumor/cancer-specific antigens include the Ras peptide and p53 peptide associated with advanced cancer, the HPV 16/18 and E6/E7 antigens associated with cervical cancer, the MUCI1-KLH antigen associated with breast carcinoma (e.g., GenBank Accession No. J03651), the CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession No. X98311), the gp100 (e.g., GenBank Accession No. S73003) or MART1 antigens associated with melanoma, and the PSA antigen associated with prostate cancer (e.g., GenBank Accession No. X14810). The p53 gene sequence is known (see, e.g., Harris et al. (1986) Mol. Cell. Biol., 6:4650-4656) and has been deposited in GenBank under accession number M14694. Thus, the proteins, nucleic acids, and/or virions of the present invention can be used as immunotherapeutic agents for the treatment of cancer, including, but not limited to, cervical cancer, breast cancer, colorectal cancer, prostate cancer, lung cancer, and melanoma.

[00179] Viral antigens are derived from known pathogens responsible for diseases including, but not limited to, measles, mumps, rubella, polio, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza virus, adenoviruses (e.g., types 4 and 7), rabies virus (e.g., GenBank Accession No. M34678), yellow fever, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue fever (e.g., GenBank Accession No. M24444), hantavirus, and human immunodeficiency virus (e.g., GenBank Accession No. U18552).

[00180] Suitable bacterial and parasitic antigens include antigens derived from known pathogens responsible for diseases including, but not limited to, diphtheria, whooping cough (e.g., GenBank Accession No. M35274), tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungal pneumonias (e.g., Haemophilus influenzae , Pneumocystis carinii , etc.), cholera, typhoid fever, plague, shigellosis, salmonellosis (e.g., GenBank Accession No. L03833), Legionnaires' disease, Lyme disease (e.g., GenBank Accession No. U59487), malaria (e.g., GenBank Accession No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. L08198), trypanosomiasis, leishmaniasis, giardiasis (e.g., GenBank Accession No. M33641), amebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinosis.

[00181] Suitable heterologous nucleic acids that encode heterologous gene products include non-translated RNAs such as an RNAi agent (as described in more detail above) (e.g., antisense RNA; siRNA; shRNA; double-stranded RNA (dsRNA)); a CRISPR agent such as Cas9 or a Cas9-like protein, crRNA-like RNA, tracrRNA-like RNA, single guide RNA and/or donor polynucleotide, etc.), a ribozyme, etc. RNAi agents can be used to inhibit gene expression. Some RNA interference agents provide a tool that can subsequently be used to inhibit gene expression (e.g., a CRISPR agent such as cas9 or a cas9-like protein).

[00182] Target genes include any gene that encodes a target gene product (RNA or protein) that is harmful (e.g., pathological), such as a target gene product that has a defective function (e.g., due to a mutation in the encoded protein sequence, due to a mutation in non-coding sequences that control the steady-state level of the gene product, etc.). Target gene products include, but are not limited to, huntingtin; hepatitis C virus; human immunodeficiency virus; amyloid precursor protein; tau; a protein that contains polyglutamine repeats; herpes virus (e.g., varicella-zoster virus); any pathological virus; and the like.

[00183] Thus, the rAAV of the present invention, which comprises a heterologous nucleic acid encoding an RNAi agent, is useful for the treatment of various disorders and pathological conditions, including, but not limited to, neurodegenerative diseases, such as trinucleotide repeat expansion diseases, such as polyglutamine repeat disease, such as Huntington's disease, spinocerebellar ataxia, spinal and bulbar muscular atrophy (SBMA), dentato-rubro-pallido-Lewis atrophy (DRPLA), etc.; an acquired pathology (e.g., a disease or syndrome manifested by an abnormal physiological, biochemical, cellular, structural or molecular biological condition), such as a viral infection, such as hepatitis that occurs or may occur as a result of HCV infection, acquired immunodeficiency syndrome resulting from HIV infection; cancer; and the like.

[00184] In many embodiments, a heterologous nucleic acid encoding an RNAi agent is operably linked to a promoter. Suitable promoters are known to those skilled in the art and include the promoter of any gene encoding a protein, such as an endogenously regulated gene or a constitutively expressed gene. For example, promoters of genes regulated by cellular physiological events, such as heat shock, oxygen levels, and/or carbon monoxide levels, such as during hypoxia, can be operably linked to a nucleic acid encoding an siRNA.

[00185] The selected heterologous nucleotide sequence, such as an EPO-encoding or nucleic acid of interest, is operably linked to regulatory elements that regulate transcription or expression of the nucleotide sequence in vivo. Such regulatory elements may comprise regulatory sequences normally associated with the selected gene (e.g., endogenous cellular regulatory elements). Alternatively, heterologous regulatory sequences may be used. Suitable heterologous regulatory sequences typically include sequences derived from sequences encoding mammalian genes or viruses. Examples include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, an endogenous cellular promoter heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, etc. In addition, sequences derived from non-viral genes such as the mouse metallothionein gene will also find use here. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA).

[00186] In some embodiments, a cell-specific or tissue-specific promoter will be operably linked to a heterologous nucleic acid encoding a heterologous gene product such that the gene product is produced selectively or preferentially in a particular cell type(s) or tissue(s). In some embodiments, an inducible promoter will be operably linked to a heterologous nucleic acid.

[00187] For example, muscle-specific and inducible promoters, enhancers, and the like can be used to deliver a gene product to a muscle cell. Such regulatory elements include, but are not limited to, those derived from the actin and myosin gene families, such as the myoD gene family; the myocyte-specific enhancer binding factor MEF-2; regulatory elements derived from the human skeletal actin gene and the cardiac actin gene; muscle creatine kinase sequence elements and the mouse creatine kinase (mCK) enhancer element; regulatory elements derived from the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene; hypoxia-inducible nuclear factors; steroid inducible elements and promoters such as the glucocorticoid response element (GRE); a fusion consensus element for RU486 induction; and elements that mediate tetracycline-regulated gene expression.

[00188] An AAV expression vector that contains a DNA molecule of interest (heterologous DNA) linked to an AAV ITR can be constructed by directly inserting the selected sequence(s) into the AAV genome that has the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, provided that a sufficient portion of the ITR remains to provide replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published January 23, 1992) and WO 93/03769 (published March 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol., 8:3988-3996; Vincent et al. (1990) Vaccines, 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology, 3:533-539; Muzyczka N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy, 5:793-801; Shelling and Smith (1994) Gene Therapy, 1:165-169; and Zhou et al. (1994) J. Exp. Med., 179:1867–1875.

[00189] Alternatively, the AAV ITRs can be excised from a viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector, using any suitable method known to those skilled in the art. For example, one suitable approach uses standard ligation techniques such as those described by Sambrook et al., supra. For example, ligation can be performed in 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 μg/ml BSA, 10-50 mM NaCl, and either 40 μM ATP, 0.01-0.02 units (Weiss). T4 DNA ligase at 0°C to 16°C (for sticky end ligation) or 1 mM ATP, 0.3-0.6 Weiss units T4 DNA ligase at 14°C (for blunt end ligation). Intermolecular sticky end ligation is typically performed at a total DNA concentration of 30-100 μg/mL (total terminal concentration 5-100 nM). AAV vectors that contain ITRs are described, for example, in U.S. Patent No. 5,139,941. In particular, several AAV vectors are described therein and are available from the American Type Culture Collection (“ATCC”) under accession numbers 53222, 53223, 53224, 53225, and 53226.

[00190] Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences located 5' and 3' of one or more selected nucleic acid sequences. Preferred codons can be used to express the chimeric gene sequence in mammalian muscle cells. The entire chimeric sequence is assembled from overlapping oligonucleotides prepared by standard techniques. See, e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

[00191] Production of infectious rAAV virions of the present invention

[00192] As an introduction, a host cell or "producer cell" is typically used to replicate and package the rAAV vector. Such a producer cell (usually a mammalian host cell) typically contains or is modified to contain several different types of components for rAAV production. The first component is a recombinant adeno-associated virus (rAAV) vector genome (or "rAAV provector") that can be replicated and packaged into vector particles by a packaging host cell. The rAAV provector typically contains a heterologous polynucleotide (or "transgene") with which it is desired to genetically alter another cell in the context of gene therapy (since packaging of such a transgene into rAAV vector particles can be effectively used to deliver the transgene to various mammalian cells). The transgene is typically flanked by two AAV inverted terminal repeats (ITRs), which contain sequences recognized during excision, replication, and packaging of the AAV vector, as well as during integration of the vector into the host cell genome.

[00193] The second component is a helper virus that can provide helper functions for AAV replication. Although adenovirus is typically used, other helper viruses can also be used, as is known in the art. Alternatively, the necessary helper virus functions can be genetically isolated from the helper virus, and the encoding genes can be used to provide the helper virus functions in trans. The AAV vector elements and the helper virus (or helper virus functions) can be introduced into the host cell either simultaneously or sequentially in any order.

[00194] The final components for AAV production that must be provided in the producer cell are "AAV packaging genes" such as the AAV rep and cap genes, which provide replication and encapsidation proteins, respectively. Several different variants of AAV packaging genes can be provided (including rep-cap cassettes and separate rep and/or cap cassettes, in which the rep and/or cap genes can be under the control of native promoters or operably linked to heterologous promoters. Such AAV packaging genes can be transiently or stably introduced into the packaging host cell, as is known in the art and is described in more detail below.

[00195] 1. rAAV Vector

[00196] A virion of an rAAV subject of the present invention comprising a heterologous DNA of interest (wherein "heterologous DNA of interest" also refers herein to "heterologous nucleic acid") can be obtained using standard techniques known to those skilled in the art. The methods typically comprise the following steps: (1) introducing an rAAV vector of the present invention into a host cell; (2) introducing an AAV helper construct into the host cell, wherein the helper construct comprises AAV coding regions capable of expression in the host cell to complement the AAV helper functions lacking in the AAV vector; (3) introducing one or more helper viruses and/or helper vectors into a host cell, wherein the helper virus and/or helper vectors provide additional functions capable of enabling efficient production of a recombinant AAV virion ("rAAV") in the host cell; and (4) culturing the host cell to produce rAAV virions. The AAV expression vector, AAV helper construct, and helper virus or helper vector(s) may be introduced into the host cell either simultaneously or sequentially using standard transfection techniques.

[00197] AAV expression vectors are constructed using known techniques to provide at least operatively linked components in the direction of transcription, regulatory elements including a transcription initiation region, DNA of interest, and a transcription termination region. The transcriptional regulatory control elements are selected to function in a mammalian muscle cell. The resulting construct, which contains operatively linked components, is linked (5' and 3') to functional AAV ITR sequences.

[00198] The nucleotide sequences of the AAV ITR regions are known. See, for example, Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. The AAV ITRs used in the vectors of the invention need not have the wild-type nucleotide sequence, and may be altered, for example, by insertion, deletion, or substitution of nucleotides. In addition, the AAV ITRs may be derived from any of several AAV serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. In addition, the 5' and 3' ITRs that flank the selected nucleotide sequence in the AAV expression vector do not need to be identical or be from the same AAV serotype or isolate, so long as they function as intended to allow excision or rescue of the sequence of interest from the host cell or vector gene and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell. The ITRs allow the vector sequence to replicate in the presence of an appropriate mixture of Rep proteins. The ITRs also allow the vector sequence to be incorporated into the capsid to generate an AAV particle.

[00199] To produce rAAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques, such as transfection. A number of transfection techniques are well known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitable transfection methods include calcium phosphate precipitation (Graham et al. (1973) Virol. 52:456-467), direct microinjection into cultured cells (Capecchi, M.R. (1980) Cell 22:479-488), electroporation. (Shigekawa et al. (1988) BioTechniques 6:742–751), liposome-mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682–690), lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413–7417), and nucleic acid delivery using high-speed microparticles (Klein et al. (1987) Nature 327:70–73).

[00200] For purposes of the present invention, suitable host cells for the production of rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells that can be used or have been used as recipients of a heterologous DNA molecule. The term includes progeny of the original cell that has been transfected. Thus, a "host cell" for the production of rAAV virions generally refers to a cell that has been transfected with an exogenous DNA sequence. Cells from the stable human cell line 293 (readily available, for example, from the American Type Culture Collection under ATCC accession number CRL1573) are used in many embodiments. In particular, the human 293 cell line is a human embryonic kidney cell line that has been transformed with adenovirus type 5 DNA fragments (Graham et al. (1977) J. Gen. Virol., 36:59) and expresses the adenoviral E1a and E1b genes (Aiello et al. (1979) Virology, 94:460). The 293 cell line is readily transfected and provides a particularly convenient platform for the production of rAAV virions.

[00201] 2. AAV Auxiliary Functions

[00202] Host cells comprising the above-described AAV expression vectors should be capable of providing AAV accessory functions to facilitate replication and encapsidation of nucleotide sequences flanked by the AAV ITRs to produce rAAV virions. AAV accessory functions are typically AAV-derived coding sequences that can be expressed to produce AAV gene products that in turn function in trans to productively replicate AAV. AAV accessory functions are used herein to supplement essential AAV functions that are lacking in the AAV expression vectors. Thus, AAV accessory functions include one or both of the essential AAV ORFs, namely the rep and cap coding regions, or functional homologs thereof. In the context of the present invention, the cap functions include one or more mutant capsid proteins, wherein at least one capsid protein comprises at least one mutation as described above.

[00203] By "AAV rep coding region" is meant the region of the AAV genome known in the art that encodes the replication proteins Rep 78, Rep 68, Rep 52, and Rep 40. These Rep expression products have been shown to have many functions, including recognition, binding, and nicking of the AAV DNA replication origin, DNA helicase activity, and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replication of the AAV genome. For a description of the AAV rep coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801. Suitable homologues of the AAV rep coding region include the rep gene of human herpesvirus 6 (HHV-6), which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology, 204:304-311).

[00204] AAV cap proteins include VP1, VP2, and VP3, wherein at least one of VP1, VP2, and VP3 comprises at least one mutation as described above.

[00205] AAV helper functions are introduced into a host cell by transfecting the host cell with an AAV helper construct either prior to or concurrently with transfection of an AAV expression vector. Thus, AAV helper constructs are used to provide at least transient expression of the AAV rep and/or cap genes to complement missing AAV functions that are required for productive AAV infection. AAV helper constructs lack the AAV ITR and are neither replicating nor packaging. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used pAAV/Ad and pIM29+45 plasmids, which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol., 63:3822-3828; and McCarty et al. (1991) J. Virol., 65:2936-2945. A number of other vectors have been described that encode Rep and/or Cap expression products. See, e.g., U.S. Patent No. 5,139,941.

[00206] Both the AAV expression vectors and the AAV helper constructs can be engineered to contain one or more optional selectable markers. Suitable markers include genes that confer resistance or sensitivity to antibiotics, impart coloration, or alter the antigenic properties of cells that have been transfected with a nucleic acid construct containing the selectable marker when the cells are cultured in an appropriate selection medium. Several selectable marker genes that can be used in practicing the methods of the present invention include the hygromycin B resistance gene (encoding aminoglycoside phosphotransferase (APH)), which provides for the selection of mammalian cells by conferring resistance to hygromycin; the neomycin phosphotransferase gene (encoding neomycin phosphotransferase), which provides for the selection of mammalian cells by conferring resistance to G418; and the like. Other suitable markers are known to those skilled in the art.

[00207] 3. Additional AAV functions

[00208] The host cell (or packaging cell) must also be endowed with the ability to provide non-AAV functions, or "accessory functions," in order to produce rAAV virions. Accessory functions are viral and/or cellular functions that are not derived from AAV, but on which AAV replication depends. Thus, accessory functions include at least those non-AAV proteins and RNAs that are required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific splicing of AAV mRNA, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. Viral-based accessory functions may be derived from any of the known helper viruses.

[00209] In particular, additional functions can be introduced and then expressed in host cells using methods known to those skilled in the art. Typically, additional functions are provided by infection of host cells with an unrelated helper virus. A number of suitable helper viruses are known, including adenoviruses; herpes viruses such as herpes simplex virus types 1 and 2; and vaccinia viruses. Non-viral additional functions, such as those provided by cell synchronization using any of a variety of known agents, will also find use here. See, e.g., Buller et al. (1981) J. Virol., 40:241-247; McPherson et al. (1985) Virology, 147:217-222; Schlehofer et al. (1986) Virology, 152:110-117.

[00210] Alternatively, additional functions can be provided using a vector with additional functions. Vectors with additional functions include nucleotide sequences that provide one or more additional functions. A vector with additional functions can be introduced into a suitable host cell to provide efficient production of the AAV virion in the host cell. Vectors with additional functions can be in the form of a plasmid, phage, transposon, cosmid, or other virus. Vectors with additional functions can also be in the form of one or more linearized DNA or RNA fragments that, when linked to appropriate regulatory elements and enzymes, can be transcribed or expressed in the host cell to provide additional functions.

[00211] Nucleic acid sequences providing additional functions can be obtained from natural sources, such as the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art. In this regard, adenovirus-derived additional functions have been extensively studied, and a number of adenovirus genes involved in additional functions have been identified and partially characterized. See, e.g., Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N. (1992) Curr. Topics. Microbiol. and Immun. 158:97-129. In particular, regions of the early adenoviral genes E1a, E2a, E4, VAI RNA, and possibly E1b are thought to be involved in providing additional functions. Janic et al. (1981) Proc. Natl. Acad. Sci. USA, 78:1925-1929. Additional functions of herpes virus have been described. See, e.g., Young et al. (1979) Prog. Med. Virol., 25:113. Additional functions have also been described from vaccinia virus. See, e.g., Carter, B.J. (1990), above, Schlehofer et al. (1986) Virology, 152:110-117.

[00212] Infection of a host cell with a helper virus or transfection of a host cell with a vector with an additional function results in expression of additional functions that transactivate the AAV helper construct to form AAV Rep and/or Cap proteins. The Rep expression products excise recombinant DNA (including DNA of interest, such as a heterologous nucleic acid) from the AAV expression vector. The Rep proteins also serve to copy the AAV genome. The expressed Cap proteins are assembled into capsids, and the recombinant AAV genome is packaged into capsids. In this manner, productive replication of AAV occurs, and the DNA is packaged into rAAV virions.

[00213] Following replication of the recombinant AAV, rAAV virions can be isolated from the host cell using a variety of conventional purification methods, such as CsCl gradient purification, affinity chromatography, and ion exchange chromatography. Additionally, if infection is used to provide additional functionality, residual helper virus can be inactivated using known methods. For example, adenovirus can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or more. Such treatment effectively inactivates only the helper virus, since AAV is very heat-stable and the adenovirus helper is heat-labile.

[00214] The resulting rAAV virions are then ready to be used for DNA delivery, such as in gene therapy applications or for delivery of a gene product to a mammalian host.

[00215] Delivery of heterologous nucleic acid

[00216] The present invention also relates to methods for delivering a heterologous nucleic acid to a target cell and/or a subject in need thereof. In some embodiments, the subject in need thereof is a human who has previously been naturally exposed to AAV and, as a result, contains anti-AAV antibodies (i.e., neutralizing antibodies to AAV). Based on the positive results of clinical trials involving delivery of an AAV gene to, for example, the liver, muscle, and retina - all tissues containing neutralizing antibodies against this carrier - there are a variety of such therapeutic uses/targets for treating diseases.

[00217] The method of the present invention typically comprises: (i) administering an effective amount of a subject rAAV virion and/or (ii) contacting a target cell with a subject virion. Typically, rAAV virions are administered to a subject using either in vivo ("direct") or in vitro ("indirect") transduction techniques. In in vitro ("indirect") transduction, a desired recipient cell (i.e., a "target cell") may be isolated from the subject, transduced with rAAV virions, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells may be used, so long as the cells do not induce an inappropriate immune response in the subject.

[00218] Suitable methods for delivering and administering transduced target cells to a subject are described. For example, cells can be transduced in vitro by combining recombinant AAV virions with cells, such as in appropriate media, and screening those cells that contain the DNA of interest using conventional techniques such as Southern blotting and/or PCR, or using selectable markers. The transduced cells can then be formulated as pharmaceutical compositions, described in more detail below, and the composition can be administered to the subject by various methods, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection.

[00219] For in vivo (i.e., "direct") delivery, rAAV virions are formulated as pharmaceutical compositions and typically administered via the parenteral route of administration (e.g., intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, intravenous, etc.).

[00220] The pharmaceutical compositions should contain a sufficient amount of genetic material to produce a therapeutically effective amount of the gene expression product of interest, i.e., an amount sufficient to suppress or alleviate the symptoms of the disease state in question, or an amount sufficient to produce the desired beneficial effect. The pharmaceutical compositions will also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the subject receiving the composition and that can be administered without causing undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included, for example, salts of inorganic acids such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH adjusting substances, etc. may be present in such carriers. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients are described in detail in various publications including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

[00221] Appropriate doses will depend on the mammal being treated (e.g., a human or a non-human primate or other mammal), the age and general health of the subject being treated, the severity of the pathological condition being treated, the particular therapeutic protein in question, the route of administration, among other factors. A suitable effective amount can be readily determined by one of skill in the art.

[00222] Thus, a "therapeutically effective amount" will be in a relatively broad range that can be determined in clinical trials. For example, for in vivo injection, i.e., injection directly into skeletal or cardiac muscle, a therapeutically effective dose will be on the order of about 10 ⁶ to about 10 ¹⁵ rAAV virions, such as about 10 ⁸ to 10 ¹² rAAV virions. For in vitro transduction, the effective amount of rAAV virions to be delivered to cells will be in the range of about 10 ⁸ to about 10 ¹³ rAAV virions. Other effective dosages can be readily determined by those skilled in the art through standard assays that establish dose-response curves.

[00223] The treatment by administration of doses may be a single dose regimen or a multiple dose regimen. Moreover, the subject may be administered as many doses as necessary. A person skilled in the art can easily determine the appropriate number of doses.

[00224] The cells of interest (i.e., "target cells") are typically mammalian cells, where the term refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo animals, laboratory animals, sports or pets, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some embodiments, the target cell is a human cell.

[00225] Target cells of interest include any cells susceptible to infection with the rAAV virion of the present invention. In some cases, such as when the method is a method of delivering a heterologous nucleic acid to a target cell, wherein the target cell may be a cell taken from a subject (e.g., a "primary" cell), or the target cell may be a tissue culture cell (e.g., from a well-known cell line).

[00226] Examples of target cells include, but are not limited to, liver cells, pancreatic cells (e.g., islet cells of Langerhans: alpha cells, beta cells, delta cells, gamma cells, and/or epsilon cells), skeletal muscle cells, cardiac muscle cells, fibroblasts, retinal cells, joint synovial cells, lung cells, T cells, neurons, glial cells, stem cells, hematopoietic progenitor cells, neural progenitor cells, endothelial cells, and tumor cells. Typical target stem cells include, but are not limited to, hematopoietic stem cells, neural crest stem cells, embryonic stem cells, induced pluripotent stem cells (iPS), mesenchymal stem cells, mesodermal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, and retinal stem cells.

[00227] The term "stem cell" is used herein to refer to a mammalian cell that has the capacity for both self-renewal and the generation of differentiated progeny (see, e.g., Morrison et al. (1997) Cell, 88:287-298). Stem cells typically also have one or more of the following properties: the capacity for asynchronous or symmetric replication, whereby the two daughter cells may have different phenotypes after division; a high capacity for self-renewal; the capacity to exist in a mitotically quiescent form; and the capacity to clonal regenerate the entire tissue in which they reside, such as the capacity of hematopoietic stem cells to reconstitute all hematopoietic lineages. As will be appreciated by those skilled in the art, "progenitor cells" differ from stem cells in that they typically do not have a high capacity for self-renewal and can often generate a more limited subset of clones in the tissue from which they originate, such as only lymphoid or erythroid clones in a hematopoietic environment. For the purposes of the present invention, the term "stem cell" encompasses both "stem cells" and "progenitor cells," which have the meanings defined above.

[00228] Stem cells can be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers, as determined by the absence of binding by specific antibodies. Stem cells can also be identified by functional assays both in vitro and in vivo, in particular assays related to the ability of stem cells to produce numerous differentiated progeny.

[00229] Suitable stem cells of interest include, but are not limited to: hematopoietic stem cells and progenitor cells derived therefrom (U.S. Patent No. 5,061,620); neural crest stem cells (see Morrison et al. (1999) Cell, 96:737-749); neural stem cells and neural progenitor cells; embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; liver stem cells, muscle stem cells, retinal stem cells, induced pluripotent stem cells (iPS cells), etc. Other hematopoietic progenitor cells of interest include cells characteristic of lymphoid lineages, such as immature T cell and B cell populations.

[00230] Purified populations of stem cells or progenitor cells can be used. For example, human hematopoietic stem cells can be positively selected using antibodies specific for CD34, thy-1; or can be negatively selected using lineage-specific markers that can include glycophorin A, CD3, CD24, CD16, CD14, CD38, CD45RA, CD36, CD2, CD19, CD56, CD66a, and CD66b; T cell markers, tumor/cancer-specific markers, etc. Markers suitable for the isolation of mesodermal stem cells include FcγRII, FcγRIII, Thy-1, CD44, VLA-4a, LFA-113, HSA, ICAM-1, CD45, Aa4.1, Sca-1, etc. Neural crest stem cells can be positively selected using low-affinity nerve growth factor receptor (LNGFR)-specific antibodies and negatively selected for sulfatide, glial fibrillary acidic protein (GFAP), myelin protein _Po , peripherin, and neurofilament markers. Human mesenchymal stem cells can be positively selected using SH2, SH3, and SH4 markers.

[00231] The target cells used may be freshly isolated, frozen, or pre-cultured. They may be embryonic, neonatal, or adult cells. Hematopoietic cells may be obtained from fetal liver, bone marrow, blood, particularly from mobilized G-CSF or GM-CSF from peripheral blood, or from any other conventional source. The manner in which the stem cells are separated from other cells of the hematopoietic or other lineage is not critical to the present disclosure. As described above, a substantially homogeneous population of stem cells or progenitor cells may be obtained by selectively isolating cells that are free of markers associated with differentiated cells while exhibiting epitope characteristics associated with stem cells.

[00232] Nucleic acids that can be delivered to a subject include any of the above heterologous nucleic acids. Proteins that can be delivered using the claimed method also include a functional fragment of any of the above proteins; and functional variants of any of the above proteins.

[00233] In some embodiments, a therapeutically effective amount of a protein is produced in a mammalian host. Whether a therapeutically effective amount of a particular protein is produced in a mammalian host using the claimed method is readily determined using assays appropriate for the particular protein. For example, if the protein is EPO, the hematocrit is measured.

[00234] If the rAAV encodes an antigenic protein, suitable antigenic proteins that can be delivered to a subject using the method of the present invention include, but are not limited to, tumor/cancer-associated antigens, autoantigens ("self" antigens), viral antigens, bacterial antigens, protozoan antigens, and allergens; and antigenic fragments thereof. In some embodiments, a cytotoxic T lymphocyte (CTL) response to an antigenic protein encoded by the rAAV is induced in the mammalian host. In other embodiments, a humoral response to an antigenic protein encoded by the rAAV is induced in the mammalian host such that antibodies specific for the antigenic protein are produced. In many embodiments, a TH1 immune response to an antigenic protein encoded by the rAAV will be induced in the mammalian host. The extent to which an immune response to an antigen protein has developed can be easily determined using well-known methods. For example, an enzyme-linked immunosorbent assay can be used to determine whether an antibody to an antigen protein has been formed. Methods for detecting antigen-specific CTLs are well known in the art. For example, a detectably labeled target cell expressing an antigen protein on its surface is used to analyze a blood sample for the presence of antigen-specific CTLs.

[00235] Whether a therapeutically effective amount of a heterologous nucleic acid (e.g., a nucleic acid encoding a polypeptide, an RNA interference agent, etc.) is delivered to a mammalian host using the claimed method can be readily determined using any suitable assay. For example, if the gene product is an RNAi agent that inhibits HIV, the viral load can be determined.

[00236] Methods for creating and identifying modified rAAV virions

[00237] The present invention relates to a method for creating and identifying a modified infectious virion of a recombinant adeno-associated virus (rAAV) that comprises a variant capsid protein comprising an amino acid sequence with at least one amino acid substitution (including deletions, insertions, etc.) compared to a parent AAV capsid protein. The parent AAV capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[00238] The method typically includes generating a library of mutant rAAV virions; and selecting the library for modified rAAV virions with altered properties compared to a parent rAAV virion. The parent rAAV virion comprises a variant AAV capsid protein that comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33. The present disclosure further relates to the library and compositions comprising the libraries.

[00239] In some embodiments, said selection step is repeated two, three, four or more times to enrich the subject AAV library for altered virion properties. In some embodiments, after selecting the AAV library, individual clones are isolated and sequenced.

[00240] Generation of a mutant AAV library

[00241] A mutant AAV library is generated that contains one or more mutations relative to a parent AAV cap gene. The parent cap gene is a cap that contains a nucleotide sequence that encodes a variant AAV capsid protein that contains an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33. Mutations in the rAAV cap gene are generated by any known method. Suitable methods for mutaging the parent AAV cap gene include, but are not limited to, a polymerase chain reaction (PCR)-based method, polynucleotide-directed mutagenesis, saturation mutagenesis, loop exchange mutagenesis, fragment shuffling mutagenesis (DNA shuffling, etc.), and the like. Methods for generating mutations are well described in the art. See, for example, Zhao et al. Nat. Biotechnol., 1998 March; 16(3):234-5; Koerber et. al.; Mol. Ther., 2008 October; 16(10):1703-9; Koerber et. al.; Mol. Ther., 2009 December; 17(12):2088-95; U.S. Patent No. 6,579,678; U.S. Patent No. 6,573,098; and U.S. Patent No. 6,582,914; all of which are incorporated herein by reference with respect to the inventive teachings relating to mutagenesis.

[00242] In some embodiments, a library of mutant AAVs containing mutations in the cap gene will be generated using a stepwise extension process. The stepwise extension process involves amplifying the cap gene using a PCR-based method. The cap gene template is primed using specific PCR primers, followed by repeated cycles of denaturation and very short annealing/polymerase-catalyzed extension. In each cycle, the growing fragments anneal to different templates based on sequence complementarity and are further extended. The denaturation, annealing, and extension cycles are repeated until full-length sequences are generated. The resulting full-length sequences include at least one mutation in the cap gene compared to the wild-type AAV cap gene.

[00243] PCR products containing AAV cap sequences that include one or more mutations are inserted into a plasmid containing a wild-type AAV genome. The result is a library of AAV cap mutants. Accordingly, the present invention provides a library of AAV cap mutant genes comprising from about 10 to about 10 ¹⁰ members and comprising mutations in the AAV cap gene. A given library member has from about one to about 50 mutations in the AAV cap gene. The library of the present invention includes from about 10 to about 10 ⁹ different members, each of which has a different mutation(s) in the AAV cap gene.

[00244] Once a library of cap mutants has been generated, viral particles are produced that can then be selected based on altered capsid properties. The plasmid DNA library is transfected into a suitable host cell (e.g., 293 cells) followed by introduction of a helper virus into the cell. Viral particles produced by the transfected host cells (rAAV library particles) are collected.

[00245] Library selection

[00246] Once the library is generated, the library is selected for a particular virion property (i.e., an altered infectivity property). Viral particles are generated as discussed above (thus creating a library of modified rAAV virions) and subjected to one or more selection steps to identify a modified rAAV virion with an altered infectivity property (as compared to an infectious rAAV virion containing a variant capsid protein) that comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33). The infectivity properties selected for may include, but are not limited to: 1) altered binding (e.g., decreased binding) to neutralizing anti-AAV antibodies; 2) increased evasion of neutralizing anti-AAV antibodies; 3) increased infectivity for a cell resistant to AAV infection; and 4) changes in heparin binding.

[00247] 1. Selection for reduced binding to neutralizing anti-AAV antibodies

[00248] In some embodiments, an AAV library of the present invention is selected for altered (e.g., reduced) binding to neutralizing antibodies that bind to and neutralize wild-type AAV virions, compared to the binding of such antibodies to wild-type AAV virions and neutralization of wild-type AAV virions (or relative to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33). The AAV library particles (AAV library virion) are contacted with neutralizing antibodies and tested for the ability of the AAV library particles to infect a permissive host cell. Typically, the AAV library particles are contacted with varying concentrations of neutralizing antibodies. The higher the concentration of neutralizing antibodies required to reduce the infectivity of the AAV library particles, the more resistant the AAV particles are to neutralization. Any convenient assay known to one of skill in the art can be used to directly measure the binding (e.g., measure the binding affinity) of the AAV library virion to neutralizing anti-AAV antibodies.

[00249] 2. Selection for increased evasion of neutralizing anti-AAV antibodies

[00250] In some embodiments, an AAV library of the present invention is selected for increased evasion of neutralizing antibodies (i.e., increased resistance to neutralizing human anti-AAV antibodies) compared to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33. The AAV library particles are contacted with target cells in the presence of neutralizing antibodies to AAV (typically neutralizing human anti-AAV). After a suitable period of time for the AAV library particles to infect the cells, a helper virus is added and the AAV library particles that successfully infected the cell(s) are collected. In some embodiments, infectivity is measured (e.g., as described above) for those virions that exhibit successful infection. In some embodiments, the cycle of infection, addition of helper virus, and collection of AAV particles is repeated one, two, three, or more times. Selection may occur with different amounts (concentrations) of neutralizing antibodies to AAV to select for different degrees of escape (e.g., each repeated round may use a higher concentration of antibodies than the previous round).

[00251] 3. Selection for increased infectivity for non-permissive cells

[00252] In some embodiments, an AAV library of the present invention is selected for increased infectivity for non-permissive cells (relative to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33). The AAV library particles are contacted with a non-permissive cell (e.g., a population of non-permissive cells). After a suitable period of time for the cells to be infected with the AAV library particles, a helper virus is added and the AAV library particles that have successfully infected the non-permissive cell(s) are collected. In some embodiments, the cycle of infection, addition of helper virus, and collection of AAV particles is repeated one, two, three, or more times.

[0253] 4. Selection for altered heparin binding

[00254] In some embodiments, the library is selected for altered heparin binding, including increased heparin binding and decreased heparin binding compared to the heparin binding of a wild-type AAV virion (or compared to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33). The AAV library particles are contacted with a heparin affinity matrix. For example, the AAV library particles are loaded onto a heparin affinity chromatography column under conditions that allow the AAV library particles to bind to heparin. Typical conditions include equilibrating the column with 0.15 M NaCl and 50 mM Tris at pH 7.5. After allowing the AAV library particle to bind to the heparin affinity matrix, the AAV library particle/heparin complex on the affinity matrix is washed with volumes of buffer containing progressively increasing concentrations of NaCl, and the eluted AAV library particles are collected at each NaCl concentration. For example, after binding of the AAV library particle/heparin complex on the affinity matrix, the AAV library particle is washed with a volume of 50 mM Tris buffer, pH 7.5, containing 200 mM NaCl, and the eluted AAV library particles are collected. The elution step is repeated with 50 mM Tris buffer, pH 7.5, containing approximately 250 mM NaCl, approximately 300 mM NaCl, approximately 350 mM, approximately 400 mM NaCl, approximately 450 mM NaCl, approximately 500 mM NaCl, approximately 550 mM NaCl, approximately 600 mM NaCl, approximately 650 mM NaCl, approximately 700 mM NaCl, or approximately 750 mM NaCl.

[00255] AAV library particles that elute at NaCl concentrations below approximately 450 mM NaCl exhibit decreased heparin binding properties compared to wild-type AAV. AAV library particles that elute at NaCl concentrations above approximately 550 mM NaCl exhibit increased heparin binding properties compared to wild-type AAV.

[00256] In some embodiments, the eluted AAV library particles are amplified by coinfecting permissive cells with a helper virus and refractionated on a heparin affinity matrix. This step can be repeated multiple times to enrich for AAV library particles with altered heparin binding properties.

[00257] In the methods herein, one or more selection steps may follow the generation of the AAV library particles. For example, in some embodiments, the method comprises selecting for increased binding to heparin followed by selecting for decreased binding to neutralizing antibodies. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies followed by selecting for increased binding to heparin. In yet other embodiments, the method comprises selecting for decreased binding to heparin followed by selecting for decreased binding to neutralizing antibodies. In yet other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies followed by selecting for decreased binding to heparin. In yet other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies followed by selecting for increased infectivity for a stem cell. In still other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies followed by selecting for increased neutralizing antibody evasion. In still other embodiments, the method comprises selecting for increased neutralizing antibody evasion followed by selecting for decreased binding to neutralizing antibodies.

[00258] Thus, the present invention provides an adeno-associated virus (AAV) library comprising a plurality of nucleic acids, each of which comprises a nucleotide sequence encoding a variant AAV capsid protein. The encoded variant AAV capsid protein comprises at least one amino acid substitution relative to the sequence shown in one of SEQ ID NOs: 10-13 and 26-33. The present invention provides a library of mutant adeno-associated virus (AAV) particles comprising a plurality of AAV particles, each of which comprises an AAV capsid protein that comprises at least one amino acid substitution relative to the sequence shown in one of SEQ ID NOs: 10-13 and 26-33. The nucleic acids encoding the mutant AAV capsid proteins are described above, as are the properties of the encoded mutant AAV capsid proteins.

[00259] The present invention also provides a library comprising at least one of the following: (i) two or more infectious rAAV virions, each comprising a variant adeno-associated virus (AAV) capsid protein and a heterologous nucleic acid; (ii) two or more isolated nucleic acids, each comprising a nucleotide sequence encoding a variant AAV capsid protein; (iii) two or more host cells, each comprising a nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein; and (iv) two or more variant AAV capsid proteins; wherein the variant AAV capsid protein of at least one library member comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence shown in one of SEQ ID NOs: 10-13 and 26-33.

[00260] Compositions and sets

[00261] The present invention also provides compositions and kits for use in the methods of the present invention. The compositions and kits of the present invention include at least one of the following: a subject rAAV infectious virion, a subject rAAV vector, a subject nucleic acid comprising a nucleotide sequence encoding a subject variant AAV capsid protein, an isolated host cell comprising a subject nucleic acid (i.e., a subject genetically modified host cell comprising a nucleic acid that comprises a nucleotide sequence encoding a subject variant AAV capsid protein); a subject library (e.g., any of the libraries described above); and a subject variant AAV capsid protein. The composition or kit may include any suitable combination of the foregoing. The composition or kit may also include a helper virus and/or a nucleic acid comprising a nucleotide sequence that encodes the helper virus. The kit may also include reagents for producing nucleic acids (i.e., "mutant" nucleic acids) encoding modified variant AAV capsid proteins.

[00262] In addition to the above components, the kits of the present invention may further include (in some embodiments) instructions for using the methods of the present invention. These instructions may be present in the claimed kits in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is printed information on a suitable carrier or substrate, such as a sheet or sheets of paper on which the information is printed, in the packaging of the kit, a package insert, and the like. Another form of these instructions is a machine-readable medium, such as a diskette, compact disc (CD), flash drive, and the like, on which the information is recorded. Another form of these instructions that may be present is a website address that can be used via the Internet to access information on a remote site.

[00263] Now that the invention has been fully described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

Examples

[00264] Example 1

[00265] To date, adeno-associated virus (AAV) gene therapy vectors have shown promising results in several clinical trials. However, circulating anti-AAV antibodies produced as a result of childhood exposure or prior administration of an AAV vector preclude the implementation of AAV gene therapy in many potential patients. The inventors have identified novel AAV variants that are capable of enhanced evasion of anti-AAV antibodies, both in vitro and in vivo. Severe selection pressure from pools of low- and high-potency human sera and human IVIG has resulted in the emergence of AAV variants capable of evading neutralization by antibodies from individual human sera, human IVIG, and mouse sera, representing the most extensively evading variants to date.

[00266] Materials and methods

[00267] Cell lines

[00268] Cell lines were cultured at 37°C and 5% _CO2 and, unless otherwise indicated, were obtained from the American Type Culture Collection (Manassas, VA). HEK293T, HeLa, and HT1080 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, Calif.) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, Calif.). CHO K1 and CHO pgsA cells were cultured in F-12K medium (ATCC) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen). Pro5 and Lec1 cells were cultured in MEM-alpha medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen).

[00269] Pools of human serum for selection

[00270] Eighteen individual human serum samples were obtained from Innovative Research, Inc. (Southfield, Mich.), and the titer of neutralizing antibodies to wild-type AAV2 was determined for each sample (Table 2). Because individual samples are likely to have variations in both antibody affinity and epitope specificity, three effective serum pools (α=A+F+G, β=B+H+M, and γ=I+J+N) were generated by mixing equal volumes of individual serum samples. Selection in the presence of these antibody variations should result in an overall increase in resistance to many preexisting human antibodies. Later selections were performed in the presence of Gamimune H, 10% human IVIG (Bayer, Elkhart Ind.) to select for resistance to an even broader range of antibodies.

[00271] Table 2: Neutralizing antibody titers in individual human serum samples

Neutralizing antibody (NAb) titers for each sample are presented as the reciprocal of the volume of serum fraction required to reduce infectivity to 37% as measured in the absence of serum. Three serum pools (α=A+F+G, β=B+H+M, and γ=I+J+N) were then created by mixing equal volumes of three individual serum samples.

[00272]

~NAb human serum Sample Titer A 500 B 275 C 200 D <75 E <75 F 350 G 425 H 450 I 200 J 500 K 172 L <75 M 2200 N 5000 O <75 P <75 Q <75 R 120

[00273] Creating a library and obtaining a virus

[00274] To generate a saturation mutagenesis library, an AAV2 cap library was generated using reduced-fidelity PCR followed by the stepwise extension process described by Zhao et al. using 5′-GCGGAAGCTTCGATCAACTACGC-3′ (SEQ ID NO: 14) and 5′-GGGGCGGCCGCAATTACAGATTACGAGTCAGGTATCTGGTG-3′ (SEQ ID NO: 15) as forward and reverse primers, respectively. Selections using pooled individual human sera identified a variant containing four point mutations (described in the Results section), which served as the basis for generating a saturation mutagenesis library. The cap gene for this variant was further mutagenized by substituting amino acids at specific sites. Primer 5'-cattNNKgaccagtctaggaactgg-3' (SEQ ID NO: 16) and the corresponding complementary reverse primer were used for mutagenesis of amino acid site R471. Primer 5' gccacaaggacgatgaagaaNNKttttttcctcagagcggggttctcatctttgggaagcaaggctcaNNKaaaacaagt gtggacattg-3' (SEQ ID NO: 17) and the corresponding complementary reverse primer were used for mutagenization of amino acid sites K532 and E548. Primer 5'-ccaacctccagagaggcNNKagacaagcagctacc-3' (SEQ ID NO: 18) and the corresponding complementary reverse primer were used for mutagenesis of amino acid site N587. Primer 5'-ccaactacaacaagtctNNKaatgtggactttactgtggacNNKaatggcgtgtatt-3' (SEQ ID NO: 19) and the corresponding complementary reverse primer were used to mutagenize the V708 and T716 amino acid sites. A library consisting of AAV2 containing randomized cap loop regions and a library containing shuffled DNA from wild-type AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9 cap genes were packaged and pooled for the initial selection steps (Koerber et al.; Mol. Ther., 2008 October, 16(10):1703-9 and Koerber et al., Mol. Ther, 2009, December, 17(12):2088-95, both of which are incorporated herein by reference in their entireties).

[00275] For the second and third rounds of evolution, random mutagenesis libraries were generated by subjecting the cap genes from the loop/shuffle exchange library and the saturation mutagenesis library to reduced-fidelity PCR using 5'-CATGGGAAAGGTGCCAGACG-3' (SEQ ID NO: 20) and 5'-ACCATCGGCAGCCATACCTG-3' (SEQ ID NO: 21) as forward and reverse primers, respectively, as described above. Replication-competent AAV libraries and recombinant AAV vectors expressing GFP under the control of the CMV promoter were packaged using HEK293T cells (ATCC) using the calcium phosphate transfection method, and the viruses were purified by iodixanol gradient centrifugation. Recombinant AAV vectors expressing GFP or luciferase under the control of the CMV promoter were further purified for in vivo use by Amicon filtration. Genomic titers of DNase resistance were determined by quantitative PCR (Excoffon et al., Proc. Natl. Acad. Sci. USA, 2009 Mar. 10; 106(10):3865-70; and Maheshri et al., Nat. Biotechnol., 2006 February; 24(2):198-204; both of which are incorporated herein by reference in their entireties).

[00276] Selection and evolution of the library

[00277] One round of selection was defined as infection of HEK293T cells with an AAV stock library (incubation for 30 min at room temperature for pooled individual human sera or for 1 h at 37°C with heat-inactivated IVIG prior to infection), followed by adenovirus rescue and collection of successful variants. Each round of evolution consisted of mutagenesis of the cap gene to generate a stock library and three rounds of selection. Three rounds of evolution were performed with each library, with clonal analysis performed between each round of evolution. Stock libraries for each round of evolution were generated as described above. Following the third round of selection, AAV cap genes were isolated from the pool of successful AAV variants and amplified using PCR. The cap genes were inserted into the recombinant AAV packaging plasmid pXX2 using NotI and HindIII. The cap genes were then sequenced at the University of California, Berkeley DNA Sequencing Facility and analyzed using Geneious software (Biomatters, Auckland, New Zealand). Three-dimensional models of the AAV2 capsid (Protein Data Bank accession number 1LP3) were visualized in the Pymol system (DeLano Scientific, San Carlos, California).

[00278] In vitro transduction assay of antibody evading variants

[00279] HEK293T cells were seeded at a density of 3 x 10 ⁴ cells/well 24 h prior to infection. Variants were incubated at 37°C for 1 h with heat-inactivated IVIG, individual human sera, or individual mouse sera prior to infection, and cells were then infected with rAAV-GFP at a genomic MOI of 2000. The percentage of GFP-positive cells was assessed 48 h post-infection using the ImageXpress Micro Cellular Imaging and Analysis System (Molecular Devices, Sunnyvale, CA) and MetaXpress Image Analysis software, version 3.1.0, Multi Wavelength Cell Scoring Application Module (Molecular Devices).

[00280] In vitro transduction assay

[00281] To determine the relative transduction efficiency of the selected mutants compared with the parental wild-type AAV serotypes, HEK293T, CHO K1, CHO pgsA (all lacking surface glycosaminoglycans), CHO Pro5 (parental line for several glycosylation mutants including Lec1 cells), CHO Lec1 (glycosylation-defective), HeLa, and HT1080 (human fibrosarcoma cell line) cells were seeded at a density of 2.5 x 10 ⁴ cells per well 24 h prior to infection. Cells were infected with rAAV1-GFP, rAAV2-GFP, rAAV6-GFP, Shuffle 100.1-GFP, Shuffle 100.3-GFP, SM 10.2-GFP, or Shuffle 100.7-GFP at MOIs ranging from 100 to 1000. The percentage of GFP-positive cells was assessed 48 h post-infection using a Beckman-Coulter Cytomics FC500 flow cytometer (Beckman-Coulter, Brea, CA).

[00282] In vivo analysis of antibody evading variants

[00283] For in vivo gene expression analysis, 8-week-old female Balb/c mice were primed with 4 mg IVIG per mouse or phosphate-buffered saline (control mice) via the tail vein 24 h prior to administration of recombinant Shuffle 100-3 (see SEQ ID NO: 12), SM 10-2 (see SEQ ID NO: 10), or AAV2 vectors. Mice were infected with 10 ¹¹ viral genomes of recombinant AAV vectors encoding luciferase under the control of the CMV promoter via the tail vein. For bioluminescence imaging, mice were anesthetized with 2% isoflurane and oxygen. D-luciferin substrate (GOLD Biotechnology, St. Louis, Mo.) was administered intraperitoneally at 500 μg/g body weight. Images were acquired using a VivoVision IVIS Lumina imager (Xenogen, Alameda, CA). Ventral images were taken for each mouse 7-10 min after substrate injection once weekly for four weeks. Five weeks after infection, blood was collected by cardiac puncture and serum was prepared, and mice were then perfused with 0.9% saline. Heart, liver, lung, kidney, spleen, brain, spinal cord, and hindlimb muscle were sampled and frozen. Frozen tissue samples were homogenized and resuspended in reporter assay lysis buffer (Promega, Mannheim, Germany) for in vitro luciferase assay. The lysate containing luciferase was clarified by centrifugation for 10 min at 10,000 g. For sample analysis, 20 μl of lysate was added to 100 μl of luciferase assay buffer, mixed, incubated for 5 min, and placed in a luminometer. The signal was integrated over 30 s at 2-s intervals and expressed as relative light units (RLU) determined by a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). The luciferase signal was normalized to total protein content determined by the bicinchoninic acid assay (Pierce).

[00284] Results

[00285] These results demonstrate that AAV can evolve to substantially overcome neutralization by anti-AAV antibodies, both in vitro and in vivo. Novel AAV variants were isolated that required 2- to 35-fold higher neutralizing antibody titers (using human IVIG) than wild-type AAV in vitro. Their ability to undergo antibody neutralization was also demonstrated by enhanced transduction in vivo in the presence of neutralizing antibodies. The identification of such novel clones resistant to anti-AAV antibodies allows for broader application of AAV-based nucleic acid delivery vector therapies (including subjects with high antibody titers who are currently ineligible for AAV gene therapy).

[00286] Generation and selection of an AAV library via directed evolution

[00287] Figure 1a shows a schematic of the directed evolution approach used to isolate novel AAV variants capable of escaping neutralization by human antibodies. Viral libraries were generated using the DNA mutagenesis methods described in the following paragraphs (Figure 1a, steps 1 and 2). During the initial selection, pools of viral libraries generated from low-fidelity PCR mutations of the AAV2 cap genes were incubated with varying dilutions of a low-activity human α-pool of sera for 30 min at room temperature prior to infection of HEK293T cells (step 3). After three rounds of selection against the low-activity human α-pool of sera (Figure 1a, steps 4 and 5), several variants with increased resistance to this pool of neutralizing sera were obtained (Figure 1a, step 6, Figure 7a). Variant 1.45 contained two point mutations (N312K, N449D) that resulted in more than 10-fold resistance to α-pool neutralization compared to wild-type AAV2.

[00288] The cap gene from variant 1.45 was subjected to additional random mutagenesis and the resulting library was selected in three additional rounds of selection against the β- and γ-pools in parallel. Since only a modest increase in antibody evasion was observed (data not shown), the recovered cap genes were pooled and subjected to further diversification by DNA shuffling and EP-PCR. Three more rounds of selection with increasing amounts of sera from both the β-pool and the γ-pool resulted in a significant increase in the amount of virus recovered from the viral library compared to wild-type AAV2 (Fig. 7b, c). Sequencing of successful cap genes from both pools revealed several low frequency mutants and one dominant mutant, variant γ4.3, which contained four point mutations (N312K, N449D, N551S, and 1698V) present in both libraries. In the presence of human IVIG, variant 1.45 showed a modest 1.2-fold increased resistance to neutralization, whereas variant γ4.3 showed a 3.1-fold increased resistance to neutralization (Fig. 7d). This observation supports the hypothesis that pools of individual human sera can be used to isolate AAV variants capable of enhanced evasion of antibodies present in the general human population.

[00289] The moderate success of the γ4.3 variant in resisting neutralization by anti-AAV antibodies prompted the development of a γ4.3 cap gene-based library. Amino acid sites R471, K532, E548, N587, V708, T716, which had previously been identified as immunogenic sites in the AAV2 capsid, were subjected to saturation mutagenesis in an attempt to find amino acid mutations that could lead to increased antibody resistance of γ4.3. This “saturation mutagenesis” library, along with a “shuffled” library consisting of random cap chimeras of the 7 parental AAV serotypes and a “loop swap” library consisting of AAV2 cap with swapped loop regions, were subjected to three additional rounds of selection in which pools of viral libraries were incubated with varying dilutions of human IVIG for one hour at 37°C prior to infection of HEK293T cells. Following infection with the AAV libraries and amplification of infectious AAV variants by adenovirus superinfection, the number of viral genomes or virus titer in each library was quantified and compared with wild-type AAV2 titers as a method for determining selection success (Fig. 1b). For each round of selection using saturation mutagenesis and loop exchange/shuffling libraries, viral pools from the 1:10 and 1:100 IVIG dilution conditions that yielded higher virus titers than wild-type AAV2 were used as the starting point for the subsequent round of selection. After three rounds of selection, successful viral cap genes were isolated and tested individually to identify the virus with the most efficient gene delivery. In addition, cap genes isolated from the third round of selection were subjected to additional rounds of lower-fidelity PCR mutagenesis, and this process was repeated to iteratively improve virus fitness.

[00290] Figure 1 shows directed evolution of AAV to achieve enhanced antibody evasion. (a) Schematic of directed evolution. 1) A viral library is generated by genetic diversification of the cap gene using multiple complementary approaches. 2) Viruses are packaged into HEK293T cells using plasmid transfection, then harvested and purified. 3) The viral library is incubated with multiple concentrations of human IVIG and introduced into HEK293T cells in vitro. 4) Successful viruses are amplified and isolated by adenovirus superinfection. 5) Successful clones are enriched by repeated selections at lower MOIs. 6) Isolated viral DNA reveals successful cap genes. 7) Successful cap genes are again mutated to serve as a new starting point for selection. (b) Selection of antibody evasion mutants from loop exchange/shuffling and saturation mutagenesis libraries. HEK293T cells were infected with the viral libraries for 24 h. Viral particles that productively infected the cells were amplified by adenovirus infection, and rescued AAV was quantified by qPCR. A 1:10 dilution of IVIG corresponds to an IVIG concentration of 10 mg/mL. Error bars indicate standard deviation (n=3).

[00291] Figure 7 shows the generation of human antibody evasion variants based on AAV2. (a) Four viral clones selected after three rounds of selection against a low stringency α pool exhibit increased resistance to 1 μl of α serum at an MOI of 1. Two additional rounds of diversification (i.e., mutagenesis and DNA shuffling) and selection (3 rounds with increasing numbers of sera) resulted in significantly increased virus recovery in the presence of large amounts of the potent (b) β and (c) γ pools. (d) In addition, two viral clones (1.45 and γ4.3) showed 1.23- and 3.10-fold increased resistance to a highly diverse pool of previously produced antibodies present with pooled human intravenous immunoglobulin (IVIg) from ~100,000 subjects compared to wild-type AAV2.

[00292] Enhanced antibody evasion of novel evolved AAV variants in vitro

[00293] Of the twelve clones selected and packaged for individual analysis from the saturation mutagenesis and loop exchange/shuffle libraries after nine rounds of screening against human IVIG, all twelve required higher neutralizing antibody titers than wild-type AAV1 and AAV2 (Figure 2a and Table 1). The Shuffle 100-3 variant (see SEQ ID NO: 12), which required a 35-fold higher concentration of IVIG in vitro for neutralization than wild-type AAV2, was still able to transduce approximately 10% of the cells in the presence of 1 mg/mL IVIG (Figure 2b). Additionally, the SM 10-2 variant from the AAV2 saturation mutagenesis library required a 7.5-fold higher concentration of WIG in vitro for neutralization than wild-type AAV2. In addition, the Shuffle 100-3 and SM 10-2 variants (see SEQ ID NO: 10) showed enhanced transduction in the presence of serum samples from individual patients excluded from a clinical trial with hemophilia B (Fig. 3) (Nathwani et al., N. Engl. J. Med., 2011, Dec. 22, 365(25):2357-65).

[00294] Figure 2 shows the neutralization profiles of antibody evasion variants. The cap genes of antibody evasion mutants isolated after three rounds of evolution were used to package recombinant AAV encoding GFP and incubated with human IVIG prior to infection of HEK293T cells. The fraction of infectious particles remaining was determined using high-concentration fluorescence imaging and normalized to the infectious titer in the absence of IVIG. Two clones from each library with resistance to IVIG are shown. Data for the other clones analyzed are presented in Table 1. (a) Neutralization curves. Error bars indicate standard deviation (n=3). (b) Representative fluorescence images from multiple dilutions of IVIG show that the mutants are capable of transducing HEK293T in the presence of high concentrations of neutralizing antibodies.

[00295] Figure 3 shows neutralization profiles of antibody escape variants. Human sera were obtained from subjects who were excluded from clinical trials with hemophilia B due to the presence of high titers of neutralizing anti-AAV antibodies. Recombinant AAV encoding GFP was incubated with individual human serum samples prior to infection of HEK293T cells. The fraction of infectious particles remaining was determined using fluorescence microscopy and normalized to the infectious titer in the absence of human serum. Error bars indicate standard deviation (n=3).

[00296] Sequence analysis of twelve clones revealed that the two variants with the highest neutralizing antibody resistance, Shuffle 100-3 (see SEQ ID NO: 12) and Shuffle 100-1 (see SEQ ID NO: 11), are nearly identical across shuffled capsids containing fragments of AAV1-4, AAV6, and AAV9 (Figure 4). The differences at amino acids 469 (from residue AAV6 to residue AAV7) and 598 (from residue AAV6 to residue AAV1) between the two variants result in a nearly 3-fold increase in neutralizing antibody titer for Shuffle 100-3 (see SEQ ID NO: 12) (Table 1). Variant Shuffle 100-7 (see SEQ ID NO: 13), which had the fourth highest resistance to neutralizing antibodies (Table 1), also has a shuffled capsid containing fragments of AAV1, AAV6, and AAV8 (Fig. 4), which is in good agreement with published data showing that wild-type AAV1 and AAV8 are able to evade anti-AAV2 antibodies. Interestingly, variant SM 10-2 (SEQ ID NO: 10) retained the point mutations acquired by variant γ4.3 and also retained wild-type residues at saturation mutagenesis sites. Variant SM 10-2 (SEQ ID NO: 10) acquired additional point mutations at the surface residue D472N and the internal residue L735Q. In Fig. Figure 4 shows the amino acid sequences of the loop exchange/shuffling and saturation mutagenesis clones. (a) Schematics of the capsid protein are shown for the two clones from each library with the highest neutralizing concentrations of IVIG. Each region is shaded according to the parental serotype from which it is derived. Black arrows denote (from left to right) the original codons of the capsid proteins VP1, VP2, and VP3. Gray arrows denote (from left to right) regions of the surface loops I, II, III, IV, and V based on the AAV2 capsid. (b) Molecular models of the complete AAV2 capsid based on the resolved structure are shown for the two clones from each library with the highest neutralizing concentrations of IVIG. Each region is shaded according to the parental serotype from which it is derived. For variant Shuffle 100-3 (see SEQ ID NO: 12): black arrows indicate differences from variant Shuffle 100-1 (see SEQ ID NO: 11). For variant SM 10-2 (see SEQ ID NO: 10): mutations N449D, D472N, N551S and 1698V are surface mutations (black).

[00297] Table 1: IVIG neutralizing antibody titers for clone libraries and parental serotypes

[00298] Human IVIG was used to neutralize recombinant AAV-GFP vectors with capsids from wild-type AAV1, AAV2, AAV8, and variants recovered from loop exchange/shuffling and saturation mutagenesis libraries. The concentration of IVIG (mg/mL) required to reduce gene delivery efficiency to 50% in the absence of IVIG is shown, compared to the concentration required to reduce delivery of AAV2. All variants analyzed required higher concentrations of IVIG than wild-type AAV1 and AAV2. Neutralizing antibody titers were determined by fitting the curves in Figure 2 to an exponential curve. SEQ ID NOs are listed as "amino acid, nucleotide."

[00299]

Table 1 Clone SEQ ID NO: Neutralizing IVIG
concentration mg/ml Fold increase in resistance relative to AAV2 AAV1 1 0.026 1,757 AAV2 2 0,015 1,000 AAV8 8 0.092 6,113 Shuffle 10-2 26, 34 0.037 2,443 Shuffle 10-6 27, 35 0.028 1,842 Shuffle 10-8 28, 36 0.084 5,583 Shuffle 100-1 11, 23 0.183 12,178 Shuffle 100-2 29, 37 0.073 4,831 Shuffle 100-3 12, 24 0.529 35,227 Shuffle 100-7 13, 25 0,090 6,025 SM 10-1 30, 38 0,071 4,732 SM 10-2 10, 22 0.113 7,519 SM 10-8 31, 39 0.051 3,409 SM 100-3 32, 40 0.074 4,941 SM 100-10 33, 41 0.066 4,393

[00300] Variants Shuffle 100-3 (see SEQ ID NO: 12), Shuffle 100-1 (see SEQ ID NO: 11), and Shuffle 100-7 (see SEQ ID NO: 13) have transduction profiles that are similar to the transduction profiles of the parental serotypes AAV1 and AAV6 (Figure 5). In addition, mutations in SM 10-2 (see SEQ ID NO: 10) do not prevent heparin dependence (as observed in the parental serotype AAV2), resulting in a profile similar to AAV2 (Figure 5).

[00301] Figure 5 shows the tropism of novel aav variants in vitro. Recombinant AAV vectors expressing green fluorescent protein were used to transduce a number of cell lines: CHO, pgsA (all lacking surface glycosaminoglycans), Pro5, Lec1 (lacking sialic acid), HEK293T, HeLa, and HT1080 (a human fibrosarcoma cell line) to profile the transduction properties of the novel AAV variants. Error bars indicate standard deviation (n=3).

[00302] Enhanced antibody evasion of novel evolved AAV variants in vivo

[00303] To determine the localization pattern of the Shuffle 100-3 and Shuffle 100-7 variants, luciferase enzyme activity was determined in various tissues of naive mice administered AAV2, Shuffle 100-3, or Shuffle 100-7 (Fig. 6A). The Shuffle 100-7 variant showed in vivo tropism similar to AAV2, except that transduction of cells in the heart was 7-fold higher, transduction in the lungs was 5-fold higher, and transduction in the liver was 4.5-fold lower. The Shuffle 100-3 variant showed more than 4-fold higher transduction of cells in the brain, more than 3-fold higher transduction in the lungs, and 27-fold higher transduction in muscle than AAV2. Analysis of sera from these mice showed that the Shuffle 100-3 variant required equal or higher in vitro serum concentrations for neutralization than AAV1 and AAV8 for sera from mice injected with AAV1, AAV2, AAV8, or Shuffle 100-3 gene delivery vectors (Fig. 11). Shuffle 100-7 required equal or higher in vitro serum concentrations for neutralization than AAV1 for sera from mice injected with AAV1, AAV2, AAV8, Shuffle 100-3, or SM 10-2 gene delivery vectors (Fig. 11). Furthermore, both variants were less neutralizable by sera from mice injected with AAV2 gene delivery vectors than all wild-type AAV serotypes tested. Interestingly, the Shuffle 100-3 variant was also less susceptible to neutralization by sera from mice immunized against it than any of the other serotypes or variants tested (Fig. 11). These data illustrate the possibility that these variants could be used in combination with wild-type or other variant AAV serotypes in applications that require multiple vector administrations.

[00304] Figure 11 shows the neutralizing antibody titers of library clones and parental serotypes in sera from immunized mice. Sera from mice injected with library clones or wild-type AAV were used to neutralize recombinant AAV-GFP vectors with capsids from wild-type AAV1, AAV2, AAV8, and variants isolated from loop exchange/shuffling and saturation mutagenesis libraries. The serum dilution required to reduce gene delivery efficiency to 50% in the absence of serum is shown.

[00305] To determine the ability of Shuffle 100-7 and Shuffle 100-3 variants to evade antibody neutralization in vivo, mice were passively immunized with human IVIG prior to administration of AAV. Shuffle 100-7 variant had significantly higher transduction of heart, liver, and muscle cells than AAV2, as measured by luciferase enzyme activity (Figure 6b). Shuffle 100-3 variant had significantly higher transduction of cells in the heart and muscle compared to AAV2 (Figure 6b).

[00306] Figure 6 shows the localization and neutralization of novel AAV variants in vivo. (a) Recombinant AAV vectors encoding luciferase were injected into the tail vein of female BALB/c mice. After 5 weeks, luciferase activity levels were determined and normalized to total protein for each sample analyzed. (b) Recombinant AAV vectors expressing luciferase were injected into the tail vein of female BALB/c mice 24 h after tail vein injection of 4 mg human IVIG. After 5 weeks, luciferase expression levels were normalized to total protein for each sample analyzed. Error bars indicate standard deviation (n=3), *=p<0.05. RLU, relative light unit.

[00307] Variant γ4.3, isolated from a reduced-fidelity PCR library of AAV2 selected from a pool of individual human sera, contained four point mutations (N312K, N449D, N551S, and I698V). Interestingly, two of these positions (N449 and N551) had previously been identified as immunogenic residues using other pools of human sera, suggesting that antigenic epitopes including these sites are targets for many different neutralizing antibodies. Thus, these sites represent interesting and valuable mutation targets. The combination of directed evolution and rational design in generating a saturation mutagenesis library resulted in the isolation of variant SM 10-2, which was capable of higher antibody resistance than both AAV1 and AAV2 in vitro. Variant SM 10-2 contains two additional point mutations (D472N and L735Q) compared to those found in variant γ4.3. The D472N mutation has previously been shown to increase capsid synthesis in HEK293 cells. Similarly, the replacement of the positively charged lysine side chain at amino acid position 735 with an uncharged glutamine side chain may contribute to capsid stabilization, as it is also present in variant Shuffle 100-7, despite being located within the assembled capsid (Fig. 4).

[00308] The generation of chimeric AAV capsids allows the generation of viral variants that can combine the desirable properties of multiple AAV serotypes. Although AAV8 and AAV9 were also shown to be significantly more resistant to neutralization by IVIG than AAV2, amino acids specific to these capsids were present only in small regions on the surface of the shuffled variants isolated during selection (Figure 4). A variant exhibiting more efficient evasion of antibody neutralization in vitro, Shuffle 100-3, exhibited in vitro tropism similar to the parental serotypes AAV1 and AAV6, but with a higher degree of infectivity than either wild-type serotype. Differences at amino acids 469 and 598 between Shuffle 100-1 and Shuffle 100-3 result in a nearly 3-fold increase in neutralizing antibody titers for Shuffle 100-3. Results from Lochrie et al. indicate that the immunogenic residues recognized by human serum and IVIG are different, suggesting that different individuals may produce different neutralizing antibodies to different sets of epitopes on the AAV capsid, and that completely evading neutralization may be challenging (Lochrie et al., J. Virol., 2006 January; 80(2):821-34). The authors' work demonstrates that using multiple rounds of directed evolution using multiple different serum pools containing different amounts and potencies of anti-AAV antibodies will result in the isolation of novel AAV variants that are capable of enhanced cellular transduction, both in vitro and in vivo, in the presence of multiple pools of anti-AAV antibodies.

[00309] Adaptive immune responses to AAV vector components in animals and humans often prevent repeated administration of AAV vectors of the same serotype, complicating gene delivery applications that require multiple vector administrations. In vitro neutralization assays using sera from mice used in biodistribution studies indicate that the variants are less neutralized by these sera than wild-type AAV (Figure 11), which may be useful for gene therapy strategies that require repeated vector administration. For example, Shuffle 100-3 is not neutralized by sera from mice injected with AAV2, and AAV2 is not neutralized by sera from mice injected with Shuffle 100-3, suggesting that this variant could be used in combination with wild-type AAV serotypes or in applications that require administration of multiple vectors. In conclusion, the inventors used directed evolution to isolate novel AAV variants that are capable of reduced neutralization by anti-AAV antibodies obtained from individual patients, pooled human serum, and mouse serum, both in vitro and in vivo.

Example 2

Identification of a capsid variant suitable for use in lung-targeted gene therapy in primates

[00310] Introduction

[00311] A directed evolution strategy was used to identify AAV capsid variants with enhanced gene delivery efficiency into non-human primate (NHP) type II (AT II) lung alveolar epithelial cells following intratracheal aerosol administration in the presence of human neutralizing antibodies (NAbs). Briefly, wild-type adeno-associated virus (AAV) cap genes were diversified using multiple approaches to generate large gene libraries that were packaged to generate viral particle libraries, and selective pressure was then applied to isolate novel variants that could overcome gene delivery barriers, including, but not limited to, anti-capsid immune responses, limited cell transduction in certain tissues, and inability to target certain cell types.

[00312] Methods

[00313] Cell lines and library production

[00314] HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured at 37°C and 5% _CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). Viral libraries were produced in HEK293T cells using triple transfection, and viruses were purified by iodixanol gradient centrifugation and filtration through an Amicon device. Genomic titers of DNase resistance were determined by quantitative PCR (qPCR).

[00315] Intratracheal injection and tissue collection

[00316] For each delivery device used in selection, one male cynomolgus macaque (macaca fascicularis), 4-6 years of age and weighing 5.5-6.0 kg, was dosed. Animals were anesthetized with ketamine at 10 mg/kg and dexmedetomidine at 15 μg/kg intramuscularly (i.m.). Five ml of the library was premixed with 1.75 mg/ml human intravenous immunoglobulin (IVIG) and administered as described below. Each animal was intubated with a 5 mm endotracheal tube, with the end of the tube positioned at the level of the clavicle (approximately 5 cm above the carina), and its position was confirmed by fluoroscopy.

[00317] The nebulizer device was connected to the distal end of the endotracheal tube and an avian respirator was used to deliver inspirations at a rate of 15 ± 1 breath/min at 20 cm _H2O . An AeroProbe® catheter (Trudell Medical International) was fitted with a piece of 0.144-inch diameter star tubing to facilitate proper placement within the endotracheal tube. The tip of the catheter was positioned directly over the end of the endotracheal tube. The AeroProbe® catheter was connected to the AeroProbe Catheter Control System and a breathing apparatus (Harvard Appartatus) was used to deliver inspirations at a rate of 20 breaths/min at 18-20 cm _H2O . After dosing was complete, each animal was extubated and 0.15 mg/kg atipamezole was administered intramuscularly to reverse sedation. Animals were visually monitored until they had fully recovered from anesthesia before being returned to their home cages.

[00318] Euthanasia was performed by trained veterinary personnel using 100 mg/kg sodium pentobarbital administered intravenously on day 15±1. The lungs, including the trachea, were removed and dissected as detailed below. DNA was isolated from AT II cells and stored at -20°C until viral genome amplification.

[00319] Isolation of alveolar epithelial type II (AT II) cells

[00320] AT II cells were isolated from non-human primate lungs as described in Fang et al., Measurement of Protein Permeability and Fluid Transport of Human Alveolar Epithelial Type II Cells Under Pathological Conditions. Humana Press, New York, NY, 2018, pp. 121-128. Briefly, lungs were washed with 500 mL of PBS containing 5 mM EDTA and 5 mM EGTA using a syringe inserted into the trachea. The lungs were then filled with 250 mL of 1.2 mg/mL elastase solution and incubated for 1 h at 37°C. The lungs were homogenized to release lung lining cells and the airways were removed. AT II cells were isolated after a series of inclusion and exclusion steps, including a Percoll gradient and CD14/CD45 Dynabeads. AT II cells were plated on collagen IV-coated inserts for 24 h prior to DNA extraction. Lysotracker and surfactant protein C assays were performed using flow cytometry and immunocytochemistry to ensure the purity of the cell isolate.

[00321] Evolution of the therapeutic vector

[00322] The vector evolution process used is shown in Fig. 12. Briefly, a library of viral capsids containing proprietary combinations of DNA mutation methods and cap genes was generated (a). The viruses were then packaged (b) such that each particle consisted of a mutant capsid surrounding a cap gene encoding that capsid and purified. The capsid library was placed under selective pressure in vivo. The tissue or cell type of interest was harvested to isolate AAV variants that successfully localized to the target. Successful viruses were recovered using PCR amplification. Successful clones were enriched by repeated selection (Step I - (c)). The selected cap genes were then subjected to proprietary repeated diversification and enriched by further selection steps to iteratively increase viral fitness (Step 2 - (d)). The variants identified as the most successful in stages 1 and 2 of vector selection were evaluated to identify capsid variants with the desired properties (e).

[00323] Motifs were considered "hit" when they met the following criteria: 1) the motif represents approximately 5% of the sequenced population in two or more consecutive rounds of selection; or 2) the motif represents at least 10% of the sequenced population in one or more rounds of selection.

[00324] Results

[00325] Pilot studies of device parameters for delivery and isolation of AT II cells

[00326] Two delivery devices, the AeroProbe® catheter (Trudell Medical International) and a proprietary CRO nebulizer, were used to ensure future compatibility with multiple clinically applicable devices. Both delivery devices were evaluated in pilot studies with Evans Blue dye delivery to ensure that ventilation parameters resulted in adequate distribution to all lung lobes and alveolar sacs. Both delivery devices demonstrated good distribution to all lobes, including the alveolar compartment, with more intense staining observed in dependent lobes.

[00327] The AT II cell isolation protocol was optimized using a total of 6 lungs from NHPs. The optimization of the protocol resulted in high yield and purity of AT II cells isolated from both NHP lungs, which were used during the evolution of the therapeutic vector.

[00328] Evolution of the therapeutic vector

[00329] Prior to Round 1 of the therapeutic vector evolution program, 37 vector libraries were synthesized, generated, and characterized. As shown in Fig. 13A, the diversity of the plasmid libraries was estimated to include between approximately 1 x 10 ⁶ and > 1 x 10 ⁸ individual unique variants per library. This represents a high-quality and highly diverse starting library of AAV variants. Each individual library was then completed to generate sufficient material for the first round of selection. As shown in Fig. 13B, all libraries were generated at a level sufficient to generate material for selection during in vivo therapeutic vector evolution.

[00330] All 37 libraries were pooled and successfully administered to NHPs via a single aerosol administration using either the AeroProbe® or a nebulizer. Prior to administration, the libraries were incubated with human intravenous immunoglobulin at a concentration of 1.75 mg/mL. This represents a high but physiologically relevant concentration of human NAbs on the lung mucosa. The library dose of 1.7 x 10 ¹² vg per NHP represents a dose that is approximately 10-fold lower than the actual maximum possible dose based on these considerations. Therefore, this represents a strong selective pressure to enable the discovery of a vector capable of transducing AT II cells in the alveolar space in the presence of NAbs. NHP lungs were recovered two weeks after administration. AT II cells were isolated from the lungs and DNA was isolated from the AT II cells.

[00331] Successful amplification of AAV capsid genomes

[00332] Amplification of capsid genes from tissue represents successful localization of library vectors into the cell type of interest. Capsids amplified using each delivery device (Fig. 14A-B) were cloned into an AAV library packaging plasmid for sequence analysis and initiation of a subsequent round of selection (if necessary).

[00333] Sequence Analysis

[00334] Sequencing was performed on individual clones in the library to determine the frequency of variants in the population. A minimum of 90 clones were sequenced for each delivery device. Variants were assessed for the presence of motifs in the sequencing data. Variants were grouped into motifs based on the presence of a unifying variation (e.g., a specific point mutation or a specific peptide insertion sequence at a constant site within the capsid) that was present in multiple sequences. A motif was further evaluated only if it represented at least 5% of the sequenced population in two or more consecutive rounds of selection or at least 10% of the sequenced population in one or more rounds of selection. A motif meeting the latter criterion is shown in Figs. 15A-15B. The selection was considered complete after the first round, since high convergence with the A101 variant (containing the capsid protein SEQ ID NO:12) was observed using both delivery devices.

[00335] Based on the above ranking criteria, capsid variant A101, comprising the capsid protein of SEQ ID NO:12, was identified as providing enhanced gene delivery efficiency to the lungs of primates following intratracheal aerosol administration in the presence of human neutralizing antibodies (NAb). A101 is a chimera composed primarily of AAV1, but also including amino acids from AAV2, AAV4, AAV6, and AAV9.

Example 3

[00336] Although initial attempts to develop AAV-based gene therapy for the lung demonstrated clinical safety, use of the AAV2 vector ultimately failed to efficiently transduce lung cells to demonstrate clinical benefit in cystic fibrosis. More recently, additional AAV serotypes, including AAV1 and AAV5, have shown improved, but still suboptimal, transduction of primate lung cells following aerosol administration. The experimental data presented below support the surprising utility of rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 as a vehicle for the efficient delivery of transgenes, such as human CFTR, into the primate lung in the presence of human neutralizing antibodies.

[00337] Recombinant AAV (rAAV) gene delivery comprising (i) a capsid containing the capsid protein of SEQ ID NO:12 and (ii) a nucleic acid comprising a nucleotide sequence encoding a reporter transgene (GFP or EGFP) operably linked to a CAG promoter was characterized (including histopathological analysis) following aerosol administration to three non-human primates (NHPs). Nebulizer delivery of rAAV resulted in robust delivery of viral genomes to all regions of the lung, including the peripheral (bronchioalveolar regions), with minimal systemic biodistribution, and rAAV mediated protein expression in all regions of the lung, including the alveoli.

[00338] Materials and methods

[00339] Neutralizing antibody assay

[00340] HEK2v6.11 cells (obtained from Johns Hopkins University) were seeded in black opaque 96-well plates at a cell density of 30,000 cells/well in Dulbecco's modified Eagle's medium (DMEM; Corning) with 1% heat-inactivated fetal bovine serum (FBS; GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Invitrogen). Cells were allowed to attach to the plate for 24 h prior to experimentation.

[00341] Each NHP serum sample was assayed at 1:10, 1:25, 1:50 dilutions. Each plate contained positive and negative controls for transduction. NHP serum samples were incubated with rAAV containing (i) a capsid containing the capsid protein of SEQ ID NO:12 and (ii) a nucleic acid containing the CAG promoter operably linked to the luciferase gene at an MOI of 1000 at 37°C for 1 h. After incubation for 1 h, each dilution of NHP serum sample plus rAAV was added to separate wells of black opaque 96-well plates containing 2V6.11 cells. Luciferase was determined using the ONE-Glo EX Luciferase Assay Kit (Promega) 48 h after transduction. Upon addition of ONE-Glo EX, cells were lysed and luciferase substrate was added to the cells in one step. Luminescence was read using a Cytation 3 microplate reader (BioTek).

[00342] Coefficients of variation (CV) and standard deviations were calculated for all dilutions of NHP serum samples and for each point on the standard curve. NHP serum samples were normalized to the positive transduction control. Neutralizing antibody titers were determined for each NHP. The neutralizing antibody titer for each NHP serum sample was defined as the lowest serum dilution that resulted in ≥50% transduction. NHPs that demonstrated ≥50% transduction at a 1:10 serum dilution were considered eligible for inclusion in the study.

[00343] Test system and immunosuppression

[00344] Three male cynomolgus macaques were included in the study. The animals ranged in age from 4 years 10 months to 9 years 8 months and weighed from 4.73 kg to 7.09 kg. The animals were immunosuppressed with methylprednisolone (20 mg/kg, intramuscularly) once weekly, beginning on day -7.

[00345] Preparation and administration of test sample

[00346] Batches of rAAV test samples containing (i) a capsid containing the capsid protein of SEQ ID NO:12 and (ii) a nucleic acid containing the CAG promoter operably linked to EGFP were thawed on ice, pooled together, and diluted in formulation buffer to deliver a final dose of 2.80 x 10 ¹² vg/kg in 5 ml to each NHP. Test sample dilutions for each animal are provided in Table 3. Animals were sedated with ketamine and dexmedetomidine. Animals were intubated such that the tip of the endotracheal tube was positioned approximately 5 cm above the carina. Animals were placed in a sitting position in a chair for administration. For each animal, 5 ml of the diluted test sample was loaded into the reservoir of an AeroEclipseII nebulizer (Trudell Medical). During administration, the pressure was adjusted to 15-20 cm _H2O . The test sample was injected at a rate of 12-24 breaths/min until visible mist was generated within 10 pulses (animals were dosed continuously for <40 min). After administration was complete, animals were extubated and sedation was removed.

[00347]

Table 3
Testing: Preparing a Sample Dose NHP number Party Body weight (kg) Total dose
(vg) Dose of solution (vg/ml) Final volume dose of the drug (ml) Stock virus
(ml) Buffer for formulation
(ml) V002969 4DER000040.01
4DER000043.01 5.41 1.51E+13 3.03E+12 5.25 2.048 3.202 V003424 4DER000040.01
4DER000043.01 4.73 1.32E+13 2.65E+12 5.25 1.791 3.459 V003062 4DER000040.01
4DER000043.01 7.09 1.99E+13 3.97E+12 5.25 2.685 2.565

[00348] In vivo observations, necropsy and tissue collection

[00349] Caged animals were observed twice daily by CRO staff from day -7 until time of necropsy. Body weights were determined weekly, and blood samples were collected at specific time points for hematology and biochemistry. Euthanasia was performed on day 57 ± 1 by trained veterinary personnel using intravenous injection of sodium pentobarbital (100 mg/kg) followed by bilateral thoracotomy and transcardial perfusion with heparinized phosphate-buffered saline. Following perfusion, samples were collected from the lungs (including trachea), brain, spinal cord (cervical, thoracic, and lumbar regions), heart (ventricular and atrial regions), liver, spleen, skeletal muscles (triceps brachii, vastus lateralis), diaphragm, and kidney. Tissue samples were collected and snap frozen for subsequent DNA and protein extraction. Additional samples were collected and fixed in 4% paraformaldehyde (lung and nerve tissues) or 10% neutral buffered formalin (peripheral tissues) for subsequent paraffin embedding and sectioning for immunofluorescence.

[00350] Large samples were taken from the trachea and lungs to obtain multiple samples for each analysis. The lungs were removed and clamped as high above the trachea as possible. The right lung was clamped twice approximately 1 mm from the main bronchus. The right lung was removed by cutting between the clamps. Sixteen samples for DNA and protein extraction were collected from areas of the right lung covering the primary/secondary bronchi, tertiary bronchi, and alveoli as shown in Fig. 16. The trachea and left lung were filled with 4% paraformaldehyde and fixed in 10 times the volume of 4% paraformaldehyde. Sections of the trachea and left lung were then prepared covering samples of the trachea, primary/secondary bronchi, tertiary bronchi, and alveoli as shown in Fig. 16.

[00351] Biodistribution of the viral genome

[00352] Biodistribution of the viral genome was determined at Charles River Laboratories, Mattawan, using a suitable assay for AAV viral genomes containing the EGFP transgene sequence. Total DNA was extracted from tissue samples using the QIAsymphony Workstation (Qiagen) and the appropriate DSP DNA mini kit. qPCR reactions were performed in 96-well plates, with each plate containing a standard curve, a set of quality control samples, and test samples. Duplicate quality control samples were prepared at high, medium, and low copy numbers/reaction against 1000 ng of NHP template DNA per reaction. Where possible, tissue DNA samples were tested at 1000 ng per reaction. If it was not possible to load the amounts specified above for a particular sample (due to too low a DNA concentration or sample volume limitations), a smaller amount of sample DNA was analyzed.

[00353] All sample reactions were performed in triplicate, and a third reaction was spiked with 200 copies of pAAV-CAG-EGFP-SV40 DNA to assess potential qPCR inhibition. If qPCR inhibition was observed, as indicated by a measured value in the third well with less than 110 copies of target DNA spiked, the DNA sample was re-analyzed in a smaller amount.

[00354] Biodistribution of protein expression

[00355] Total protein was extracted from tissue samples using a softMACS tissue homogenizer (Miltenyi Biotec) and appropriate reagents. EGFP was quantified using a GFP ELISA kit (Abcam), and total protein was quantified using a Pierce BCA protein assay kit (ThermoFisher). For both GFP and total protein, reactions were performed in triplicate, and a standard curve was generated for each set.

[00356] Immunofluorescence imaging

[00357] Tissues were processed to obtain paraffin blocks using Sakura VIP 5 using the standard Seventh Wave program for canine, NHP, and porcine tissues. Sections were prepared at 10 μm thickness at Seventh Wave and stored at 4°C. Immunohistochemistry was performed on 6 lung sections (including alveolar and bronchial regions) and 2 tracheal sections from each experimental animal (n=3) and an additional control animal. Paraffin sections were dehydrated using a standard paraffin antibody staining protocol. Briefly, sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol in water (100%, 90%, 70%, 50%, and 30%) followed by washes with PBS. Antigen retrieval was performed prior to antibody staining using a combination of heat-induced retrieval (HEIR) and pressure. Sections were incubated for 10 min in boiling sodium citrate buffer, then incubated for 3 min under pressure. Sections were cooled to room temperature prior to antibody staining. Following antigen retrieval, sections were stained with primary chicken polyclonal anti-GFP antibody (Abcam #13970) at 1:1000 dilution, secondary goat anti-chicken IgY antibody (Abcam #175779) at 1:1000 dilution, and DAPI. Fluorescence imaging was performed using a Zeiss AxioObserver microscope. Anti-GFP signal was acquired in the red-most channel (647 nm) at 1000 ms. The DAPI signal was detected in the blue channel (355 nm) at 100 ms. All images were processed with Zeiss ZenPro software using the same parameters and pixel intensity values in the anti-GFP channel.

[00358] Histopathological analysis

[00359] Tissue processing, embedding, sectioning, and hematoxylin and eosin staining were performed at Seventh Wave. Tissues were processed for paraffin blocks using a Sakura VIP 5 using a standard program for canine, NHP, and porcine tissues. Sections were prepared at 4 μm thickness and stained with hematoxylin and eosin (H&E) using a Leica XL automated stainer. Histopathological analysis was performed on 16 lung sections, 4 trachea sections, and 1 carina section from each experimental animal (n=3) and an additional control animal, with the histopathologist blinded to the processing condition. Sections were scored for character and grade using a standard scoring system.

[00360] Computerized systems

[00361] For screening of serum neutralizing antibodies, data were acquired and analyzed using a Cytation 3 microplate reader (Biotek), Gen5plus software version 3.03.14, and Microsoft Excel version 15.32.

[00362] To quantify viral genomes in tissue samples, data were acquired and analyzed using the QuantStudio 7 Flex Real-Time PCR System, QuantStudio v1.4 Real-Time PCR Software, Microsoft Excel, and GraphPad Prism version 8.1.2.

[00363] To quantify EGFP expression in tissue samples, data were acquired and analyzed using a Cytation 3 microplate reader (Biotek), Gen5plus software version 3.03.14, Microsoft Excel version 15.32, and GraphPad Prism version 8.1.2.

[00364] For representative immunofluorescence imaging, images were acquired using a Zeiss Axio Observer z1 microscope and ZenPro software. Images were processed using ZenPro software. Images were transferred to Microsoft PowerPoint version 15.32 for presentation.

[00365] All data analysis and compilation was performed on a Macbook Pro running OSX (10.12.6).

[00366] Results and discussion

[00367] Screening for neutralizing anti-AAV antibodies identifies NHPs for inclusion in the study

[00368] A neutralizing antibody assay was used to assess levels of neutralizing antibodies against the AAV capsid containing the capsid protein of SEQ ID NO:12 in the serum of non-human primates (NHPs). The neutralizing antibody titer was determined for each NHP serum sample. An animal was considered seronegative and eligible for inclusion in the study if transduction was observed at a level of ≥50% at a 1:10 serum dilution.

[00369] A total of 20 NHP serum samples were evaluated in four 96-well plates. Assay acceptance criteria were established for 1) the coefficient of variation (CV) for the standard curve, 2) the CV for unknown serum samples, and 3) the percent deviation from the actual input protein for the standard curve. Acceptable CV values for the standard curve were set at < 25%, but the actual CV values did not exceed 5%. Acceptable CVs for unknown serum samples within the limit of quantification were set at < 30%, but the actual CV values did not exceed 19%. Acceptable percent deviation from the input protein for the standard curve was set at < 25%, but the actual percent deviation did not exceed 19%. All plates met all assay acceptance criteria, and the data obtained from these plates were used for the evaluation. Overall, 11 (55%) of the NHP serum samples analyzed were seronegative for the capsid containing capsid protein SEQ ID NO:12 (Fig. 17). The NHPs with the three highest transduction percentages at a 1:10 serum dilution were selected for inclusion in the study. All selected NHPs demonstrated transduction higher than that observed in the absence of NHP serum. This observation has been noted in previous studies and likely results from a favorable interaction with an unknown serum protein. The numbers of selected NHPs and the transduction percentages at a 1:10 serum dilution are presented in Table 4.

[00370]

Table 4
NHPs included in the study NHP number % transduction at 1:10 dilution V003272 BLQ V003281 88 V003270 BLQ V003516 113 V003062 147 V003125 140 V003511 BLQ V003421 110 V003433 BLQ V002964 BLQ V002996 BLQ V003002 107 V002969 160 V003015 36 V003119 60 V002851 BLQ V003101 137 V003083 BLQ V003424 160 V003251 124

[00371] Nebulized delivery of variant capsid containing capsid protein of SEQ ID NO:12 is well tolerated by NHP

[00372] The study design is presented in Table 5. All animals recovered to normal after administration of the test sample and remained alive until the scheduled necropsy dates. No significant clinical pathology was observed in any animal at any time point during the intravital portion of the study. Minor changes outside the reference ranges for hematological and biochemical parameters were observed in some animals at some time points, but these changes were interpreted as normal physiological fluctuations. No significant pathology was observed during macroscopic examination at necropsy.

[00373]

Table 5
Generalized study design # Path & Dose Dose Intravital study Fabric collection Analysis 3 Aerosol
(AeroEclipse II) 2.80×10 ¹²
vg/kg 8 weeks lung
heart
liver
skeletal muscles (triceps brachii, quadriceps, diaphragm)
bud
spleen
CNS (brain, spinal cord) qPCR
ELISA (lung & qPCR+ tissue)
IHC (lung & ELISA+ tissue)

[00374] Variant capsid comprising capsid protein of SEQ ID NO:12 mediates robust gene delivery to all regions of the lung

[00375] Viral genomes were quantified using qPCR for all necropsy specimens to determine the genomic biodistribution of rAAV containing the variant capsid (containing the capsid protein SEQ ID NO:12) and nucleic acid containing the EGFP transgene operably linked to the CAG promoter.

[00376] High amounts of viral genomes, ranging from approximately ¹⁰⁴ to ¹⁰⁵ vg/μg, were observed in all 48 lung samples (n=16 samples per NHP; n=3 NHPs), which represented samples from the alveolar sacs, tertiary bronchi, and primary/secondary bronchi (Fig. 18). No significant differences in the amounts of viral genomes were observed across lung lobes or lung regions across all three animals. A small number of heart and liver samples, 3 (of 15) heart samples from NHP V003424 and 10 (of 10) liver samples from NHP V002969, contained detected viral genomes. However, the amount of viral genomes present per µg DNA was 1,000-10,000 times lower than the amount of viral genomes present in the lung. Genome content in all skeletal muscle (triceps brachii, vastus lateralis), diaphragm, kidney, spleen, brain, and spinal cord samples tested were below the limit of quantification (BLQ). Thus, delivery of rAAV by nebulizer results in robust delivery of viral genomes to all regions of the lung with minimal systemic exposure.

[00377] Variant capsid (containing capsid protein of SEQ ID NO:12) mediates protein expression in all regions of the lung and transduces multiple cell types

[00378] EGFP protein expression was quantified for all samples for which qPCR results demonstrated the presence of viral genomes above the lower limit of quantification of the assay. This set of samples included all lung samples from all three NHPs, 3 (of 15) heart samples from NHP V003424, and 10 (of 10) liver samples from NHP V002969. EGFP expression was noted in all 48 lung samples (n=16 samples per NHP; n=3 NHPs), which represented samples from alveolar sacs, tertiary bronchi, and primary/secondary bronchi (Fig. 19). Expression was highest for animal V002969 in all regions and in all lung lobes. There were no significant differences in EGFP protein expression levels between lung lobes for all three animals. Overall, alveolar samples contained intermediate to above-average amounts of EGFP, but this trend was not statistically significant. EGFP protein expression was detected in qPCR+ liver samples from NHP V002969, but expression levels were orders of magnitude lower than those in the lung.

[00379] These results are consistent with genome biodistribution data showing that liver genome localization was orders of magnitude lower than lung genome localization (Figure 18). No detectable EGFP protein expression was detected in qPCR+ heart samples from NHP V003424. These results indicate that delivery of rAAV using a nebulizer containing a capsid with the capsid protein of SEQ ID NO:12 and a nucleic acid encoding the transgene mediates protein expression in all regions of the lung.

[00380] Immunofluorescence imaging was performed to further determine the extent of cellular transduction in different regions of the lung. Sections representing 6 lung sections (including alveolar and bronchial regions) and 2 tracheal sections from each experimental animal (n=3) and an additional control animal were scanned, and representative images were acquired for each region. Overall, EGFP expression was highest in animal V002969 across all regions and lobes of the lung, consistent with the observed relative expression across animals as determined by ELISA. In the trachea and bronchi, EGFP expression was observed predominantly in ciliated epithelial cells (Figure 20). High EGFP expression was observed in the alveoli (Figure 20), but ATI and ATII cells could not be detected without the use of specific cell markers. These results demonstrate that nebulized delivery of rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 and a nucleic acid encoding the transgene mediates protein expression in all regions of the lung.

[00381] Administration of rAAV containing a capsid containing a capsid protein of SEQ ID NO:12 is safe and does not result in inflammation of lung tissue

[00382] Animals were observed in their housing cages twice daily throughout the in vivo portion of the study, beginning one week prior to dosing. There were no significant clinical signs of pathology in any animal throughout the in vivo portion of the study. Hematology and biochemistry of blood samples were performed biweekly throughout the in vivo portion of the study and one week prior to dosing. Although some individual hematology and/or biochemistry values were outside the reference range, these values were largely interpreted as physiological fluctuations.

[00383] Following necropsy, histopathologic analysis of lung tissue was performed and compared with controls (non-treated). Focal hemorrhage, black pigment, and minimal mononuclear cell infiltrates in the alveolar spaces were observed in all animals and are common incidental findings in monkeys. Additionally, submucosal lymphoid infiltrates in the trachea and inflammation on the mucosal surface of the carina are also likely incidental and unrelated to the test specimen. None of the observations were considered unfavorable. No findings in treated animals were different or more severe than those observed in the control animal.

[00384] Conclusions

rAAV containing a capsid with a variant capsid protein of SEQ ID NO:12 and a nucleic acid encoding a reporter transgene (EGFP) was characterized by aerosol delivery of the reporter gene to cynomolgus monkeys. Sera were pre-screened to identify animals that were seronegative for previously produced neutralizing antibodies to the test sample capsids. Animals (n=3) received the maximal possible dose of rAAV delivered using the AeroEclipseII device, a clinically relevant active nebulizer.

[00385] High amounts of viral genome (by qPCR) and resulting high EGFP expression (by ELISA and immunostaining) were observed in all lung specimens from all 3 NHPs included in the study, which represented alveolar sac, tertiary bronchus, and primary/secondary bronchus specimens. A small number of heart and liver specimens had low but detectable levels of viral genome, and no viral genome was detected in all other tissue specimens. These data demonstrate that delivery of rAAV via nebulizer results in robust delivery of viral genomes to all regions of the lung with minimal systemic biodistribution, and rAAV mediates protein expression in all regions of the lung, including the alveoli. rAAV delivery and EGFP expression were stable among animals, with uniform distribution across multiple bronchial and alveolar levels and even distribution across upper, middle, and lower compartments. Protein expression data are consistent with genome biodistribution data showing that liver genome localization was several orders of magnitude lower than lung genome localization.

[00386] Additional experiments were conducted to determine the potency and specificity for AECII cells in the lungs of primates. If additional specificity beyond that inherent to rAAV containing a capsid containing the capsid protein of SEQ ID NO: 12 is desired, a cell-specific promoter is used to direct expression of the transgene of interest. The experiments described herein demonstrate that rAAV containing the capsid protein of SEQ ID NO: 12 can be used to safely and efficiently deliver a CFTR transgene to the lungs of subjects with cystic fibrosis and to deliver therapeutic genes for the treatment of other lung diseases.

Example 4

[00387] Summary

[00388] A cell culture model of alveolar epithelial cell type II (AECII) cells from freshly isolated non-human primate lungs and human donor lungs rejected for transplant was established. This model was used to characterize rAAV containing a capsid containing the variant capsid protein SEQ ID NO:12, identified as a target for AECII cells. The transduction efficiency of rAAV and the wild-type AAV5 serotype was assessed in AECII cell culture at the air-liquid interface (ALI). rAAV containing a capsid containing the variant capsid protein SEQ ID NO:12 showed enhanced transduction of AECII cells compared to AAV5 after apical transduction at a multiplicity of infection (MOI) of 35,000. rAAV also showed high resistance to human anti-AAV antibodies.

[00389] To confirm that the rAAV capsid targets AECII cells and that infectivity is transferred to human AECII cells, AECII cells were isolated from NHP lungs and human donor lungs rejected for transplantation and cultured at an air-liquid interface to mimic the lung environment. The transduction efficiency of rAAV was determined by expression of a CAG promoter-driven enhanced green fluorescent protein (eGFP) reporter at specific time points post-infection and compared to AAV5.CAG-eGFP. These data demonstrate that rAAV variants containing a capsid containing the variant capsid protein of SEQ ID NO:12 are more infectious than naturally occurring serotypes (i.e., are more efficient transducers than the AAV5 capsid), potentially providing improved treatment for genetic diseases.

[00390] One common problem in preclinical and clinical studies of gene therapy with AAV is that previously produced neutralizing antibodies can inhibit successful transduction. To assess the ability of rAAV containing a capsid containing the variant capsid protein of SEQ ID NO:12 to evade neutralizing antibodies in the human population compared to wild-type AAV, rAAV and wild-type AAV1, AAV2, AAV5, AAV8, and AAV9 were assayed against human IVIG in an in vitro luciferase assay. The data presented herein demonstrate that rAAV containing a capsid containing the variant capsid protein of SEQ ID NO:12 is resistant to neutralizing antibodies when exposed to human IVIG (4-fold greater than AAV5 and 32-fold greater than AAV2), a critical component for the aerosol delivery processing and selection pressures used in the therapeutic vector evolution process.

[00391] Materials and methods

[00392] Isolation of type II alveolar epithelial cells of the lungs

[00393] Non-human primate (NHP, Cynomolgus macaque, CRO) lungs or human donor lungs rejected for transplantation (Donor Network West, donor number: AGES430) were used to isolate alveolar epithelial cell type II (AECII) cells as described by Fang et al. ^1,2 . For NHP cell isolation, the entire lung was used; for human donor lungs, the right middle lobe was excised and used for cell isolation. Bronchi were rinsed with calcium- and magnesium-free PBS (ThermoFisher) containing EDTA (Sigma) and EGTA (Sigma), followed by elastase loading (Worthington Chemicals). The tissue was incubated for 1 h at 37°C. The lung was homogenized and the trachea and bronchi were removed. The cell homogenate was passed through layers of gauze to remove large remaining tissue fragments. Cells were passed successively through 100 μm and 20 μm meshes. Cells were loaded onto a two-step Percoll density gradient (ThermoFisher) (70% and 30%) and centrifuged at 1800 rpm for 20 min. The intervening layer was removed, centrifuged and washed twice with PBS lacking calcium or magnesium. Monocytes and macrophages were removed using CD14 and CD45 Dynabeads (ThermoFisher). Remaining cells were incubated overnight at 37°C in IgG-coated plates to remove T cells. The following day, non-adherent cells were collected from the plates, centrifuged and placed in hypotonic red blood cell lysis solution (ACK Lysis Buffer 1:10, ThermoFisher). The resulting cells were seeded onto human collagen IV coated inserts (Sigma, Human Placental Collagen IV, 18-24 h at 25°C) at a density of 1.5× ¹⁰⁶ cells/ ^cm2 and incubated at 37°C, 5% _CO2 . Cells were cultured in airway epithelial cell basal medium (ATCC) with commercial supplements, fetal bovine serum (10%, HyClone, ThermoFisher) and insulin, transferrin and selenium (1:200, ThermoFisher). One day after seeding, the inserts were washed twice with PBS to remove non-adherent cells. The top of the insert was kept dry to allow the formation of an air-liquid interface (ALI).

[00394] LysoTracker staining

[00395] Cells that reached ALI were analyzed by LysoTracker staining in culture. LysoTracker (ThermoFisher) is an indicator dye that is taken up by highly acidic components of living cells, such as lysosomes and lamellar bodies in AECII cells. It is commonly used to label AECII cells.

[00396] LysoTracker on adherent cells and microscopy

[00397] The cells on the inserts to be stained were washed twice with PBS. LysoTracker concentrate was diluted with medium (1:1000). 100 μl of diluted LysoTracker was added to the live cell staining insert. The cells were incubated for 5 min at 37°C. After incubation, the insert was washed three times with PBS and visualized on a Zeiss Axio Observer D.1 fluorescence microscope. An unstained well was used as a control and to set the exposure.

[00398] LysoTracker in cell suspension and flow cytometry

[00399] Cells were incubated with 0.05% trypsin-EDTA (ThermoFisher) for 10 min at 37°C. Trypsin was inactivated with soybean trypsin inhibitor (ThermoFisher), cells were collected from the inserts and centrifuged at 300×g for 4 min. LysoTracker concentrate was diluted in medium (1:1000). 100 μl of diluted LysoTracker was added to each cell pellet and vortexed at half speed. A sample of unstained cells was used as a control and for gating in flow cytometry. Cells were incubated for 5 min at 37°C. After incubation, cells were centrifuged and washed twice with PBS. Cells were resuspended in PBS and analyzed on a BD Accuri C6 Plus flow cytometer. The LysoTracker-positive population was identified as a right-shifted population compared to the unstained control.

[00400] Enabling EdU

[00401] Cell proliferation was determined using the Click-iT EdU Alexa Fluor kit (ThermoFisher) according to the manufacturer's instructions. Briefly, cells were treated with EdU for 2 h, washed twice with PBS, and fixed with 4% paraformaldehyde (15 min at 4°C). A Click-iT reaction mixture containing Alexa Fluor azide was prepared and incubated with cells for 30 min at 25°C. Following the reaction, cells were washed twice with PBS and counterstained with DAPI for 10 min at 25°C. Cells were visualized using a Zeiss Axio Observer D.1 fluorescence microscope.

[00402] Vector transduction

[00403] NHP AECII cells were transduced 2 days after plating. Human AECII cells were transduced 24 hours after plating. On the day of transduction, three inserts were incubated with 0.05% trypsin-EDTA (ThermoFisher) for 10 min at 37°C. Trypsin was inactivated using soybean trypsin inhibitor (ThermoFisher). Cells were harvested from the inserts and counted on a hemocytometer. The average cell number was determined per insert and used to calculate the total number of viral genomes required for each insert. A multiplicity of infection (MOI) of 35,000 was used for all experiments in a total volume of 100 μl per insert. Cells were treated apically for 48 h with rAAV containing a capsid with the capsid protein of SEQ ID NO:12 and the GFP gene operably linked to the CAG promoter or native AAV serotype 5 containing the GFP gene operably linked to the CAG promoter. At 2 days post infection, virus was removed from the inserts to re-establish the air-liquid interface. At 3 days post infection, NHP cells were harvested for analysis, for a total of five days in culture. At 6 and 10 days post infection, human cells were harvested for analysis, for a total of seven and eleven days in culture.

[00404] Immunocytochemistry (ICC)

[00405] Cells were washed twice with PBS and fixed with 4% paraformaldehyde (15 min at 4°C). Cells were blocked with 5% goat serum and 2% bovine serum albumin (BSA) in 0.2% Triton X-100 in PBS for 30 min. Cells were incubated with anti-surfactant protein C antibody or control IgG (MilliporeSigma, 1:100) for 2 h at 25°C. The primary antibody was washed three times with 0.2% Triton in PBS, followed by incubation with secondary antibody (goat anti-rabbit conjugated to Alexa Fluor 555, 1:500) for 30 min at 25°C. Cells were counterstained with DAPI for 10 min at 25°C and washed three times with PBS. Cells were visualized on a Zeiss Axio Observer D.1 fluorescence microscope with control IgG to set the exposure.

[00406] Flow cytometry

[00407] Post-transduction cells were detached using 0.05% trypsin-EDTA (ThermoFisher) for 10 min at 37°C. Trypsin was inactivated with soybean trypsin inhibitor (ThermoFisher), cells were collected from the inserts and centrifuged at 300×g for 4 min. Cells were resuspended in PBS and analyzed on a BD Accuri C6 Plus flow cytometer. Transduced (eGFP positive) cells were identified as a right-shifted population compared to the untransduced control.

[00408] Neutralizing antibody resistance assay

[00409] HEK 2V6.11 cells (obtained from Johns Hopkins University) were seeded in 96-well plates at a density of 3 x 10 ⁴ cells per well. Twenty-four hours after seeding, rAAV containing (i) a capsid containing the capsid protein of SEQ ID NO: 12 (“A101”) and (ii) a luciferase transgene operably linked to the CAG promoter and wild-type AAV (serotypes AAV1, AAV2, AAV5, AAV8, and AAV9 carrying the luciferase reporter transgene) were incubated at 37°C for 1 hour with five dilutions of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 human intravenous immunoglobulin (IVIG) prior to infection. Cells were then infected with rAAV and wild-type AAV at an MOI of 1000. Each plate contained positive and negative transduction controls. The positive control was either rAAV or wild-type AAV in the absence of IVIG. The negative transduction control was serum-free medium or AAV.CAG-luciferase. Luciferase activity was measured 48 h post-infection using a Cytation 3 plate reader (Biotek).

[00410] Computerized systems

[00411] FloJo, LLC software was used to analyze flow cytometry data.

[00412] Microsoft Excel was used to calculate means and standard deviations from FloJo output and to plot histograms.

[00413] For serum neutralizing antibody screening, data were acquired and analyzed using a Cytation 3 microplate reader (Biotek), Gen5plus software version 3.03.14, and Microsoft Excel.

[00414] Results and discussion

[00415] Characteristics of the AECII ALI Culture of Non-Human Primates

[00416] Alveolar epithelial lung type II (AECII) cells were isolated from non-human primate (NHP) lung and cultured at the air-liquid interface (ALI). Cells were cultured on collagen-coated inserts and analyzed for the presence of AECII specific markers, LysoTracker dye, and surfactant protein-C (SPC). Cells at days 1 and 5 contained greater than 90% LysoTracker positive cells as determined by ICC and quantified by flow cytometry (Figures 21A and 21B). Cultured AECII cells were also analyzed for SPC, a mature AECII marker, at days 1 and 5 of culture. Cells expressed SPC as determined by ICC at the time points (Figure 21C). To further characterize the AECII culture system, cell mitosis was examined over time. Mitosis was analyzed by EdU incorporation followed by a Click-iT reaction with an azide-containing fluorescent dye.

[00417] Mitosis was analyzed from day 2 to day 5 by fluorescence microscopy and nuclear staining (Fig. 21D). Mitosis was highest at 2 days after plating; as the cells were maintained in culture, the number of cells in mitosis decreased, as evidenced by the lack of EdU incorporation into nuclei.

[00418] Characterization of rAAV containing a capsid with a variant capsid protein of SEQ ID NO:12 in the non-human primate AECII ALI culture system

[00419] Two days after seeding, AECII cells isolated from NHPs were transduced with rAAV containing a capsid with the variant capsid protein of SEQ ID NO:12 and a GFP transgene under the control of the CAG promoter or with AAV containing a wild-type AAV5 capsid and a GFP transgene under the control of the CAG promoter at an MOI of 35,000. At 3 days post-infection, for a total of 5 days in culture, the cells were analyzed for eGFP expression using ICC and flow cytometry. rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 yielded a significantly higher percentage of eGFP-positive cells (~30%) compared to AAV5 (~8%), indicating higher transduction efficiency (Figures 22A and 22B).

[00420] Characteristics of Human AECII ALI Culture

[00421] Human right middle lung lobe obtained from Donor Network West (donor number: AGES430) was dissected out of the remaining lungs and AECII cells were isolated. Cells were cultured on collagen-coated inserts and ALI was generated. Two markers for AECII, LysoTracker and SPC protein expression, were analyzed to determine purity. LysoTracker was analyzed over time from fresh isolate (day 0) to day 11 in culture. LysoTracker-positive cells were approximately 80% through day 7. The LysoTracker-positive cell population decreased to approximately 50% by day 11 as AECII transdifferentiated to AECI (Figures 23A and 23B). Cells also showed high SPC expression at the time points analyzed (Figure 23C).

[00422] Mitosis was analyzed over time as in the NHP AECII system. A similar result was observed, with the highest number of cells in mitosis occurring during the first 3 days in culture and decreasing over time (Fig. 23D).

[00423] Characterization of 4D-A101 in the Human AECII ALI Culture System

[00424] One day after seeding, human AECII cells were infected with rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 or AAV5 containing the eGFP transgene at a multiplicity of infection of 35,000. At 6 and 10 days post-infection, cells were visualized for eGFP expression as an indicator of transduction efficiency. rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 showed higher levels of eGFP-positive cells at both day 6 and day 10 post-infection compared to AAV5 (Fig. 24). The cells retained LysoTracker staining, indicative of the AECII cell phenotype.

[00425] Neutralizing antibody resistance assay

[00426] The relative light units (RLU) of each AAV luciferase vector at each IVIG dilution were normalized to the positive transduction control, AAV luciferase vector only. After normalization, each vector at each IVIG dilution was transduced and expressed as percent transduction. High percent transduction correlates with low neutralization, and low percent transduction correlates with high neutralization. The neutralizing antibody titer, defined as the lowest dilution at which it achieved greater than 50% transduction, was then determined for each AAV luciferase vector. Overall, 4D-A101 demonstrated increased resistance to neutralizing antibodies compared to each wild-type AAV tested (Figure 25, illustrating percent transduction of HEK2v6.11 cells in the presence of human IVIG). 4D-A101 demonstrated a greater than 32-fold increase in antibody evasion compared to wild-type AAV1, AAV2, AAV8, and AAV9 and a 4-fold increase in antibody evasion compared to wild-type AAV5 (Table 6).

[00427] Table 6. Neutralizing antibody titers for wild-type AAV1, AAV2, AAV5, AAV8, AAV9, and 4D-A101. The dilution of IVIG required to reduce luciferase activity to 50% compared to no IVIG is shown.

AAV Dilution of neutralizing IVIG Fold increase relative to AAV2 AAV1 >1:1600 1 AAV2 >1:1600 -- AAV5 1:200 8 AAV8 >1:1600 1 AAV9 >1:1600 1 A101 1:50 32

[00428] Conclusions

[00429] AECII cells were isolated and cultured in ALI and maintained high purity as determined by Lysotracker staining and SPC expression. rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 demonstrated higher transduction efficiency compared to AAV5 in both NHP and human AECII ALI culture systems.

[00430] The ability of rAAV containing a capsid containing the capsid protein of SEQ ID NO:12 to evade neutralizing antibodies present in the human IVIG pool has been demonstrated, increasing its therapeutic potential for patients. Based on these results, it is likely that a much smaller proportion of the target patient population will be excluded from treatment due to the presence of previously produced neutralizing antibodies.

Example 5

[00431] Formulation screening studies were conducted to select and characterize optimal formulation buffers for solubility, delivery, and safety of recombinant AAV particles comprising a capsid comprising the VP1 capsid protein of SEQ ID NO:12, the VP2 capsid protein comprising amino acids 138-736 of SEQ ID NO:12, and the VP3 capsid comprising amino acids 203-736 of SEQ ID NO:12 and a nucleic acid encoding a transgene (e.g., a reporter gene such as luciferase or green fluorescent protein, or a therapeutic transgene such as CFTR).

[00432] All studies were performed with capsids (containing VP1 SEQ ID NO:12) purified by affinity (AVB) and anion exchange (CIMQA) chromatography. Initial studies were performed with purified material previously formulated in DPBS with 0.005% Pluronic. These lots were either diluted or buffer exchanged into the appropriate buffer systems. Later studies were performed with lots buffer exchanged directly into the appropriate buffer systems immediately after CIMQA purification.

[00433] Stability under freeze-thaw cycles

[00434] Small aliquots of samples (≤ 100 µl) containing ~2-3 x 10 ¹³ viral genomes/ml were transferred to 1.5 ml polypropylene tubes and subjected to 5 or 10 cycles of snap freezing (i.e., rapid cooling at -80°C) followed by thawing at room temperature until ice crystals disappeared. In a separate study, a volume of ≥ 5 ml, containing approximately ~6 x 10 ¹¹ viral genomes/ml, was transferred to a 15 ml polypropylene Falcon tube and placed at -80°C for approximately 1 h (until freezing), and then thawed for approximately 1 h at room temperature until ice crystals were observed with gentle agitation. This process was repeated 2 more times (3X FT). The sample was stored overnight at room temperature after the third freeze-thaw cycle.

[00435] Accelerated stability evaluation at 5 ° C, room temperature, 37 ° C and 40 ° C/75% RH

[00436] Various samples with concentrations ranging from approximately 1×10 ¹² to 6×10 ¹³ viral genomes per ml were aliquoted (≤ 100 μl), transferred to 1.5 ml polypropylene tubes or 2.0 ml polypropylene cryovials and stored at 5°C, room temperature, 37°C, and 40°C/75% relative humidity for the specified time in each assay protocol. Sample tubes were placed in ventilated storage boxes, and Parafilm was typically used to minimize the effects of evaporation during sample storage.

[00437] Stability under stirring

[00438] Small aliquots of samples (≤ 100 µl) containing ~2-3×10 ¹³ viral genomes per ml were transferred to 1.5 ml polypropylene tubes and placed in a sample box that was attached to a vortex mixer set at 1500 rpm. Samples were mixed at room temperature for 2-4 days.

[00439] State of charge modeling

[00440] An Excel-based charge state calculator (online download; Gale Rhodes, University of Southern Maine) was modified to automatically extract and quantify ionizable and labile residues. The net charge of a capsid protein monomer (e.g., VP1 and VP3) was determined by adding the partial negative and partial positive charges for each ionizable species at each pH range (≤ 1 pH unit):

[00441] Contribution of negative charge = number of residues * -1 * (10^-(pKa-pH)/((10^-(pKa-pH))+1)

[00442] Positive charge contribution = number of residues * (10^(pKa-pH)/((10^(pKa-pH))+1)

Ionizable group pKa Charge N-terminus 8 Positive: no charge if the group is acetylated C-end 3.1 Negative Asp 4.4 Negative Arg 12 Positive Cys 8.5 Negative: no charge if there is a Cys-Cys bond Glu 4.4 Negative His 6.5 Positive Tyr 10 Negative Lys 9.8 Positive

[00443] The specified isoelectric point (pI) is the pH value at which the net charge has been mathematically determined to be closest to 0.

[00444] Extinction coefficient at 280 nm (theoretical)

[00445] ProtParam (web.expasy.org/protparam/) was used to determine the molar mass of VP1, VP2, and VP3 proteins based on the known primary sequences. The molar extinction coefficient at 280 nm was calculated using the following equation: (M-1 cm-1) = (#Trp)(5500) + (#Tyr)(1490) + (#cystine)(125). Note: VP1, VP2, and VP3 all contain 5 cysteine residues, but according to the literature, AAV cysteines do not form disulfide bonds (SS=cystine). Abs280nm ^0.1% (or absorbance units for a 1 mg/mL solution) was then calculated by dividing the molar extinction coefficient by the molar mass.

A101VP1 A101 VP3 Total amount of balances 736 543 Molar mass (g/mol) 81370,5 59560,2 Number of cystine residues (S-S bond) 0 0 Number of tyrosine residues 30 24 Amount of tryptophan residues 15 12 Molar extinction coefficient at 280 nm (M ^-8 cm ^-8 ) 127200 101760 Abs _280nm ^0.1% 1.56 1.71

[00446] Absorbance at 280 nm (A280) and turbidity by UV spectrophotometry

[00446] Approximately 30 μl of sample (or buffer) was transferred to a 3 mm quartz cuvette and UV spectra (250-400 nm; 1 nm interval) were recorded on a NanoDrop spectrophotometer. Alternatively, multiple test samples (or buffers) were transferred to a UV Star 96-well microtiter plate and UV spectra (250-400 nm; 1 nm interval) were recorded on a Cytation plate reader. The optical path length of the microtiter plate sample wells was determined empirically by measuring the absorbance at 977 and 900 nm and the following equation was used:

[00448] Buffer and sample spectra were normalized to a 1 cm pathlength cell by dividing the optical density (OD) measured at each wavelength by the pathlength of the sample cuvette or well. Normalized spectra were averaged, with duplicates analyzed. Sample spectra were background corrected by subtracting the normalized optical density values for the corresponding blank buffer control. The contribution of light scattering at 280 nm (LS280) was calculated using the logarithmic extrapolation obtained for the non-absorbing UV region (~300-400 nm) and subtracted from OD280 -LS280=A280. The A280 corrected light scatter was multiplied by the sample dilution factor (if necessary) and divided by the theoretical extinction coefficient of VP3 (Abs280nm ^0.1% = 1.71) to yield the estimated capsid protein concentration in mg/mL protein. However, this method is prone to overestimation of capsid protein due to DNA uptake. Alternatively, A280 values are presented in this report without further transformation to avoid overestimation of capsid protein due to DNA uptake. OD350 values were originally used to semi-quantitatively monitor changes in light scatter or turbidity resulting from self-association. However, 350/A280 values are also presented in this report to normalize the scatter to the amount of capsid in the soluble fraction.

[00449] pH

[00450] Small or large volume pH probes were used to measure the pH of the sample or buffer, respectively. The pH probes were calibrated using pre-packaged Mettler Toledo pH standards of pH 4.01, 7.01, 10.01.

[00451] Osmolality at Freezing Point Depression

[00452] 15 µL of a reference solution (e.g., 290 mOsm/kg) or sample was transferred to the freezer chamber of an Advanced Instruments Freezing Point Depression Osmometer. The sample was supercooled until freezing and the temperature was monitored until a plateau was observed. The plateau temperature was used to calculate osmolality according to the following equation: 1 mOsm solute/kg water = 1.858 millidegrees (m°C) ↓ freezing point.

[00453] Hydrodynamic Size and Polydispersity Using Dynamic Light Scattering

[00454] Approximately 30 µl of sample was loaded into a clean 3 mm cuvette (ZN2112) and multiple scans (n=2 or 3) were performed on a Malvern Zetasizer Ultra (dispersant=water, sample type=protein). Scan data were averaged to plot intensity (%) and volume (%). The former was used during early formulation screening as the low level, HMW species are easier to observe by intensity (LS ~ diameter ⁶ ~ Mw ² ). Scan data were also averaged to obtain polydispersity values using Cumulants Fits, mean size by intensity (10-100 dnm), mean size by volume (10-100 dnm), area % by intensity (10-100 dnm), and area % by volume (10-100 dnm) where appropriate.

[00455] Subvisible Particle Sizing with Horizon Background Membrane Imaging (BMI)

[00456] Background membrane imaging (BMI) was performed using the Horizon Particle Analysis System. An empty 96-well plate was loaded onto the Horizon system and a background image was acquired. The plate was then transferred to a vacuum collector. Approximately 20-30 μL of sample was loaded into each well. A blotter paper and blotter adapter were attached to the vacuum collector and the sample plate was moved to the top of the stack. The vacuum was applied to remove any remaining liquid at the bottom of the wells. The sample plate was then transferred to the Horizon instrument. Particle counts and images (2-10 μm, 10-25 μm, >25 μm overall) were acquired.

[00457] Titer using droplet digital polymerase chain reaction (ddPCR)

[00458] Frozen 5-10 μl aliquots were used to determine AAV genome concentration by ddPCR according to SOP-AD-018. rAAV samples containing the capsid protein of SEQ ID NO:12 were diluted in Dulbecco's calcium magnesium phosphate buffered saline containing 0.02% Pluronic™ F-68 nonionic surfactant (DPBS+0.02% F68) mixed with DNase I enzyme to digest any unencapsulated DNA and further diluted with DPBS+0.02% F68 to bring the test sample within the dynamic range of the assay. The DNase treated sample was then mixed with ddPCR Supermix primers and SV40 (or CFTR)/FAM labeled probes. Twenty microliters of the reaction mixture were then split into droplets using a Bio-Rad QX200 Auto DG droplet generator, subjected to PCR, and then read on a Bio-Rad QX200 droplet reader, which measures the fluorescent signal of each droplet individually. Data were analyzed using Bio-Rad QuantaSoft software, which utilizes Poisson statistical analysis of positive and negative droplets to provide absolute quantification of target sequences. No-template controls were used to establish a negative baseline for samples. ddPCR without DNase I enzyme treatment was also performed on individual samples.

[00459] Chemical purity using polyacrylamide gel electrophoresis (PAGE)

[00460] Frozen aliquots of 5-10 μl were subjected to chemical purity analysis according to SOP-AD-028 Rev. 00 (Determination of Capsid Purity Using SDS-PAGE with Krypton™ Staining) or SOP-AD-002 (Determination of Capsid Purity Using Silver Staining LDS-PAGE). Briefly, approximately 1.00×10 ¹⁰ (SOP-AD-028) or 1.00×10 ⁹ (SOP-AD-002) viral genomes were mixed with 4× LDS sample buffer, 10× sample reducing agent, water, and heated at 95°C for 10 min. NuPAGE™ 4-12% Bis-Tris gel was placed in a gel box containing an upper chamber (1× SDS buffer + antioxidant) and a lower chamber (1× SDS buffer). Heat-denatured (and reduced) reaction mixtures were loaded onto the gel. The gel box lid was attached and the electrodes were connected to an external power source. Power was then applied according to the manufacturer's instructions. Current flow was stopped when the sample dye front was > ¾ of the length of the full gel. Gels were washed and stained according to the manufacturer's instructions. Gel visualization was performed on a ChemiDoc MP Imager.

[00461] Functional activity of green fluorescent protein (GFP) expression

[00462] 15 μl of frozen aliquots were analyzed for green fluorescent protein (GFP) expression. HEK2v6.11 cells were transduced at 10,000 MOI and harvested 72 h post-transduction. GFP expression was visualized by fluorescence microscopy and quantified by flow cytometry.

[00463] A significant loss (~50%) of rAAV containing the VP1 capsid of SEQ ID NO:12 was observed during concentration to ≥ 1× ¹⁰¹³ viral genomes/mL (vg/mL) and buffer exchange of purified AAV into Dulbecco's phosphate-buffered saline formulation buffer (Dulbecco's calcium-magnesium phosphate-buffered saline containing 0.05% Pluronic™ F-68 nonionic surfactant (DPBS+0.05% F68)). The experiments described here identified the underlying cause of the loss and described an improved formulation buffer.

[00464] The pI values for VP1 of SEQ ID NO:12, VP2, and VP3 are determined to be 6.7, 7.4, and 6.8, respectively. VP1 has a higher number of charged residues compared to VP2 and VP3, which likely explains the theoretical differences in net charge observed at pH below 5 and above pH 9 (Figure 26). However, the net charge between pH 5 and pH 9 appears to be largely the same for VP1 and VP3, while some subtle differences are predicted for VP2. The monomers of VP1, VP2, and VP3 contain aspartic acid (D), asparagine (N), methionine (M), and free cysteine (C) residues. Therefore, VP1 and VP3 monomers are predicted to be sensitive to aspartic acid shuffling at low pH, deamidation at neutral/basic pH, and oxidation and disulfide shuffling at high pH.

[00465] The pH of DBPS is approximately 7.0. This value is very close to the pI value or theoretical minimum solubility for the VP1 and VP3 proteins. Therefore, it has been suggested that pH may play a role in the physical instability observed during concentration and buffer exchange in tangential flow filtration.

[00466] pH vs. Solubility

[00467] rAAV stock containing the VP1 capsid of SEQ ID NO:12 and GFP-encoding nucleic acid (~2× ¹⁰¹³ vg/mL in DPBS+0.005% Pluronic F68) was thawed and diluted 10-fold to approximately 2× ¹⁰¹² vg/mL in various buffers (pH 4-8) of both low ionic strength (~14 mM NaCl) and physiological ionic strength (~150 mM NaCl). The low ionic strength formulations showed a pH-dependent increase in high molecular weight (HMW) as pH increased from 4 to 8. This pH dependence was significantly reduced in the presence of 150 mM NaCl.

[00467] The samples were then stored at room temperature and then analyzed by UV spectrophotometry the following day (T=1 day at room temperature). The UV absorbance at 280 nm (A280) is plotted versus pH in Figure 27. The low ionic strength samples showed a very sharp decrease in A280 signal (i.e., indicating a reduced concentration of rAAV) above pH 5, consistent with the physical instability observed by DLS. All samples containing 150 mM NaCl showed similar A280 values. These data indicate that lower pH and addition of salt result in increased solubility. Details on the formulation, sample pH, and absorbance at A280 (corrected for light scattering and path length) can be found below:

[00469] These results suggest that both ionic strength and pH influence the solution behavior of rAAV. Therefore, a subsequent study was conducted to further evaluate the solubility limits of rAAV at pH values between 5 and 8 in the presence of sodium chloride (ionic strength ~0.15 M) or trisodium citrate (high ionic strength). rAAV stock containing the VP1 capsid SEQ ID NO:12 and the luciferase-encoding nucleic acid (1.76× ¹⁰¹³ vg/mL in DPBS+0.005% Pluronic F68) was thawed, buffer exchanged, and concentrated in various buffers (Table 7) to a target level of approximately 3× ¹⁰¹³ vg/mL. Process yields were determined by dividing the recovered viral genomes by the viral genomes in the stock. Decreased recoveries were observed for the DPBS+0.005% F68 and 10 mM Tris, pH 8+150 mM NaCl+0.005% F68 formulations. These lower yields also appeared to be consistent with slower buffer exchange and concentration rates.

Table 7

Summary results of pH vs. solubility study of A101-Luc (after filtration through 0.2 µm filter)

[00470] Samples containing sodium chloride showed a similar pH dependence, i.e. ↓ solubility at ↑ pH, for titer and absorbance at A280. The high ionic strength formulation (10 mM Tris, pH 8+100 mM sodium citrate+0.005% F68) showed the highest solubility of the formulations tested, further demonstrating the effect of ionic strength. Samples profiled through a 0.2 μm filter showed a pH dependence, i.e. ↑ O.D.350 at lower pH. However, the 350/A280 values were plotted to represent physical instability relative to the amount of rAAV in the soluble fraction. The 350/A280 value showed instability at pH 5. However, an inflection point was observed at pH 6 (10 mM citrate, pH 6+150 mM NaCl+0.005% F68), which appears to represent a balance between high solubility and physical stability. The high ionic strength formulation showed comparable turbidity to the pH 6 formulation. As shown in Fig. 35 (consistent with data in Table 7), the lower pH shows increased solubility in the presence of 0.15 M NaCl (higher solubility at pH 5 and 6 compared to pH 7 and 8 when the salt concentration is ~150 mM NaCl) and clearly demonstrates that increased ionic strength is required to “recover” solubility at higher pH. These results ultimately led to the use of the pH 6 citrate formulation.

[00471] DLS (dynamic light scattering) showed variable levels of high molecular weight species in the concentrated samples, except for the high ionic strength formulation (10 mM Tris, pH 8 + 100 mM trisodium citrate + 0.005% F68). However, the amount of high molecular weight species was significantly reduced after filtration through a 0.2 μm filter, with the exception of persistent submicron species observed in the 10 mM sodium acetate, pH 5 + 150 mM NaCl + 0.005% F68 formulation. This is consistent with the increase in 350/A280 observed for the same sample.

[00472] The results of this study suggest that it is possible to formulate rAAV at levels ≥ 3 x 10 ¹³ vg/mL when the pH ≤ 6 or when the ionic strength increases above 0.15 M. However, at low pH (e.g., pH 5), there may be a risk of introducing soluble high molecular weight species, and higher pH formulations may require a concentration of ionic strength modifier (e.g., sodium citrate) that is higher than that present in other approved inhalation products according to the FDA Inactive Ingredient Database. Therefore, a subsequent study was aimed at narrowing the pH range of the formulation (6-8) and also evaluating the effects of sodium citrate over a concentration range of 20 to 100 mM.

[00473] rAAV stock containing the VP1 capsid of SEQ ID NO:12 and GFP-encoding nucleic acid (8.75× ¹⁰¹² vg/mL in DPBS+0.005% Pluronic F68) was thawed, buffer exchanged, and concentrated in various buffers (Table 8; all formulations also included 0.005% F68) to a target level of approximately 3× ¹⁰¹³ vg/mL. Buffer pH and osmolality values (without rAAV) can be found in Table 9. Formulated samples were filtered through a 0.2 μm filter and aliquoted into polypropylene tubes. 1 aliquot of each formulation was used for measurement at T=0 and then subjected to 4 days of shaking at room temperature at 1500 rpm. Separate aliquots were used to evaluate the effects of freeze-thaw cycling (5/10× FT cycles), room temperature storage (T=13 days), and 28-day storage (15 days at 2-8°C followed by 13 days at room temperature). rAAV stock, 8.46× ¹⁰¹² vg/mL in DPBS+0.005% F68, was evaluated under selected conditions.

[00474]

[00475]

Table 9

pH and osmolality of the buffer (mOsm/kg)

[00476] The ddPCR titer assay was performed on a small scale due to the large number of formulations and conditions to be screened. However, at T=0, sample titers ranged from approximately 2.3 to 2.9 x ¹⁰¹³ vg/mL (Table 10). This low variability is presumed to be due to the small scale of the process, as most conditions met or exceeded recoveries > 90%. The exceptions were fPD_2019_006_003, fPD_2019_006_005, and fPD_2019_006_007, which showed lower recoveries. Sample titer was also measured after 28 days of storage (15 days at 2-8°C, then 13 days at room temperature). All formulations maintained titer ≥ 100% relative to T=0.

[00477]

Table 10

Titer at T=0 and recovery (%)

N/A - not available

[00478] Little or no change in volume % hydrodynamic size was observed for any of the formulations under the conditions tested. Therefore, superpositions were created for the intensity % hydrodynamic size, which is typically more sensitive to the presence of small molecular weight species. fPD_2019_006-05 and fPD_2019_006-10 showed an increase in peak width after 4 days of stirring at 1500 rpm. This could also be observed in the polydispersity plot of the sample. The polydispersity at T=0 of the starting material (fPD_2019_006-13) was higher than any of the 12 formulations screened, further indicating an improvement over the starting DPBS formulation.

[00479] Table 9 also includes the A280 values as well as the % signal remaining at A280 relative to T=0. Most formulations retained ≥ 95% of their respective signals at A280, with the exception of fPD_2019_006-11 and fPD_2019_006-13 (DPBS control) after 4 days of shaking at 1500 rpm, and fPD_2019_006-03 and fPD_2019_006-08, which showed a slight decrease after 28 days of storage.

[00480] Significantly higher particle counts were observed for the agitated samples compared to the other test conditions, suggesting that the agitation duration may have been overly aggressive. However, the formulations appeared to vary in their response to the different stressors applied. For example, fPD_2019_006-05 contained elevated particle counts >10 μm after agitation, while fPD_2019_006-09 and fPD_2019_006-10 showed elevated particle counts after 10 freeze-thaw cycles.

[00481] The titer and physical stability results obtained in the fPD_2019_006 study were assessed using a semi-quantitative weighing system, and fPD_2019_006-01 (20 mM citrate, pH 6 + 125 mM NaCl + 0.005% F68), fPD_2019_006-02 (50 mM citrate, pH 6 + 70 mM NaCl + 0.005% F68), fPD_2019_006-08 (10 mM phosphate, pH 5 + 50 mM NaCl + 50 mM trisodium citrate + 0.005% F68), and fPD_2019_006-12 (10 mM Tris, pH 8 + 100 mM trisodium citrate+0.005% F68), which all showed favourable characteristics, need to be investigated further. After this decision was made, the remaining aliquots of the 28-day samples were analyzed by electrophoresis with krypton staining. VP1, VP2 and VP3 were present in all samples tested. Low molecular weight (LMW) bands were also observed to varying degrees; the most prominent of these is fPD_2019_006-01. Unfortunately, the values at T=0 were not available for comparison, so it was unclear whether these LMW bands were degradation products or process impurities. Therefore, the chemical stability was assessed in subsequent studies.

[00482] rAAV containing the VP1 capsid of SEQ ID NO:12 and the CFTR pool-encoding nucleic acid, CIMQA (lot #dPD_2020_001, 7.92× ¹⁰¹¹ vg/mL) was buffer exchanged and concentrated directly in the 4 buffers specified in fPD_2019_006 to a target level of approximately 3× ¹⁰¹³ vg/mL. rAAV containing the VP1 capsid of SEQ ID NO:12 and the nucleic acid encoding GFP (lot #4DER000057 g/mL) in DPBS+0.005% F68 was buffer exchanged and concentrated in 20 mM citrate, pH 6+125 mM NaCl+0.005% F68 to a target level of approximately 6E13 vg/mL. The rAAV-CFTR and rAAV-GFP compositions were then aliquoted into polypropylene tubes and subjected to 10 freeze-thaw cycles, 40 h agitation at 1500 rpm, or 40 h storage at 40°C.

[00483]

Table 11

Design of the formulation screening study

[00484] rAAV-CFTR titers, T=0, ranged from 1.7 to 1.9× ¹⁰¹³ vg/mL. They were ≥30% lower than the target of 3× ¹⁰¹³ vg/mL, which was due to the small working volume rather than solubility limitations. The pH 7 and pH 8 formulations (fPD_2020_001-02 and fPD_2020_001-03) showed approximately 4% loss upon exposure to mixing stress, while no loss was observed for the pH 6 formulations (fPD_2020_001-01 and fPD_2020_001-04). This trend towards stability was significantly enhanced at 40°C, as the pH 7 and pH 8 formulations showed ≥90% loss, whereas the loss at pH 6 was <30%. All four rAAV-CFTR preparations retained ≥100% titer after 10 freeze-thaw cycles. rAAV-GFP (fPD_2020_001-05, 5.3E13 vg/mL at T=0) was highly stable at pH 6 with no loss upon agitation and <3% loss upon storage at 40°C over 10 freeze-thaw cycles. It is suggested that the difference in relative stability between rAAV-CFTR and rAAV-GFP formulated in the same buffer is related to the transgene size. However, further studies are required in the future.

[00485] fPD_2020_001-02 (pH 7) and fPD_2020_001-03 (pH 8) showed higher signal loss at A280 compared to pH 6 samples during storage at 40°C. This is consistent with the trend observed for titer, but to a lesser extent. This is likely due to the increase in empty capsids (and free DNA) during storage at 40°C. Empty capsids and DNA will still absorb UV light, but the latter will be susceptible to DNase treatment. The A280 results for the rAAV-GFP formulation (fPD_2020_001-05) are also consistent with the titer analysis; minimal to no change.

[00486]

Table 12

Absorption at 280 nm (A280)

[00487] Hydrodynamic size versus intensity plots showed an increase in peak width for all rAAV-CFTR samples at 40°C. High molecular weight species were detected at low levels in fPD_2020_001-01. However, the large differences in titer and UV may indicate the absence of this peak in less stable preparations due to precipitation, surface adsorption, or changes in the empty/full capsid ratio. Size differences were less obvious in volume, but polydispersity also appeared to be very sensitive to small differences. Consistent with other testing methods, rAAV-GFP showed virtually no change.

[00488] Storage at 40°C resulted in a high number of 2-10 μm particles in the A101-GFP formulation (fPD_2020_001-05). This was expected to be concentration dependent (i.e., rAAV-GFP titer was approximately 3-fold higher than rAAV-CFTR samples). Interestingly, this sample showed relatively few particles > 10 μm, possibly indicating that the formulation prevents the formation of larger aggregates. rAAV-CFTR prepared in the same buffer (fPD_2020_001-01) showed sensitivity to agitation and heating, but did not appear to produce particles > 25 μm. Other rAAV-CFTR formulations showed little tendency for particles > 25 μm.

[00489] No significant differences were observed at T=0, 40 h at 1500 rpm, or after 10X FT. However, the pH 7 (fPD_2020_001-02) and pH 8 (fPD_2020_001-03) formulations showed increased levels of low molecular weight species and were overloaded. In contrast, fPD_2020_001-01 showed the presence of some high molecular weight bands after storage at 40°C. However, it was unclear whether this was an artifact related to the sample preparation or the staining procedure (e.g., silver staining is considered a non-quantitative assay). Fortunately, Western blot analysis was performed on the same samples. The Western blot results clearly showed that the pH 7 and pH 8 samples were overloaded (titer-based) at 40°C. This further supports the idea that the disproportionate loss in titer compared to uptake at A280 is likely due to an increase in empty capsids. For the 40°C fPD_2020_001-01 sample, no high molecular weight bands were observed by Western blot.

[00490] Much of the formulation screening effort has focused on improving the physical stability of rAAV and, to a lesser extent, monitoring chemical stability. However, an important part has not yet been addressed, namely functional activity. Therefore, the functional activity of rAAV-GFP was assessed in the three formulations tested in study fPD_2020_001. rAAV-GFP (1.58 a 10 ¹³ vg/mL) in DPBS+0.005% F68 was buffer exchanged and concentrated in 20 mM citrate, pH 6+125 mM NaCl+0.005% F68 (fPD_2020_002-01), 10 mM potassium phosphate, pH 7+50 mM citrate+50 mM NaCl+0.005% F68 (fPD_2020_002-02), or 50 mM citrate, pH 6+70 mM NaCl+0.005% F68 (fPD_2020_002-03) to a target level of approximately 3×10 ¹³ vg/mL. The rAAV-GFP formulations were then aliquoted into polypropylene tubes and stored at room temperature for 13 days. Titer was measured at T=0 (Table 12) and values were applied to both T=0 and T=13-day samples (i.e., the same loading volume for functional testing).

[00491]

Table 13

fPD_2020_002: A101-GFP functional activity study

[00492] The hydrodynamic size was evaluated at T=0. No differences were observed for the three formulations tested. No loss of signal at A280 (Table 14) was observed when stored for 13 days at room temperature.

[00493]

[00494] Fluorescence microscopy and flow cytometry results showed that all rAAV-GFP samples exhibited green fluorescent protein expression, while no fluorescence was observed for the solvent. No difference was observed in the % of GFP-positive cells after 13 days of storage at room temperature, indicating that all three preparations retained the functional activity of rAAV.

[00495] Based on the results of studies fPD_2020_001 and fPD_2020_002, a decision was made to perform a final evaluation of two formulations with citrate, pH 6. Purified rAAV-CFTR (4D130109) containing 20 mM citrate, pH 6+125 mM NaCl+0.005% F68 (fPD_2020_003-01, lot number dPD_2020_007) and 50 mM citrate, pH 6+85 mM NaCl+0.005% F68 (fPD_3-3-2020_007)02, lot number dPD_2020_007) were thawed and aliquoted (~100 µL) into polypropylene tubes. Two aliquots of each formulation (n=2) were used for measurements at T=0. The remaining vials were placed at 40°C/75% RH and retrieved after 4 h (n=2), 20 h (n=2), or 44 h (n=2). The 20 mM citrate, pH 6 formulation (fPD_2020_003-01) used the same buffer that showed good stability in previous studies. The 50 mM citrate formulation was similar to the 50 mM citrate, pH 6 formulation determined in previous studies, with a slight modification; the NaCl concentration was increased from 70 mM to 85 mM to increase the tonicity of the solution. The osmolality of the 20 mM and 50 mM citrate buffers, pH 6, were 289 and 295 mOsm/kg, respectively.

[00496]

[00497] Titer analysis using ddPCR (n=2) was performed with and without DNase pretreatment. A measurable difference was observed between the two methods, suggesting either the presence of free capsid DNA or DNase-sensitive capsids (e.g., disrupted or chemically damaged). However, titer loss at 40°C/75% RH was comparable for the two formulations.

[00498] Increased signal loss at A280 was observed for fPD_2020_003-02 (50 mM citrate formulation, pH 6), possibly indicating salting out (or precipitation) of some of the soluble fraction. Interestingly, turbidity increased in fPD_2020_003-01, further indicating the presence of “disrupted” species that were retained in solution at the lower citrate concentration.

[00499] The DLS results were correlated with the turbidity data. Sample fPD_2020_003-01 showed detectable levels of high molecular weight species in intensity at 20 and 44 h at 40°C/75% RH, while fPD_2020_003-02 was devoid of these species. This peak explains the differences observed in % peak area and polydispersity.

[00500] Samples were also analyzed for chemical purity using PAGE. The following observations were made. Cleavage products were detected at T=0 for both formulations. These are presumably process impurities carried over during subsequent purification. Low level cleavage products began to form over a prolonged period at 40°C/75% RH. The chemical purity of both formulations was comparable.

[00501] Based on these results, it was determined that the increased citrate level may result in a small increase in the physical stability of rAAV-CFTR at 40°C/75% RH. However, it was unclear whether this was a true protective effect or whether the simultaneous loss in A280 suggested that physically unstable species were salting out. Additionally, 50 mM citrate did not show a reduction in titer loss compared to the 20 mM citrate formulation, and both formulations appeared to have a similar increase in low molecular weight species as determined by PAGE. Thus, it was determined that the 50 mM citrate formulation did not provide sufficient improvement to warrant a higher citrate concentration, which may be less tolerable in an inhaled drug product.

[00502] Thus, the formulation with 20 mM citrate, pH 6 + 125 mM NaCl + 0.005% F68 was identified as the preferred formulation for the pilot toxicity/dose-finding study in NHP. Preliminary requirements for toxicity test samples can be found in Table 16. A study (fPD_2020_006) was conducted to evaluate the freeze-thaw stability of low doses (~6×10 ¹¹ vg/mL).

[00503]

Table 16

Requirements for a pilot test sample for a NHP toxicity study

[00504] rAAV-CFTR (4D130109, lot# 4DER000060.02, ~3× ¹⁰¹³ vg/mL) in 20 mM citrate, pH 6+125 mM NaCl+0.005% F68 was diluted in the same formulation buffer to a target of approximately 6× ¹⁰¹¹ vg/mL (low dose in 5.5 mL total volume). A 100 µL aliquot was removed for T=0 titer and DLS. The remaining material was placed in a -80°C freezer for 1 h. The tube was removed and allowed to thaw at room temperature for 1 h. The thawed tube was gently inverted to mix and a 100 µl aliquot was removed for DLS and titration (1X freeze-thaw). This process was repeated two more times. After the third freeze-thaw cycle, the material was left overnight at room temperature.

[00505] The titer showed very little change over 3 freeze-thaw cycles (3× FT), while a small decrease in titer (~10%) was observed when the 3X FT sample was stored at room temperature overnight. However, this value was still within the variation range of the ddPCR assay and was therefore considered a worst-case scenario.

[00506] No significant difference in physical stability was observed for the freeze-thawed samples using DLS. Smaller aliquots (~50 µL) of the 0, 2X, and 3X FT samples were also measured after an additional hour of storage at room temperature (2 h after complete thawing). No changes were observed in these samples.

Example 6

In Vitro and Ex Vivo Characterization of 4D-710 in Human Cells

[00507] The ability of rAAV containing (i) the capsid of SEQ ID NO:12 and (ii) a heterologous nucleic acid containing the nucleotide sequence of SEQ ID NO:45 (4D-710) to transduce human cells and deliver a codon-optimized human CFTR transgene of SEQ ID NO:43 (cohCFTRΔR, encoding a functional CFTR gene with amino acids 708-759 deleted) was assessed. Human cells were HEK2v6.11, 16HBE14o-G542X (available through the Cystic Fibrosis Foundation), and ex vivo ALI non-CF cultures. Protein expression, membrane localization of the protein, and mRNA expression of the therapeutic transgene cassette were assessed.

[00508] Materials and Methods - SDS-PAGE and Western Blotting . Cells were lysed for Western blot analysis in PBS containing 1% v/v NP-40 for 30 min at 4°C before removing insoluble material. For Western blotting, equal volumes of lysate containing 1X dye and reducing agent (Thermo Bolt) were heated at 50°C for 10 min, then loaded onto a 4%-12% Bis-Tris-acrylamide gel (Thermo Bolt) and run at 250 V for 45 min. The gel was semi-dry transferred to 0.2 μM nitrocellulose (BIO-RAD) using the "High MW" transfer setting of the BIO-RAD Trans-Blot Turbo (10 min, 1.3A). The blot was then briefly washed with ultrapure water and then blocked with 1X iBind Flex (Thermo) for 10 min. The blot was then incubated in 1X iBindFlex containing anti-CFTR antibody (Ab660, CFF/UNC 1:500) or anti-tubulin antibody (E-7, DSHB 1:1000) for 2 h at room temperature with gentle shaking. The blots were then washed with PBS (Corning) containing 0.01% Tween-20 (Sigma) for 5 min with shaking; this washing step was repeated two more times. The blots were then incubated in anti-mouse HRP secondary antibody (R&D systems, 1:5000) in 1X iBind Flex for 1 h at room temperature. The washing steps were then repeated with PBS-0.01% Tween 20. The washed blots were then coated with DuraWest HRP Detection Substrate (Thermo) for 5 min, and the blots were then visualized using the BIO-RAD ChemiDoc imaging system.

[00509] Materials and Methods - Immunocytochemistry . For immunocytochemistry, 2v6.11 cells were cultured on glass slides with poly-L-lysine-coated wells, 16HBE14o- were cultured on Transwell inserts coated with both bovine collagen I and human plasma fibronectin. At 2 days (2v6.11) or 4 days (16HBE14o-) post-transduction, cells were fixed in 4% PFA for 20 min at room temperature in the dark. Cells were then permeabilized with 0.2% Triton-X in PBS (without calcium and magnesium) and incubated at 4°C overnight with CFTR antibody MM13-4 (EMD Millipore, 1:100) in blocking buffer. The blocking buffer consisted of 0.2% Triton-X, 5% goat serum, and 2% bovine serum albumin in PBS. After washing away the antibodies with PBS-Triton, a secondary goat anti-mouse antibody conjugated with Alexa Fluor-647 (Invitrogen, 1:500) was added to the blocking buffer for one hour at room temperature in the dark. The secondary antibody was then washed away. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) in PBS, then coverslipped with Prolong Gold (Invitrogen), and then visualized. Cells were visualized with a Zeiss Axio Observer.D1 fluorescence microscope. Image processing was performed using Zeiss Zen 2 software (Carl Zeiss Microscopy LLC, White Plains, NY).

[00510] Materials and Methods - RNA Extraction and Droplet Digital PCR. RNA was isolated from cells using the RNeasy Micro Qiagen kit (Thermo Fisher Scientific) and cDNA was prepared using the Maxima H minus cDNA Synthesis Kit (Thermo Fisher). cDNA samples were prepared by making serial dilutions in 1× TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0). Samples were then placed in a master mix containing the ddPCR supermix and either a primer/probe set targeting CFTRΔR (SEQ ID NO:43) or the endogenous CFTR gene (Thermo Fisher). Samples were then split into nanoliter droplets using an automated droplet generator (BIO-RAD), subjected to endpoint PCR analysis in the C1000 Sensor Thermal Cycler (BIO-RAD), and read on a QX200 Droplet Reader (BIO-RAD), which measures each droplet individually for fluorescent signal. Data were then analyzed using BIO-RAD QuantaSoft software, which utilizes Poisson statistical analysis of positive (droplets containing a fluorescent signal above a threshold) and negative (droplets with a fluorescent signal below a threshold) droplets to provide absolute quantification of the target sequence(s). Further analysis was performed using Microsoft Excel.

[00511] Results

[00512] In a proof-of-concept approach, initial experimental transgene expression and membrane localization of the protein following transduction with 4D-710 were assessed by transducing the HEK2v6.11 cell line, a relatively simple system (compared to the more complex human cell model at the air-liquid interface). HEK2v6.11 cells were derived from the human embryonic kidney cell line, HEK-293T, expressing the ORF6 protein of human adenovirus E4 induced by ponasterone A. Multiple AAV serotypes are capable of high transduction efficiency in this cell line. Briefly, HEK2v6.11 were cultured in DMEM (Gibco) with 10% FBS and 1% penicillin/streptomycin. After fusion, cells were treated at different MOIs for 48 h. Ponasterone was added at a concentration of 1 μg/ml to increase expression from the AAV vector. Cells were analyzed by Western blotting or immunocytochemistry and transduced with 4D-710 for 24 h and analyzed by Western blotting or immunocytochemistry (ICC) 48 h post transduction. Dose-dependent transduction of 4D-710 in HEK2v6.11 cells was demonstrated by Western blotting at increasing multiplicities of infection (MOI) (Fig. 28A). Band B, which corresponds to the ER-core glycosylated form of the protein (150 kDa), and band C, reflecting the fully glycosylated mature form of CFTR (170-180 kDa), can be seen, suggesting proper protein translation and cellular processing. Representative immunofluorescence images further demonstrate dose-dependent transduction (Fig. 28B), cytoplasmic (Fig. 28C), and membrane (Fig. 28C) expression of CFTR protein. Untransduced controls show that endogenous CFTR protein is not detected, indicating that the detected protein is the transduced CFTRΔR transgene. Thus, not only are immature and mature forms of CFTRΔR produced, but membrane expression of CFTRΔR protein was also observed using ICC (using anti-CFTR antibody and anti-F-actin antibody to stain the cell membrane).

[00513] Transduction of the human lung cell line 4D-710 was assessed. 16HBE14o-G542X cells are an immortalized human bronchial epithelial (HBE) cell line (originally immortalized with an origin of replication-deficient SV40 plasmid) containing a CFTR null mutation (G542X, generated by CRISPR-based gene editing) and does not express CFTR. 16HBE14o-G542X cells were cultured in MEM (Gibco 11095-072) supplemented with 10% FBS (Gibco 26140-079) and 1% penicillin/streptomycin (Gibco 15140-122). Cells were seeded on Transwell inserts at 9,000 cells per insert. Medium was added to both the top (100 μl) and bottom (500 μl). On day 3 post-seeding, cells were treated at different MOIs apically for 72 h. Cells were analyzed by immunocytochemistry or droplet digital reverse transcription-polymerase chain reaction (RT-ddPCR) using primers and probes specific for codon-optimized CFTRΔR mRNA (4D-710 transgene). To detect exogenous CFTR protein, transduction was performed in parallel with doxorubicin treatment (1 μM) for 24 h in the culture medium. To assess transcript levels of the codon-optimized CFTRΔR transgene following transduction with 4D-710 into human bronchial epithelial cells, 16HBE14o-G542X cultures were transduced with 4D-710 at increasing MOIs. To further confirm transduction of HBEs with 4D-710, RT-ddPCR was performed to determine CFTRΔR mRNA copy number. Figure 29A shows the dose-response curve for CFTRΔR mRNA copy number for increasing MOIs. In the presence of doxorubicin, transduced cells were identified using immunocytochemistry (Fig. 29B), where more cells were transduced at an MOI of 50,000 compared to an MOI of 35,000. Thus, dose-dependent transduction of human bronchial epithelial cells and stable expression of the CFTR transgene in these disease-relevant human cells were demonstrated.

[00514] Air-liquid interface (ALI) cell cultures were prepared from healthy (non-CF) lungs and, as such, have endogenous CFTR expression. Endogenous CFTR was distinguished from expression of the truncated 4D-710 CFTR transgene using RT-ddPCR using primers and probes specific for endogenous CFTR or specific for CFTRΔR FTRΔR mRNA. Standard ex vivo ALI cell culture from non-CF lungs represents the best in vitro model of the in vivo pulmonary system (these cultures are complexes with non-homogeneous cell types (such as the in vivo lung)). Briefly, ex vivo ALI cultures of non-CF human lungs were seeded on type IV collagen-coated Corning Transwell inserts and cultured basally in PneumaCult ALI medium (StemCell Technologies 05040) supplemented with Pen/Strep gene (Gibco 15140-122), gentamicin, and amphotericin B according to the manufacturer's protocol. At 30 days post-seeding, when ALI cultures were mature, cultures were treated at various MOIs apically with 0.625 μM idarubicin for 24 h in the culture medium. Seven days post-transduction, cells were analyzed by RT-ddPCR using primers and probes specific for endogenous CFTR or specific for CFTRΔR mRNA. To assess transcript levels of the codon-optimized CFTRΔR transgene following ex vivo transduction, healthy lung ALI cultures were transduced with 4D-710 at MOIs of 50,000 and 100,000 in the presence of idarubicin. RNA was isolated 7 days post-transduction and cDNA was synthesized. RT-ddPCR was performed on the prepared samples and transcript levels per droplet were determined as copies/μl. Quantification yielded the number of droplets above a specified threshold containing the transcript of a given primer/probe set. Two primer/probe sets were designed to specifically differentiate the codon-optimized human CFTRΔR transgene from the endogenous human CFTR gene. Expression from untransduced ALI cultures was below the limit of quantification of CFTRΔR transcript, as expected (Fig. 30). Following transduction with 4D-710, cells showed a dose-dependent increase in CFTRΔR transcript (Fig. 30). At 50,000 MOI, CFTRΔR transcript levels were ~80% of endogenous CFTR levels (Fig. 30). At 100,000 MOI, CFTRΔR transcript levels approached and in some cases exceeded endogenous CFTR levels (Fig. 30). These data demonstrate that 4D-710 can transduce ex vivo lung ALI cultures with dose-dependent expression of the CFTRΔR transgene and that at high MOI, CFTRΔR mRNA is increased relative to endogenous CFTR.

[00515] Conclusions: 4D-710 is a recombinant AAV gene replacement therapy product intended for the treatment of cystic fibrosis caused by CFTR mutations, a respiratory disease of the lungs, preferably by single dose delivery to the lungs as an aerosol. 4D-710 comprises a capsid protein of SEQ ID NO:12, identified by directed evolution as unexpectedly useful for the delivery of therapeutic gene products to the lungs, and a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 (the nucleotide sequence of SEQ ID NO:45 comprises a codon-optimized human cystic fibrosis gene therapy payload (SEQ ID NO:43) operably linked to the CMV173 promoter (SEQ ID NO:44)). In vitro and ex vivo data in human cells showed that 4D-710 restores human CFTR transcript and transgene expression in human bronchial epithelial cells containing the cystic fibrosis class I mutation and in ALI healthy lung cultures. Furthermore, CFTR protein expressed following 4D-710 transduction was expressed, post-translationally glycosylated, and localized to the cytoplasm and cell membrane in HEK2v6.11 cells. These data demonstrate that 4D-710 is able to transduce a human lung cell line and deliver detectable CFTRΔR mRNA, resulting in dose-dependent CFTR protein production and membrane localization of expressed CFTR protein. Combined with the data from Example 3 and Example 7 demonstrating safe, robust, and widespread transduction and expression of the transgene in the lungs of primates following aerosol delivery with minimal systemic exposure, these data represent a significant advance over existing AAV serotypes for the development of gene therapies for cystic fibrosis and other lung diseases.

Example 7

Dose range and pilot safety study of aerosol delivery of 4D-710 to the lungs of cynomolgus monkeys

[00516] An in vivo study was initiated to test the safety and transduction activity of the therapeutic product 4D-710 in a pilot study in cynomolgus macaques to evaluate the ability of engineered viral vectors to transduce cells and express the CFTRΔR transgene in the airways, lungs, and other tissues following a single aerosol administration over a range of doses. As discussed in Example 6, 4D-710 is an rAAV comprising the capsid protein of SEQ ID NO:12 and the heterologous nucleic acid of SEQ ID NO:45. The heterologous nucleic acid component of 4D-710 comprises the nucleotide sequence of SEQ ID NO:43, a codon-optimized human CFTRΔR transgene operably linked to the CMV173 promoter of SEQ ID NO:44. Expression and function of the putative therapeutic transgene cassette were characterized in vivo in a primate, cynomolgus macaque. Serum was prescreened to identify animals that were seronegative for previously produced neutralizing antibodies to test drug capsids. Animals received vehicle (formulation buffer only), 3 x 10 ¹² vg 4D-710, or 3 x 10 ¹³ vg 4D-710 delivered endoscopically just below the larynx using an AeroEclipseII device (Trudell Medical) to ensure optimal delivery to the distal lung. After 8 weeks, selected tissues (lung, heart, skeletal muscle, liver, kidney, pancreas, spleen, brain, spinal cord, tracheobronchial lymph nodes, and testes) were collected for analysis. Viral genomes were quantified by qPCR. Tissue samples that contained detectable viral genomes were assessed for CFTRΔR transcript using RT-qPCR, and lung samples were sectioned and visualized for CFTR protein expression using IHC.

[00517] The data presented below demonstrate that delivery of 4D-710 via nebulizer resulted in robust delivery of viral genomes to all regions of the lung, including peripheral (bronchioalveolar) regions, with minimal systemic biodistribution, and 4D-710 mediated CFTRΔR transcript and protein expression in all regions of the lung. There were no reports of adverse safety events in animals administered vehicle or 4D-710 test drug ( ^3x1012 and ^3x1013 vg).

[00518] Materials and methods

[00519] Neutralizing antibody assay

[00520] HEK2v6.11 cells (obtained from Johns Hopkins University) were seeded in black opaque 96-well plates at a cell density of 30,000 cells/well in Dulbecco's modified Eagle's medium (DMEM; Corning) with 1% heat-inactivated fetal bovine serum (FBS; GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Invitrogen). Cells were allowed to attach to the plate for 24 h prior to experimentation.

[00521] Each non-human primate (NHP) serum sample was assayed at 1:10, 1:25, 1:50 dilutions. Each plate contained positive and negative controls for transduction. NHP serum samples were incubated with virus at 37°C for 1 h. Each well was infected with 4D-A101.CAG-luciferase (rAAV containing the capsid protein of SEQ ID NO:12 and a heterologous nucleic acid encoding luciferase operably linked to the CAG promoter) at a multiplicity of infection of 1000. After incubation for 1 h, each NHP serum sample plus the 4D-A101.CAG-luciferase dilution were added to separate wells of black opaque 96-well plates containing HEK2v6.11 cells. Luciferase was determined using the ONE-Glo EX Luciferase Assay Kit (Promega) 48 h after transduction. Upon addition of ONE-Glo EX, cells were lysed and luciferase substrate was added to the cells in one step. Luminescence was read using a Cytation 3 microplate reader (BioTek).

[00522] Coefficients of variation (CV) and standard deviations were calculated for all dilutions of NHP serum samples and each standard curve point. NHP serum samples were normalized to the positive transduction control. Each NHP was assigned a neutralizing antibody titer. The neutralizing antibody titer for each NHP serum sample was defined as the lowest serum dilution that demonstrated ~50% transduction. NHPs that demonstrated ~50% transduction at a 1:10 serum dilution were considered for inclusion in the study.

[00523] Test system and immunosuppression

[00524] Seven male cynomolgus macaques were included in the study. Animals ranged in age from 4.7 to 4.9 years and weighed between 3.8 and 6.0 kg. Animals received immunosuppression with methylprednisolone (10 mg/kg, intramuscularly) once weekly beginning on day -7 until one week prior to euthanasia and tissue sampling (day 49). Additional experimental details are provided in the contract research organization (CRO) study report (preclinical study report pending).

[00525] Preparation and administration of test sample

[00526] Batches of test article (TA) 4D-710 and vehicle were diluted in formulation buffer to deliver final doses of 3× ¹⁰¹² vg and 3× ¹⁰¹³ vg 5 mL. TA was thawed at room temperature for >1 h and the tubes were gently inverted 5 times before adding 5 mL to the delivery device (AeroEclipseII).

[00527] Animals were anesthetized with Telazol (IM, 7-8 mg/kg) and a cuffed endotracheal tube (size 4.5) was inserted into the trachea just below the larynx and secured to prevent slippage during transfer and placement of the animal in the chair. The animals were then placed in the chair in an upright position to connect the endotracheal tube to the aerosol generating system. Animals were covered with blankets and a Bair Hugger device for external heating, and heart rate and oxygen saturation were monitored during exposure to and recovery from anesthesia.

[00528] The aerosol generation and delivery system used an AeroEclipse II nebulizer (BAN, Trudell Medical International, Canada) to generate aerosol. A total of five (5) ml of control or test drug was administered to anesthetized animals through an endotracheal tube. The nebulizer was operated in continuous aerosol generation mode by switching the knob on the nebulizer cap. In addition, the vents inside the nebulizer cap were plugged.

[00529] Animal Observations

[00530] Twice daily observations were made at the animal housing cage in accordance with the SOP for the Care and Maintenance of Domestic Non-Human Primates. Animals were continuously observed by research staff on treatment and procedure days. Key observations included, but were not limited to, signs of nausea, lethargy, respiratory distress and cyanosis, discoloration of mucous membranes, vomiting, and irregular discharge from bodily orifices or blood in feces/urine.

[00531] Blood sampling

[00532] Animals were fasted overnight with free access to drinking water prior to any blood collection and fed immediately after the procedure. Blood samples for clinical pathology and antibody titer and immunogenicity analysis were collected by femoral vein venipuncture. Samples (1 ml in EDTA) for hematology and complete cell count were sent to an external reference laboratory (IDEXX) on the day of collection for analysis within 24-48 h of collection. Samples for clinical chemistry (1 ml in SST) were analyzed in the CRO pathology laboratory on the day of collection.

[00533] Bronchoalveolar lavage (BAL)

[00534] Animals were fasted overnight and anesthetized with ketamine hydrochloride followed by isoflurane inhalation according to the SOP for Anesthesia in Non-Human Primates. An appropriately sized cuffed endotracheal tube was inserted just proximal to the carina to allow insertion of a pediatric fiberoptic bronchoscope to perform BAL according to the SOP for Lung Lavage and/or Bronchoscopy in Non-Human Primates ACL-1536. Samples from both sides of the lung were collected at baseline (day -7) and 4 weeks post-treatment. Three 10 mL aliquots of Dulbecco's PBS were instilled into the appropriate side of the lung through the bronchoscope and sequentially aspirated. Lavage isolates from the first wash were stored separately, and the 2nd and 3rd washes were pooled at the time of collection and stored on wet ice until processed within 2 h of collection. Pooled cells from all washes were counted and slides were prepared for differential cell counts using morphological criteria in CRO.

[00535] Euthanasia and necropsy

[00536] For euthanasia, animals were anesthetized with ketamine (11.0 to 12.4 mg/kg, IM) followed by intravenous injection of euthanasia solution in accordance with CRO SOP, Large Animal Euthanasia. After death was confirmed, trained personnel performed a necropsy in accordance with CRO SOP, Non-Rodent Necropsy Procedure, and tissues were collected, weighed, preserved, and examined. Tissues were processed as appropriate for DNA and RNA or prepared for histopathological analysis.

[00537] Animals were perfused with heparinized saline prior to collection of any tissue. Samples were collected from the lung and trachea, skeletal muscle (diaphragm, triceps brachii, and vastus lateralis), heart, liver, spleen, pancreas, testis, kidney, brain, tracheobronchial lymph nodes, and spinal cord. Each tissue was divided into different regions, and multiple samples were collected from each region. Macroscopic examination of major peripheral organs and tissue samples collected from any identified pathological areas were performed. Tissue samples were collected and snap frozen for subsequent DNA analysis or stored in RNALater for subsequent RNA extraction. Additional samples were collected and fixed in 10% neutral buffered formalin for subsequent paraffin embedding and sectioning for immunohistochemistry.

[00538] Larger samples were collected from the trachea and lungs to obtain multiple samples for each analysis. The lungs were collected and clamped as high above the trachea as possible. The right lung was clamped twice at approximately 1 mm intervals on the main bronchi. The right lung was removed by cutting between the clamps. Sixteen samples for DNA and RNA extraction were collected from areas of the right lung covering the primary/secondary bronchi, tertiary bronchi, and alveoli as shown in Fig. 16. The trachea and left lung were filled with fixative and fixed in 10% neutral buffered formalin. Sections of the trachea and left lung were then prepared to cover samples of the trachea, primary/secondary bronchi, tertiary bronchi, and alveoli as described in Fig. 16.

[00539] Biodistribution of the viral genome and transgene expression

[00540] A real-time quantitative polymerase chain reaction (qPCR) and a real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay were developed for quantitative analysis in NHP tissues. Quantification of viral genomes in the lungs of NHPs from each group was performed by qPCR and RT-qPCR using primers and probes against (and specific for) the codon-optimized transgene (SEQ ID NO:43). Viral titers measured in tissue were expressed as copies per μg DNA (BLQ=50), and transgene transcript expressed as copies per reaction with 250 ng RNA (BLQ=25).

[00541] Immunohistochemistry was performed on tracheal and left lung tissue samples, including tracheal, primary/secondary bronchus, tertiary bronchus, and alveolar samples as shown in Fig. 16, from all animals. CFTR IHC analysis was performed using the Roche Discovery ULTRA automated stain and detection reagents, CC1S antigen retrieval protocol and tyramide amplification kit, and mouse anti-CFTR monoclonal antibody (Abcam ab270238 M3A7).

[00542] Results

[00543] Screening for neutralizing anti-AAV antibodies identifies NHPs for inclusion in the study

[00544] A neutralizing antibody assay was used to assess the levels of neutralizing antibodies against the 4D-A101 capsid (containing the capsid protein of SEQ ID NO:12) in NHP serum. The neutralizing antibody titer was determined for each NHP serum sample. An animal was considered seronegative and eligible for inclusion in the study if transduction was ≥50% at a 1:10 serum dilution.

[00545] A total of 50 NHP serum samples were analyzed. Assay acceptance criteria were established for 1) the coefficient of variation (CV) of the standard curve, 2) the CV of unknown serum samples, and 3) the percent deviation from the actual input protein for the standard curve. Acceptable values for the coefficients of variation for the standard curve were defined as <25%. Acceptable CVs for unknown serum samples within the limit of quantification were defined as <30%. Acceptable percent deviation from the input protein for the standard curve was defined as <25%. All plates met all assay acceptance criteria, and the data obtained on these plates were used for the evaluation. Overall, 19 (38%) of the NHP serum samples analyzed were seronegative for 4D-A101 (Fig. 31). NHPs with the highest percentage of transduction at a 1:10 serum dilution and that had passed the health screening at the supplier were selected for inclusion in the study. The selected NHP identifiers and percentage of transduction at a 1:10 dilution are shown in Table 17:

Table 17
NHPs included in the study Number
analysis NHP ID # % transduction at 1:10 1 2DRPL2-39C-B-A BLQ 2 2DRPL2-4C-E 10 3 2DRPZ2-18C-G BLQ 4 2DRPZ2-26C-H BLQ 5 2DRPZ2-27C-F 115 6 2DRPZ2-9C-I 96 7 2DRPZ4-16C-B BLQ 8 2DRPZ6-30C-G 100 9 DPC15-39A-K BLQ 10 DPL8-26A-L 74 11 DRP8BL-22K-E 58 12 DRPL13-9A-K 112 13 DRPL3-37D-B 102 14 DRPL4-2D-C 89 15 DRPL5-17D-B 11 16 DRPL7-31D-A 97 17 DRPS1-31C-G BLQ 18 DRPS15-53B-A 118 19 DRPS7-11B-H BLQ 20 DRPZ10-19B-I BLQ 21 DRPZ11-18C-E 40 22 DRPZ12-21A-D-E BLQ 23 DRPZ12-33A-L BLQ 24 DRPZ13-44A-I BLQ 25 DRPZ1-5D-D 78 26 DRPZ16-12C-A BLQ 27 DRPZ16-20B-A-C 12 28 DRPZ16-2B-F 78 29 DRPZ2-24D-A BLQ 30 DRPZ31-13C-G BLQ 31 DRPZ33-36C-A 25 32 DRPZ34-7C-A BLQ 33 DRPZ3-9C-H 56 34 DRPZ40-18C-C 20 35 DRPZ40-22C-C 69 36 DRPZ4-17B-G BLQ 37 DRPZ4-36B-F 112 38 DRPZ5-15D-C 92 39 DRPZ5-62A-E 70 40 DRPZ6-37D-C BLQ 41 DRPZ7-105B-K 49 42 DRPZ7-28B-I BLQ 43 DRPZ7-77B-J BLQ 44 DRPZ9-16C-G BLQ 45 DRPZ9-86C-C BLQ 46 PRPL2-49C6-A 93 47 PRPL2-74C6-A 32 48 PRPL7-21C6-A BLQ 49 PRPL9-33C6-A 116

[00546] Delivery of 4D-710 using a nebulizer is well tolerated in NHPs

[00547] The study design is presented in Table 18:

Table 18
Generalized study design Group Processing Route of administration #animals Dose (vg) Volume (ml) Necropsy 1 Control/Carrier Inhalation 1 absent 5 Day 57 2 4D-710 3 3×10 ¹² 5 Day 55/56/57 3 4D-710 3 3×10 ¹³ 5 Day 55/56/57 Fabric collection Analysis Lung
Tracheobronchial lymph node
Heart
Liver
Skeletal muscle (triceps brachii, quadriceps, diaphragm)
Bud
Spleen
Pancreas
Brain
Spinal cord qPCR
RT-qPCR (lung & qPCR+ tissue)
IHC (lung)

[00548] No animals died or were prematurely euthanized during the course of this study. No animals showed any signs of distress or health problems related to TA exposure during the entire study period. No loss of appetite or changes in feeding behavior were observed during processing and sample collection. There were no treatment-related changes in body weight during this study.

[00549] Hematology and complete cell count samples were submitted to an external reference laboratory (IDEXX) on the day of collection and analyzed no more than 48 hours after collection. Samples for biochemistry endpoints were analyzed by the CRO pathology laboratory on the day of collection. Some of the biochemistry endpoints changed over time, but no differences were observed between the vehicle group compared to both dose levels of the 4D-710 group at any time point. Similarly, no significant changes in hematology or white blood cell count were observed from baseline levels at 8 weeks post-inhalation treatment.

[00550] The differential cell ratio and cell count in lavage fluid were determined for the right and left sides of the lavage, and all data are presented as the mean values for both sides. Total cell count and differential cell ratio in BALF did not differ between treatment groups measured at day 28 compared to baseline levels, and due to variability and small sample size, further conclusions cannot be drawn.

[00551] Organ weights and organ weights normalized to body weight were determined on the day of euthanasia for all treatment groups. Organ weights and body weight-normalized weights did not differ between treatment groups.

[00552] No major pathologies were reported during macroscopic examination at autopsy. Microscopic examination revealed that there were no processing-related observations in the tissues examined.

[00553] 4D-710 mediates robust gene delivery to all regions of the lung

[00554] Viral genomes were quantified in a tiered approach on necropsy specimens to determine the genomic biodistribution of 4D-710. All lung specimens from vehicle, 3 x 10 ¹² vg, and 3 x 10 ¹³ vg treated animals were analyzed. Brain, spinal cord (cervical, thoracic, and lumbar), heart, liver, spleen, pancreas, kidney, skeletal muscle (triceps brachii, vastus lateralis, diaphragm), tracheobronchial lymph nodes, and testes were analyzed from all animals following administration of 3 x 10 ¹³ vg. Tissues that had viral genome levels above the BLQ were then analyzed at the lower dose of 3 x 10 ¹² vg (data pending). Data are presented as viral genomes per µg DNA.

[00555] The qPCR results indicate uniform delivery of virus to different regions of the lung, which represent samples from the alveolar sacs (distal), tertiary bronchi (medial), and primary/secondary bronchi (proximal), with the exception of samples labeled R6. This sample was difficult to collect due to its proximity to the carina and therefore a site for separation and bridging of the right and left sides of the lung (see Fig. 16). The mean copy numbers for the remaining sample locations for the 3 x 10 ¹² vg and 3 x 10 ¹³ vg dose groups were 10 ⁴ and 10 ⁵ , respectively (Fig. 32A). At least a 10-fold difference between the low and high dose groups is well consistent with a 10-fold difference in treatment dose. For all animals in the same treatment group, no significant differences were observed in the amount of viral genomes in different lung lobes or different lung regions (Fig. 32B).

[00556] Systemic exposure beyond the lungs was assessed in animals administered 3 x 10 ¹³ vg of 4D-710 (Figure 32C). Animals had detectable viral genomes of ~10 ⁴ vg/μg present in the tracheobronchial lymph nodes (Figure 32C). Pathological findings in these animals are normal for this tissue. One NHP had detectable viral genomes in the spleen (Figure 32C), with pathology considered normal. All other brain, spinal cord, heart, liver, pancreas, kidney, skeletal muscle, and testis samples tested had viral genome levels below the lower limit of quantification (Figure 32C). No pathologies associated with the test sample were reported. Thus, delivery of 4D-710 with a nebulizer results in safe and reliable delivery of viral genomes to all regions of the lung with minimal extrapulmonary systemic exposure.

[00557] 4D-710 mediates transgene expression in all lung regions

[00558] CFTRΔR transcript was quantified using a tiered approach on necropsy samples to determine 4D-710 transgene expression. All lung samples from animals receiving vehicle at 3 x ¹⁰¹² vg and 3 x ¹⁰¹³ vg were analyzed. Brain, spinal cord, heart, liver, spleen, pancreas, kidney, skeletal muscle, tracheobronchial lymph nodes, and testes from animals receiving the 3 x ¹⁰¹³ vg dose were analyzed (data pending). Tissues that had transcript levels above the BLQ were then analyzed at the lower dose of 3 x ¹⁰¹² vg (data pending). Data are presented as copies per 250 ng RNA reaction and are plotted as copies per μg RNA.

[00559] Results of RT-qPCR analysis indicate successful transduction and transcript expression in the lung. In animals dosed at 3 x 10 ¹³ vg, 44 of 48 lung samples exceeded BLQ with ~10 ³ copies per μg RNA, with no vehicle samples above BLQ (Figure 33A). Three of the four BLQ samples were collected from the same R6 sampling site, where collection was technically challenging, and one NHP had an additional BLQ sample. Transduction was noted in a variety of lung regions (Figure 33B), representing samples from the alveolar sacs (distal), tertiary bronchi (medial), and primary/secondary bronchi (proximal), with the exception of samples labeled R6 (see rationale above). In contrast, in lung samples from animals treated with the lower dose of 3× ¹⁰¹² vg, 41 of 48 samples were BLQ. These data suggest that the 3× ¹⁰¹³ vg dose of 4D-710 is capable of quantitatively converting NHP lung tissue and expressing the therapeutic CFTRΔR transcript.

[00560] CFTR protein expression detected using immunohistochemistry showed increased CFTR expression in 4D-710-treated NHP lung compared to vehicle in tracheal epithelium, bronchial epithelium, and alveolar sections (Figure 34A). Highly increased CFTR expression was observed in all animals treated with 3 x 10 ¹³ vg (Figure 34B) compared to vehicle (Figure 34A). These results demonstrate that delivery of 4D-710 via nebulizer mediates protein expression in all regions of the lung.

[00561] Conclusions

[00562] The therapeutic product 4D-710 was characterized by aerosol delivery to cynomolgus macaques. Serum was pre-screened to identify animals that were seronegative for previously produced neutralizing antibodies to the capsid of the test sample 4D-A101. Animals received either vehicle (formulation buffer), a 3 x 10 ¹² vg dose of 4D-710, or a 3 x 10 ¹³ vg dose of 4D-710 delivered via the AeroEclipseII device.

[00563] In this study, high amounts of viral genomes and, as a result, CFTRΔR transcript expression and CFTR protein expression were observed in NHP lung samples, which included alveolar sacs, tertiary bronchi, and primary/secondary bronchi. One spleen sample contained a small amount of viral genomes, but they were detectable, and all other tissue samples had no detectable viral genomes. These data demonstrate that delivery of 4D-710 via nebulizer results in safe and reliable delivery of viral genomes to all regions of the primate lung (trachea, alveoli, and bronchial epithelium) with minimal systemic biodistribution. These data also demonstrate that 4D-710 mediates stable expression of CFTRΔR mRNA and protein product in all regions of the lung, including the alveoli, confirming the utility of rAAVs with capsids containing the capsid protein of SEQ ID NO:12 for lung delivery of a vector for the treatment of various lung diseases and, in particular, supporting the use of 4D-710 (containing the capsid protein of SEQ ID NO:12 and the heterologous nucleic acid of SEQ ID NO:45) for the delivery of a biologically active therapeutic CFTR transgene for the treatment of cystic fibrosis in human patients.

[00564] Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adapt a particular situation, material, composition of matter, process, method step or steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

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List of sequences

<110> 4D Molecular Therapeutics, Inc.

<120> OPTIONS, COMPOSITIONS BASED ON ADENO-ASSOCIATED VIRUS AND METHODS FOR

PULMONARY DELIVERY

<130> 090400-5013 WO

<150> US 63/016,246

<151> 2020-04-27

<150> US 63/088,432

<151> 2020-10-06

<160> 45

<170> PatentIn version 3.5

<210> 1

<211> 736

<212> PROTEIN

<213> adeno-associated virus 1

<400> 1

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

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Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

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Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 2

<211> 735

<212> PROTEIN

<213> adeno-associated virus 2

<400> 2

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 3

<211> 736

<212> PROTEIN

<213> adeno-associated virus 3

<400> 3

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Gly

130 135 140

Ala Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Arg Gly Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser

450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn

485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn

500 505 510

Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln

565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr

580 585 590

Thr Gly Thr Val Asn His Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 4

<211> 734

<212> PROTEIN

<213> adeno-associated virus 4

<400> 4

Met Thr Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu

1 5 10 15

Gly Val Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys

20 25 30

Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Val

50 55 60

Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln

65 70 75 80

Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Leu

115 120 125

Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro Gly Lys Lys Arg Pro

130 135 140

Leu Ile Glu Ser Pro Gln Gln Pro Asp Ser Ser Thr Gly Ile Gly Lys

145 150 155 160

Lys Gly Lys Gln Pro Ala Lys Lys Lys Leu Val Phe Glu Asp Glu Thr

165 170 175

Gly Ala Gly Asp Gly Pro Pro Glu Gly Ser Thr Ser Gly Ala Met Ser

180 185 190

Asp Asp Ser Glu Met Arg Ala Ala Ala Gly Gly Ala Ala Val Glu Gly

195 200 205

Gly Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys

210 215 220

Asp Ser Thr Trp Ser Glu Gly His Val Thr Thr Thr Ser Thr Arg Thr

225 230 235 240

Trp Val Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys Arg Leu Gly Glu

245 250 255

Ser Leu Gln Ser Asn Thr Tyr Asn Gly Phe Ser Thr Pro Trp Gly Tyr

260 265 270

Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln

275 280 285

Arg Leu Ile Asn Asn Asn Trp Gly Met Arg Pro Lys Ala Met Arg Val

290 295 300

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Thr Ser Asn Gly Glu

305 310 315 320

Thr Thr Val Ala Asn Asn Leu Thr Ser Thr Val Gln Ile Phe Ala Asp

325 330 335

Ser Ser Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gln Glu Gly Ser

340 345 350

Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro Gln Tyr Gly Tyr

355 360 365

Cys Gly Leu Val Thr Gly Asn Thr Ser Gln Gln Gln Thr Asp Arg Asn

370 375 380

Ala Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly

385 390 395 400

Asn Asn Phe Glu Ile Thr Tyr Ser Phe Glu Lys Val Pro Phe His Ser

405 410 415

Met Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile

420 425 430

Asp Gln Tyr Leu Trp Gly Leu Gln Ser Thr Thr Thr Gly Thr Thr Leu

435 440 445

Asn Ala Gly Thr Ala Thr Thr Asn Phe Thr Lys Leu Arg Pro Thr Asn

450 455 460

Phe Ser Asn Phe Lys Lys Asn Trp Leu Pro Gly Pro Ser Ile Lys Gln

465 470 475 480

Gln Gly Phe Ser Lys Thr Ala Asn Gln Asn Tyr Lys Ile Pro Ala Thr

485 490 495

Gly Ser Asp Ser Leu Ile Lys Tyr Glu Thr His Ser Thr Leu Asp Gly

500 505 510

Arg Trp Ser Ala Leu Thr Pro Gly Pro Pro Met Ala Thr Ala Gly Pro

515 520 525

Ala Asp Ser Lys Phe Ser Asn Ser Gln Leu Ile Phe Ala Gly Pro Lys

530 535 540

Gln Asn Gly Asn Thr Ala Thr Val Pro Gly Thr Leu Ile Phe Thr Ser

545 550 555 560

Glu Glu Glu Leu Ala Ala Thr Asn Ala Thr Asp Thr Asp Met Trp Gly

565 570 575

Asn Leu Pro Gly Gly Asp Gln Ser Asn Ser Asn Leu Pro Thr Val Asp

580 585 590

Arg Leu Thr Ala Leu Gly Ala Val Pro Gly Met Val Trp Gln Asn Arg

595 600 605

Asp Ile Tyr Tyr Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp

610 615 620

Gly His Phe His Pro Ser Pro Leu Ile Gly Gly Phe Gly Leu Lys His

625 630 635 640

Pro Pro Pro Gln Ile Phe Ile Lys Asn Thr Pro Val Pro Ala Asn Pro

645 650 655

Ala Thr Thr Phe Ser Ser Thr Pro Val Asn Ser Phe Ile Thr Gln Tyr

660 665 670

Ser Thr Gly Gln Val Ser Val Gln Ile Asp Trp Glu Ile Gln Lys Glu

675 680 685

Arg Ser Lys Arg Trp Asn Pro Glu Val Gln Phe Thr Ser Asn Tyr Gly

690 695 700

Gln Gln Asn Ser Leu Leu Trp Ala Pro Asp Ala Ala Gly Lys Tyr Thr

705 710 715 720

Glu Pro Arg Ala Ile Gly Thr Arg Tyr Leu Thr His His Leu

725 730

<210> 5

<211> 724

<212> PROTEIN

<213> adeno-associated virus 5

<400> 5

Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu

1 5 10 15

Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys

20 25 30

Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val

50 55 60

Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu

65 70 75 80

Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile

130 135 140

Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser

145 150 155 160

Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln

165 170 175

Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr

180 185 190

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

195 200 205

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

210 215 220

Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro

225 230 235 240

Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp

245 250 255

Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr

260 265 270

Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln

275 280 285

Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val

290 295 300

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr

305 310 315 320

Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp

325 330 335

Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys

340 345 350

Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr

355 360 365

Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser

370 375 380

Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn

385 390 395 400

Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser

405 410 415

Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp

420 425 430

Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln

435 440 445

Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp

450 455 460

Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly

465 470 475 480

Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu

485 490 495

Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr

500 505 510

Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile

515 520 525

Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu

530 535 540

Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg

545 550 555 560

Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser

565 570 575

Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro

580 585 590

Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp

595 600 605

Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met

610 615 620

Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn

625 630 635 640

Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser

645 650 655

Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu

660 665 670

Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln

675 680 685

Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp

690 695 700

Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu

705 710 715 720

Thr Arg Pro Leu

<210> 6

<211> 736

<212> PROTEIN

<213> adeno-associated virus 6

<400> 6

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 7

<211> 735

<212> PROTEIN

<213> adeno-associated virus 7

<400> 7

Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu

1 5 10 15

Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro Lys

20 25 30

Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro Val

50 55 60

Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln

65 70 75 80

Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Leu

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Ala Lys Lys Arg Pro

130 135 140

Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile Gly

145 150 155 160

Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Ser Val Gly Ser Gly Thr Val Ala Ala Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Glu Thr Ala Gly Ser Thr Asn Asp Asn Thr

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Lys Leu Arg Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Ile Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ser Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr Ser Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ala Arg

435 440 445

Thr Gln Ser Asn Pro Gly Gly Thr Ala Gly Asn Arg Glu Leu Gln Phe

450 455 460

Tyr Gln Gly Gly Pro Ser Thr Met Ala Glu Gln Ala Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Phe Arg Gln Gln Arg Val Ser Lys Thr Leu Asp Gln

485 490 495

Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu

500 505 510

Asn Gly Arg Asn Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr His

515 520 525

Lys Asp Asp Glu Asp Arg Phe Phe Pro Ser Ser Gly Val Leu Ile Phe

530 535 540

Gly Lys Thr Gly Ala Thr Asn Lys Thr Thr Leu Glu Asn Val Leu Met

545 550 555 560

Thr Asn Glu Glu Glu Ile Arg Pro Thr Asn Pro Val Ala Thr Glu Glu

565 570 575

Tyr Gly Ile Val Ser Ser Asn Leu Gln Ala Ala Asn Thr Ala Ala Gln

580 585 590

Thr Gln Val Val Asn Asn Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Glu Val Phe Thr Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Phe Glu Lys Gln Thr Gly Val Asp Phe Ala Val Asp Ser Gln Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn

725 730 735

<210> 8

<211> 738

<212> PROTEIN

<213> adeno-associated virus 8

<400> 8

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr

405 410 415

Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly

450 455 460

Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile

530 535 540

Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala

580 585 590

Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 9

<211> 736

<212> PROTEIN

<213> adeno-associated virus 9

<400> 9

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 10

<211> 735

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 10

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Lys Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asp Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asn Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Ser Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Gln

725 730 735

<210> 11

<211> 736

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 11

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Leu Ile Glu Ser Pro Gln Gln Pro Asp Ser Ser Thr Gly Ile Gly

145 150 155 160

Lys Lys Gly Lys Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Asp Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 12

<211> 736

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 12

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Leu Ile Glu Ser Pro Gln Gln Pro Asp Ser Ser Thr Gly Ile Gly

145 150 155 160

Lys Lys Gly Lys Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Asp Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Thr Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 13

<211> 736

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 13

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Ile Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Gln

725 730 735

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial nucleic acid sequence

<400> 14

gcggaagctt cgatcaacta cgc 23

<210> 15

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 15

ggggcggccg caattacaga ttacgagtca ggtatctggt g 41

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<220>

<221> misc_feature

<222> (5)..(6)

<223> n is a, c, g, or t

<400> 16

cattnnkgac cagtctagga actgg 25

<210> 17

<211> 90

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<220>

<221> misc_feature

<222> (21)..(22)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (69)..(70)

<223> n is a, c, g, or t

<400> 17

gccacaagga cgatgaagaa nnkttttttc ctcagagcgg ggttctcatc tttgggaagc 60

aaggctcann kaaaacaagt gtggacattg 90

<210> 18

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<220>

<221> misc_feature

<222> (18)..(19)

<223> n is a, c, g, or t

<400> 18

ccaacctcca gagaggcnnk agacaagcag ctacc 35

<210> 19

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<220>

<221> misc_feature

<222> (18)..(19)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (42)..(43)

<223> n is a, c, g, or t

<400> 19

ccaactacaa caagtctnnk aatgtggact ttactgtgga cnnkaatggc gtgtatt 57

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 20

catgggaaag gtgccagacg 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 21

accatcggca gccatacctg 20

<210> 22

<211> 2208

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 22

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctatgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggaacgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaagttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggtaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacagacact ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cggaaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca agtgtggaca ttgaaaaggt catgattaca 1680

gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740

accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800

cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860

attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920

caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980

ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040

gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga agttcagtac 2100

acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160

tcagagcctc gccccattgg caccagatac ctgactcgta atcagtaa 2208

<210> 23

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 23

atggctgctg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggtga caacccctac ctcaagtaca accacgccga cgcggagttc 300

cagcagcggc ttcagggcga cacatcgttt gggggcaacc tcggcagagc agtcttccag 360

gccaaaaaga gggttcttga acctcttggt ctggttgagc aagcgggtga gacggctcct 420

ggaaagaaga gaccgttgat tgaatccccc cagcagcccg actcctccac gggtatcggc 480

aaaaaaggca agcagccggc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggtgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgcacctg ggccttgccc acctacaata accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg acttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaaactctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc agactatcag ctcccgtacg tgctcgggtc ggctcacgag 1080

ggctgcctcc cgccgttccc agcagacgtc ttcatggtgc cacagtatgg atacctcacc 1140

ctgaacaacg ggagtcaggc agtaggacgc tcttcatttt actgcctgga gtactttcct 1200

tctcagatgc tgcgtaccgg aaacaacttt accttcagct acacttttga ggacgttcct 1260

ttccacagca gctacgctca cagccagagt ctggaccgtc tcatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccggggggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac cttaatgggc gtgaatctat aatcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

agacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc catttgggcc 1860

aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920

aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980

gagttttcag ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt 2040

gtggaaattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt tacctcaccc gtcccctgta a 2211

<210> 24

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 24

atggctgctg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggtga caacccctac ctcaagtaca accacgccga cgcggagttc 300

cagcagcggc ttcagggcga cacatcgttt gggggcaacc tcggcagagc agtcttccag 360

gccaaaaaga gggttcttga acctcttggt ctggttgagc aagcgggtga gacggctcct 420

ggaaagaaga gaccgttgat tgaatccccc cagcagcccg actcctccac gggtatcggc 480

aaaaaaggca agcagccggc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggtgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgcacctg ggccttgccc acctacaata accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg acttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaaactctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc agactatcag ctcccgtacg tgctcgggtc ggctcacgag 1080

ggctgcctcc cgccgttccc agcagacgtc ttcatggtgc cacagtatgg atacctcacc 1140

ctgaacaacg ggagtcaggc agtaggacgc tcttcatttt actgcctgga gtactttcct 1200

tctcagatgc tgcgtaccgg aaacaacttt accttcagct acacttttga ggacgttcct 1260

ttccacagca gctacgctca cagccagagt ctggaccgtc tcatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccggggggtc tccaactggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac cttaatgggc gtgaatctat aatcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

agacgaag aggaaatcaa agccactaac cccgtggcca ctgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgccatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactc 1920

aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980

gagttttcag ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttca ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt tacctcaccc gtcccctgta a 2211

<210> 25

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 25

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg cgctgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120

gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggatgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcaatcgcca caagagccag actcctcctc gggcatcggc 480

aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgaacatg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt cgacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca actggggatt ccggcccaag agactcagct tcaagctctt caacatccag 960

gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgat 1320

caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaat 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca taaagacgac gaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680

agacgaag aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg 1740

gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca tgctatggga 1800

gcattacctg gcatggtgtg gcaagataga gacgtgtacc tgcagggtcc catttgggcc 1860

aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920

aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980

gagttttcag ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac attgatttca ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt tacctcaccc gtccccagta a 2211

<210> 26

<211> 736

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 26

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Cys Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 27

<211> 736

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 27

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Val Asn Gln Gln Lys Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Thr Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 28

<211> 723

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 28

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Val Asn Gln Gln Lys Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Gln Val

305 310 315 320

Lys Glu Thr Thr Asp Val Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr

325 330 335

Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly

340 345 350

Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met

355 360 365

Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser Gln Ala Val

370 375 380

Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu

385 390 395 400

Arg Thr Gly Asn Asn Phe Thr Ser Tyr Thr Phe Glu Asp Val Pro Phe

405 410 415

His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro

420 425 430

Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr Gln Asn Gln Ser

435 440 445

Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser Arg Gly Ser Pro Thr

450 455 460

Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg

465 470 475 480

Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn Asn Asn Ser Asn Phe

485 490 495

Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn Gly Arg Glu Ser Ile

500 505 510

Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys

515 520 525

Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly

530 535 540

Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile Thr Asp Glu Glu Ala

545 550 555 560

Thr Asn Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala Val Asn Leu

565 570 575

Gln Ser Ser Pro Ala Thr Asp Val His Ala Met Gly Ala Leu Pro Gly

580 585 590

Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala

595 600 605

Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly

610 615 620

Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr

625 630 635 640

Pro Val Pro Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala

645 650 655

Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu

660 665 670

Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln

675 680 685

Tyr Thr Ser Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp

690 695 700

Asn Asn Gly Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu

705 710 715 720

Thr Arg Pro

<210> 29

<211> 736

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 29

Met Ala Ser Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Arg Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 30

<211> 736

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 30

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 31

<211> 735

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 31

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Lys Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asp Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Ser Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 32

<211> 735

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 32

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asn Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Lys Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Arg Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asp Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Ser Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 33

<211> 735

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic amino acid sequence

<400> 33

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Lys Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asp Ala Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Ser Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 34

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 34

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120

gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggatgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac cttcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga gggttctcga acctctcggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcattggc 480

aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgcacctg ggccttgccc acctacaata accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg acttcaacag attccactgc cacttttcac cacgtgactg gcaaagactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcagtacgg ctacctaacg 1140

ctcaacaatg gcagccaggc agtgggacgg tcatcctttt actgcctgga atatttccca 1200

tcgcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gtgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac cttaatgggc gtgaatctat aatcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

agacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920

aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980

gagttttcag ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccaac gttgatttca ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt tacctcaccc gtcccctgta a 2211

<210> 35

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 35

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gaatggtggg acttgaaacc tggagccccg aaacccaaag tcaaccagca aaagcaggac 120

aacgctcggg gtcttgtgct tccgggttac aaatacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac cttcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc ttggacgagc agtcttccag 360

gccaagaaga gggttctcga accttttggt ctggttgagg aaggtgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcattggc 480

aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgcacctg ggccttgccc acctacaata accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg acttcaacag attccactgc cacttttcac cacgtgactg gcaaagactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaggc agtgggacgg tcatcctttt actgcctgga atatttccca 1200

tcgcagatgc tgagaacggg caataacttt accttcagct acacttttga ggacgttcct 1260

ttccacagca gctacgctca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccaactggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac cttaatgggc gtgaatctat aatcaaccct 1560

ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

agacgaag aggaaatcaa agccactaac cccgtggcca ctgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgccatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttca ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt tacctcaccc gtcccctgta a 2211

<210> 36

<211> 2208

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<220>

<221> misc_feature

<222> (955)..(955)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (971)..(972)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (981)..(981)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (986)..(986)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1235)..(1235)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1694)..(1696)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1698)..(1698)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1763)..(1765)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1778)..(1778)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (2204)..(2204)

<223> n is a, c, g, or t

<400> 36

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gaatggtggg acttgaaacc tggagccccg aaacccaaag tcaaccagca aaagcaggac 120

aacgctcggg gtcttgtgct tccgggttac aaatacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac cttcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc ttggacgagc agtcttccag 360

gccaagaaga gggttctcga accttttggt ctggttgagg aaggtgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcattggc 480

aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgaacatg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaataaca attggggatt ccggcccaag agactcaact tcaaactctt caacntccaa 960

gtcaaggagg nnacgacgaa ngatgncgtc acaaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaggc agtgggacgg tcatcctttt actgcctgga atatttccca 1200

tcgcagatgc tgagaacggg caataacttt acctncagct acacttttga ggacgttcct 1260

ttccacagca gctacgctca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccaactggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac cttaatgggc gtgaatctat aatcaaccct 1560

ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

agacgaag agannncnaa gccactaacc ccgtggccac tgaaagattt gggactgtgg 1740

cagtcaatct ccaagcagca cannnaccct gcgaccgnag atgtgcatgc catgggagcc 1800

ttacctggaa tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa 1860

attcctcaca cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag 1920

cacccgcctc ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag 1980

ttttcggcta caaagtttgc ttcattcatc acccagtatt ccacaggaca agtgagcgtg 2040

gagattgaat gggagctgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat 2100

acatctaact atgcaaaatc tgccaacgtt gatttcactg tggacaacaa tggactttat 2160

actgagcctc gccccattgg cacccgttac ctcacccgtc cccngtaa 2208

<210> 37

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 37

atggcttccg atggttatct tccagattgg ctcgaggaca acctctctga gggcatccgc 60

gagtggtggg acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120

gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggatgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca gagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaaga gggttctcga accttttggt ctggttgagg aaggtgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcattggc 480

aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgaacatg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg atttcaacag attccactgc catttctcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaaactctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcagtacgg ctacctaacg 1140

ctcaacaatg gcagccaggc agtgggacgg tcatcctttt actgcctgga atatttccca 1200

tcgcagatgc tgagaacggg caataacttt accttcagct acaccttcga ggacgtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccggggggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac cttaatgggc gtgaatctat aatcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

agacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc catttgggcc 1860

aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attctactgg ccaagtcagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttca ctgtggacaa caatggactt 2160

tatactgagc ctcgtcccat tggcacccgt tacctcaccc gtcccctgta a 2211

<210> 38

<211> 2211

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 38

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcatcggc 480

aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgaacatg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt cgacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaata actggggatt ccggcccaag agactcagct tcaagctctt caacatccag 960

gtcaaggagg tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgat 1320

caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaat 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680

acggacgaag aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg 1740

gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca tgctatggga 1800

gcattacctg gcatggtgtg gcaagataga gacgtgtacc tgcagggtcc catttgggcc 1860

aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920

aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980

gagttttcag ctacaaagtt tgcttcattc atcactcaat actccacagg acaagtgagc 2040

gtggaaattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttca ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt tacctcaccc gtcccctgta a 2211

<210> 39

<211> 2208

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 39

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctatgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg atccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaagttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtatgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggtaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacagacact ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca agtgtggaca ttgaaaaggt catgattaca 1680

gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740

accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800

cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860

attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920

caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980

ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040

gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga agttcagtac 2100

acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160

tcagagcctc gccccattgg caccagatac ctgactcgta atctgtaa 2208

<210> 40

<211> 2208

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 40

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctatgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcaaac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaagttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcgggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggtaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacagacact ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca agtgtggaca ttgaaaaggt catgattaca 1680

gacgaagagg aaatcaggac gaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740

accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800

cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860

attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920

caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980

ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040

gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga agttcagtac 2100

acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160

acagagcctc gccccattgg caccagatac ctgactcgta atctgtaa 2208

<210> 41

<211> 2208

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 41

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctatgac 240

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480

aaggcgggtc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540

tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600

aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660

gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900

aacaacaact ggggattccg acccaagaga ctcaagttca agctctttaa cattcaagtc 960

aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020

caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080

tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140

aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200

cagatgctgc gtaccggtaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260

cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320

tacctgtatt acttgagcag aacagacgct ccaagtggaa ccaccacgca gtcaaggctt 1380

cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440

ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500

tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560

ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620

atctttggga agcaaggctc agagaaaaca agtgtggaca ttgaaaaggt catgattaca 1680

gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740

accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800

cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860

attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920

caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980

ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040

gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga agttcagtac 2100

acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160

tcagagcctc gccccattgg caccagatac ctgactcgta atctgtaa 2208

<210> 42

<211> 4443

<212> DNA

<213> Homo sapiens

<400> 42

atgcagaggt cgcctctgga aaaggccagc gttgtctcca aacttttttt cagctggacc 60

agaccaattt tgaggaaagg atacagacag cgcctggaat tgtcagacat ataccaaatc 120

ccttctgttg attctgctga caatctatct gaaaaattgg aaagagaatg ggatagagag 180

ctggcttcaa agaaaaatcc taaactcatt aatgcccttc ggcgatgttt tttctggaga 240

tttatgttct atggaatctt tttatattta ggggaagtca ccaaagcagt acagcctctc 300

ttactgggaa gaatcatagc ttcctatgac ccggataaca aggaggaacg ctctatcgcg 360

atttatctag gcataggctt atgccttctc tttattgtga ggacactgct cctacaccca 420

gccatttttg gccttcatca cattggaatg cagatgagaa tagctatgtt tagtttgatt 480

tataagaaga ctttaaagct gtcaagccgt gttctagata aaataagtat tggacaactt 540

gttagtctcc tttccaacaa cctgaacaaa tttgatgaag gacttgcatt ggcacatttc 600

gtgtggatcg ctcctttgca agtggcactc ctcatggggc taatctggga gttgttacag 660

gcgtctgcct tctgtggact tggtttcctg atagtccttg ccctttttca ggctgggcta 720

gggagaatga tgatgaagta cagagatcag agagctggga agatcagtga aagacttgtg 780

attacctcag aaatgattga aaatatccaa tctgttaagg catactgctg ggaagaagca 840

atggaaaaaa tgattgaaaa cttaagacaa acagaactga aactgactcg gaaggcagcc 900

tatgtgagat acttcaatag ctcagccttc ttcttctcag ggttctttgt ggtgttttta 960

tctgtgcttc cctatgcact aatcaaagga atcatcctcc ggaaaatatt caccaccatc 1020

tcattctgca ttgttctgcg catggcggtc actcggcaat ttccctgggc tgtacaaaca 1080

tggtatgact ctcttggagc aataaacaaa atacaggatt tcttacaaaa gcaagaatat 1140

aagacattgg aatataactt aacgactaca gaagtagtga tggagaatgt aacagccttc 1200

tgggaggagg gatttgggga attatttgag aaagcaaaac aaaacaataa caatagaaaa 1260

acttctaatg gtgatgacag cctcttcttc agtaatttct cacttcttgg tactcctgtc 1320

ctgaaagata ttaatttcaa gatagaaaga ggacagttgt tggcggttgc tggatccact 1380

ggagcaggca agacttcact tctaatggtg attatgggag aactggagcc ttcagaggt 1440

aaaattaagc acagtggaag aatttcattc tgttctcagt tttcctggat tatgcctggc 1500

accattaaag aaaatatcat ctttggtgtt tcctatgatg aatatagata cagaagcgtc 1560

atcaaagcat gccaactaga agaggacatc tccaagtttg cagagaaaga caatatagtt 1620

cttggagaag gtggaatcac actgagtgga ggtcaacgag caagaatttc tttagcaaga 1680

gcagtataca aagatgctga tttgtattta ttagactctc cttttggata cctagatgtt 1740

ttaacagaaa aagaaatatt tgaaagctgt gtctgtaaac tgatggctaa caaaactagg 1800

attttggtca cttctaaaat ggaacattta aagaaagctg acaaaatatt aattttgcat 1860

gaaggtagca gctattttta tgggacattt tcagaactcc aaaatctaca gccagacttt 1920

agctcaaaac tcatgggatg tgattctttc gaccaattta gtgcagaaag aagaaattca 1980

atcctaactg agaccttaca ccgtttctca ttagaaggag atgctcctgt ctcctggaca 2040

gaaacaaaaa aacaatcttt taaacagact ggagagtttg gggaaaaaag gaagaattct 2100

attctcaatc caatcaactc tatacgaaaa ttttccattg tgcaaaagac tcccttacaa 2160

atgaatggca tcgaagagga ttctgatgag cctttagaga gaaggctgtc cttagtacca 2220

gattctgagc agggagaggc gatactgcct cgcatcagcg tgatcagcac tggccccacg 2280

cttcaggcac gaaggaggca gtctgtcctg aacctgatga cacactcagt taaccaaggt 2340

cagaacattc accgaaagac aacagcatcc acacgaaaag tgtcactggc ccctcaggca 2400

aacttgactg aactggatat atattcaaga aggttatctc aagaaactgg cttggaaata 2460

agtgaagaaa ttaacgaaga agacttaaag gagtgctttt ttgatgatat ggagagcata 2520

ccagcagtga ctacatggaa cacatacctt cgatatatta ctgtccacaa gagcttaatt 2580

tttgtgctaa tttggtgctt agtaattttt ctggcagagg tggctgcttc tttggttgtg 2640

ctgtggctcc ttggaaacac tcctcttcaa gacaaaggga atagtactca tagtagaaat 2700

aacagctatg cagtgattat caccagcacc agttcgtatt atgtgtttta catttacgtg 2760

ggagtagccg acactttgct tgctatggga ttcttcagag gtctaccact ggtgcatact 2820

ctaatcacag tgtcgaaaat tttacaccac aaaatgttac attctgttct tcaagcacct 2880

atgtcaaccc tcaacacgtt gaaagcaggt gggattctta atagattctc caaagatata 2940

gcaattttgg atgaccttct gcctcttacc atatttgact tcatccagtt gttattaatt 3000

gtgattggag ctatagcagt tgtcgcagtt ttacaaccct acatctttgt tgcaacagtg 3060

ccagtgatag tggcttttat tatgttgaga gcatatttcc tccaaacctc acagcaactc 3120

aaacaactgg aatctgaagg caggagtcca attttcactc atcttgttac aagcttaaaa 3180

ggactatgga cacttcgtgc cttcggacgg cagccttact ttgaaactct gttccacaaa 3240

gctctgaatt tacatactgc caactggttc ttgtacctgt caacactgcg ctggttccaa 3300

atgagaatag aaatgatttt tgtcatcttc ttcattgctg ttaccttcat ttccattta 3360

acaacaggag aaggagaagg aagagttggt attatcctga ctttagccat gaatatcatg 3420

agtacattgc agtgggctgt aaactccagc atagatgtgg atagcttgat gcgatctgtg 3480

agccgagtct ttaagttcat tgacatgcca acagaaggta aacctaccaa gtcaaccaaa 3540

ccatacaaga atggccaact ctcgaaagtt atgattattg agaattcaca cgtgaagaaa 3600

gatgacatct ggccctcagg gggccaaatg actgtcaaag atctcacagc aaaatacaca 3660

gaaggtggaa atgccatatt agagaacatt tccttctcaa taagtcctgg ccagaggtg 3720

ggcctcttgg gaagaactgg atcagggaag agtactttgt tatcagcttt tttgagacta 3780

ctgaacactg aaggagaaat cgatcgat ggtgtgtctt gggattcaat aactttgcaa 3840

cagtggagga aagcctttgg agtgatacca cagaaagtat ttattttttc tggaacattt 3900

agaaaaaact tggatcccta tgaacagtgg agtgatcaag aaatatggaa agttgcagat 3960

gaggttgggc tcagatctgt gatagaacag tttcctggga agcttgactt tgtccttgtg 4020

gatgggggct gtgtcctaag ccatggccac aagcagttga tgtgcttggc tagatctgtt 4080

ctcagtaagg cgaagatctt gctgcttgat gaacccagtg ctcatttgga tccagtaaca 4140

taccaaataa ttagaagaac tctaaaacaa gcatttgctg attgcacagt aattctctgt 4200

gaacacagga tagaagcaat gctggaatgc caacaatttt tggtcataga agagaacaaa 4260

gtgcggcagt acgattccat ccagaaactg ctgaacgaga ggagcctctt ccggcaagcc 4320

atcagcccct ccgacagggt gaagctcttt ccccaccgga actcaagcaa gtgcaagtct 4380

aagccccaga ttgctgctct gaaagaggag acagaagaag aggtgcaaga tacaaggctt 4440

tag 4443

<210> 43

<211> 3464

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 43

atgcagcgca gcccactgga gaaggcaagc gtggtgtcca agctgttctt ttcctggacc 60

aggcctatcc tgaggaaggg atacaggcag cggctggagc tgagcgacat ctatcagatc 120

ccttctgtgg acagcgccga taatctgtcc gagaagctgg agagagagtg ggatagggag 180

ctggcctcta agaagaaccc aaagctgatc aatgccctgc ggagatgctt cttttggcgg 240

ttcatgttct acggcatctt cctgtatctg ggcgaggtga ccaaggccgt gcagccactg 300

ctgctgggca gaatcatcgc ctcttacgac cccgataaca aggaggagag gagcatcgcc 360

atctatctgg gcatcggcct gtgcctgctg tttatcgtga ggacactgct gctgcaccca 420

gccatcttcg gcctgcacca catcggcatg cagatgagaa tcgccatgtt cagcctgatc 480

tacaagaaga ccctgaagct gagctccagg gtgctggaca agatctccat cggccagctg 540

gtgtccctgc tgtctaacaa tctgaacaag tttgatgagg gactggccct ggcacacttc 600

gtgtggatcg caccactgca ggtggccctg ctgatggggcc tgatctggga gctgctgcag 660

gcaagcgcct tttgcggact gggcttcctg atcgtgctgg ccctgttcca ggcaggactg 720

ggacgcatga tgatgaagta cagagaccag agggccggca agatctctga gcggctggtc 780

atcaccagcg agatgatcga gaacatccag tccgtgaagg cctattgttg ggaggaggcc 840

atggagaaga tgatcgagaa tctgcgccag acagagctga agctgaccag aaaggccgcc 900

tacgtgaggt acttcaactc tagcgccttc tttttctctg gctttttcgt ggtgttcctg 960

agcgtgctgc catacgccct gatcaagggc atcatcctgc ggaagatctt taccacaatc 1020

tccttctgca tcgtgctgag aatggccgtg acaaggcagt ttccctgggc cgtgcagacc 1080

tggtatgact ctctgggcgc catcaataag atccaggatt tcctgcagaa gcaggagtac 1140

aagacactgg agtataacct gaccacaacc gaggtggtca tggagaatgt gaccgccttc 1200

tgggaggagg gctttggcga gctgttcgag aaggccaagc agaacaataa caatcgcaag 1260

acatctaacg gcgacgatag cctgtttttc agcaattttt ccctgctggg cacccccgtg 1320

ctgaaggaca tcaacttcaa gatcgagagg ggacagctgc tggcagtggc aggctccaca 1380

ggcgccggca agacctctct gctgatgatg atcatgggcg agctggagcc aagcgaggc 1440

aagatcaagc actccggccg gatctctttt tgcagccagt tctcctggat catgcccggc 1500

accatcaagg agaatatcat ctttggcgtg tcctacgatg agtacagata taggtctgtg 1560

atcaaggcct gtcagctgga ggaggacatc agcaagttcg ccgagaagga taacatcgtg 1620

ctgggcgagg gcggcatcac actgagcgga ggacagaggg caaggatctc cctggccaga 1680

gccgtgtaca aggacgccga tctgtatctg ctggacagcc cctttggcta tctggatgtg 1740

ctgaccgaga aggagatctt cgagtcctgc gtgtgcaagc tgatggccaa taagacaagg 1800

atcctggtga cctctaagat ggagcacctg aagaaggccg acaagatcct gatcctgcac 1860

gagggctcct cttactttta tggcacattc agcgagctgc agaatctgca gcctgacttc 1920

agctccaagc tgatgggctg tgactccttt gatcagttct ctgccgagag gcgcaactcc 1980

atcctgacag agaccctgca cagattctct ctggagggcg acgcacccgt gagctggaca 2040

gagaccaaga agcagtcctt taagcagacc ggcgagttcg gcgagaagag gaagaattct 2100

atcctgaacc ctatcaatag cacactgcag gcccggagaa ggcagtctgt gctgaacctg 2160

atgaccaca gcgtgaacca gggccagaat atccacagaa agacaaccgc cagcacaagg 2220

aaggtgtccc tggcacctca ggcaaacctg accgagctgg acatctactc ccgccggctg 2280

tctcaggaga ccggactgga gatctctgag gagatcaatg aggaggatct gaaggagtgc 2340

tttttcgacg atatggagag catcccagcc gtgacaacct ggaacacata cctgcgctat 2400

atcaccgtgc acaagtccct gatctttgtg ctgatctggt gtctggtcat cttcctggca 2460

gaggtggcag catctctggt ggtgctgtgg ctgctgggca acacacccct gcaggacaag 2520

ggcaattcta cccacagccg caacaattcc tacgccgtga tcatcacatc tacctctagc 2580

tactacgtgt tctacatcta tgtgggcgtg gccgatacac tgctggccat gggctttttc 2640

cggggcctgc ccctggtgca cacactgatc accgtgagca agatcctgca ccacaagatg 2700

ctgcacagcg tgctgcaggc ccctatgtcc acactgaaca ccctgaaggc cggcggcatc 2760

ctgaatcggt tttccaagga catcgccatc ctggacgatc tgctgcctct gaccatcttt 2820

gatttcatcc agctgctgct gatcgtgatc ggagcaatcg cagtggtggc cgtgctgcag 2880

ccttacatct tcgtggccac agtgccagtg atcgtggcct ttatcatgct gcgcgcctat 2940

ttcctgcaga ccagccagca gctgaagcag ctggagagcg agggccggtc ccctatcttt 3000

acacacctgg tgacctccct gaagggactg tggacactga gggccttcgg ccggcagcca 3060

tactttgaga ccctgttcca caaggccctg aacctgcaca cagccaattg gtttctgtat 3120

ctgagcaccc tgcgctggtt tcagatgcgg atcgagatga tcttcgtgat ctttttcatc 3180

gccgtgacct tcatctccat cctgacaacc ggagagggag agggaagagt gggaatcatc 3240

ctgacactgg ccatgaacat catgtctacc ctgcagtggg ccgtgaattc ctctatcgac 3300

gtggatagcc tgatgagatc tgtgagcagg gtgtttaagt tcatcgacat gccccacagag 3360

ggcaagccta caaagagcac caagccatac aagaacggcc agctgtccaa agtgatgatc 3420

atcgagaatt ctcacgtgaa gaaggacgat atctggccat ccgg 3464

<210> 44

<211> 173

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 44

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 60

aaaatcaacg ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg 120

gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgt 173

<210> 45

<211> 4874

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleic acid sequence

<400> 45

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcctgcggc cgcactcacg gggatttcca agtctccacc 180

ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 240

gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 300

taagcagagc tcgtttagtg aaccgtcaga attctcgagt gatcgaaaga gcctgctaaa 360

gcaaaaaaga agtcaccatg cagcgcagcc cactggagaa ggcaagcgtg gtgtccaagc 420

tgttcttttc ctggaccagg cctatcctga ggaagggata caggcagcgg ctggagctga 480

gcgacatcta tcagatccct tctgtggaca gcgccgataa tctgtccgag aagctggaga 540

gagagtggga tagggagctg gcctctaaga agaacccaaa gctgatcaat gccctgcgga 600

gatgcttctt ttggcggttc atgttctacg gcatcttcct gtatctgggc gaggtgacca 660

aggccgtgca gccactgctg ctgggcagaa tcatcgcctc ttacgacccc gataacaagg 720

aggagaggag catcgccatc tatctgggca tcggcctgtg cctgctgttt atcgtgagga 780

cactgctgct gcacccagcc atcttcggcc tgcaccacat cggcatgcag atgagaatcg 840

ccatgttcag cctgatctac aagaagaccc tgaagctgag ctccagggtg ctggacaaga 900

tctccatcgg ccagctggtg tccctgctgt ctaacaatct gaacaagttt gatgagggac 960

tggccctggc acacttcgtg tggatcgcac cactgcaggt ggccctgctg atgggcctga 1020

tctgggagct gctgcaggca agcgcctttt gcggactggg cttcctgatc gtgctggccc 1080

tgttccaggc aggactggga cgcatgatga tgaagtacag agaccagagg gccggcaaga 1140

tctctgagcg gctggtcatc accagcgaga tgatcgagaa catccagtcc gtgaaggcct 1200

attgttggga ggaggccatg gagaagatga tcgagaatct gcgccagaca gagctgaagc 1260

tgaccagaaa ggccgcctac gtgaggtact tcaactctag cgccttcttt ttctctggct 1320

ttttcgtggt gttcctgagc gtgctgccat acgccctgat caagggcatc atcctgcgga 1380

agatctttac cacaatctcc ttctgcatcg tgctgagaat ggccgtgaca aggcagtttc 1440

cctgggccgt gcagacctgg tatgactctc tgggcgccat caataagatc caggatttcc 1500

tgcagaagca ggagtacaag acactggagt ataacctgac cacaaccgag gtggtcatgg 1560

agaatgtgac cgccttctgg gaggagggct ttggcgagct gttcgagaag gccaagcaga 1620

acaataacaa tcgcaagaca tctaacggcg acgatagcct gtttttcagc aatttttccc 1680

tgctgggcac ccccgtgctg aaggacatca acttcaagat cgagagggga cagctgctgg 1740

cagtggcagg ctccacaggc gccggcaaga cctctctgct gatgatgatc atgggcgagc 1800

tggagccaag cgagggcaag atcaagcact ccggccggat ctctttttgc agccagttct 1860

cctggatcat gcccggcacc atcaaggaga atatcatctt tggcgtgtcc tacgatgagt 1920

acagatatag gtctgtgatc aaggcctgtc agctggagga ggacatcagc aagttcgccg 1980

agaaggataa catcgtgctg ggcgagggcg gcatcacact gagcggagga cagagggcaa 2040

ggatctccct ggccagagcc gtgtacaagg acgccgatct gtatctgctg gacagcccct 2100

ttggctatct ggatgtgctg accgagaagg agatcttcga gtcctgcgtg tgcaagctga 2160

tggccaataa gacaaggatc ctggtgacct ctaagatgga gcacctgaag aaggccgaca 2220

agatcctgat cctgcacgag ggctcctctt acttttatgg cacattcagc gagctgcaga 2280

atctgcagcc tgacttcagc tccaagctga tgggctgtga ctcctttgat cagttctctg 2340

ccgagaggcg caactccatc ctgacagaga ccctgcacag attctctctg gagggcgacg 2400

cacccgtgag ctggacagag accaagaagc agtcctttaa gcagaccggc gagttcggcg 2460

agaagaggaa gaattctatc ctgaacccta tcaatagcac actgcaggcc cggagaaggc 2520

agtctgtgct gaacctgatg acccacagcg tgaaccaggg ccagaatatc cacagaaaga 2580

caaccgccag cacaaggaag gtgtccctgg cacctcaggc aaacctgacc gagctggaca 2640

tctactcccg ccggctgtct caggagaccg gactggagat ctctgaggag atcaatgagg 2700

aggatctgaa ggagtgcttt ttcgacgata tggagagcat cccagccgtg acaacctgga 2760

acacatacct gcgctatatc accgtgcaca agtccctgat ctttgtgctg atctggtgtc 2820

tggtcatctt cctggcagag gtggcagcat ctctggtggt gctgtggctg ctgggcaaca 2880

cacccctgca ggacaagggc aattctaccc acagccgcaa caattcctac gccgtgatca 2940

tcacatctac ctctagctac tacgtgttct acatctatgt gggcgtggcc gatacactgc 3000

tggccatggg ctttttccgg ggcctgcccc tggtgcacac actgatcacc gtgagcaaga 3060

tcctgcacca caagatgctg cacagcgtgc tgcaggcccc tatgtccaca ctgaacaccc 3120

tgaaggccgg cggcatcctg aatcggtttt ccaaggacat cgccatcctg gacgatctgc 3180

tgcctctgac catctttgat ttcatccagc tgctgctgat cgtgatcgga gcaatcgcag 3240

tggtggccgt gctgcagcct tacatcttcg tggccacagt gccagtgatc gtggccttta 3300

tcatgctgcg cgcctatttc ctgcagacca gccagcagct gaagcagctg gagagcgagg 3360

gccggtcccc tatctttaca cacctggtga cctccctgaa gggactgtgg acactgaggg 3420

ccttcggccg gcagccatac tttgagaccc tgttccacaa ggccctgaac ctgcacacag 3480

ccaattggtt tctgtatctg agcaccctgc gctggtttca gatgcggatc gagatgatct 3540

tcgtgatctt tttcatcgcc gtgaccttca tctccatcct gacaaccgga gagggagagg 3600

gaagagtggg aatcatcctg acactggcca tgaacatcat gtctaccctg cagtgggccg 3660

tgaattcctc tatcgacgtg gatagcctga tgagatctgt gagcagggtg tttaagttca 3720

tcgacatgcc cacagagggc aagcctacaa agagcaccaa gccatacaag aacggccagc 3780

tgtccaaagt gatgatcatc gagaattctc acgtgaagaa ggacgatatc tggccatccg 3840

gaggacagat gaccgtgaag gatctgacag ccaagtatac cgagggcggc aacgccatcc 3900

tggagaatat ctccttttct atcagccctg gacagagggt gggactgctg ggacggacag 3960

gctccggcaa gtctaccctg ctgagcgcct tcctgaggct gctgaataca gagggcgaga 4020

tccagatcga cggcgtgagc tgggattcca tcaccctgca gcagtggaga aaggcctttg 4080

gcgtgatccc tcagaaggtg tttatcttct ccggcacctt caggaagaac ctggacccat 4140

acgagcagtg gtctgatcag gagatctgga aggtggccga cgaagtgggc ctgagatctg 4200

tgatcgagca gtttccaggc aagctggact tcgtgctggt ggatggagga tgcgtgctga 4260

gccacggaca caagcagctg atgtgcctgg ccaggtctgt gctgagcaag gccaagatcc 4320

tgctgctgga cgagccaagc gcccacctgg atcccgtgac ataccagatc atcagaagga 4380

ccctgaagca ggcctttgcc gattgcaccg tgatcctgtg cgagcaccgc atcgaggcca 4440

tgctggagtg ccagcagttc ctggtcatcg aggagaacaa ggtgcggcag tatgacagca 4500

tccagaagct gctgaatgag cggagcctgt ttcggcaggc catctccccc tctgatcgcg 4560

tgaagctgtt ccctcaccgg aacagctcca agtgtaagtc caagccccag atcgccgccc 4620

tgaaggagga gacagaggag gaggtgcagg acaccagact gtgaaataaa acatctttat 4680

tttcattaca tctgtgtgtt ggttttttgt gtgaacaacg gccggccgga ggaaccccta 4740

gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgcccgggca 4800

aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga 4860

gagggagtgg ccaa 4874

<---

Claims (30)

1. A method for delivering a heterologous nucleic acid to a lung cell in a subject, comprising administering to the subject a composition comprising a recombinant adeno-associated virus (rAAV) vector, wherein said rAAV vector comprises (i) a capsid comprising a capsid protein comprising the amino acid sequence presented as SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 and comprising Thr at amino acid 469 and Ala at amino acid 598, based on the amino acid numbering presented in SEQ ID NO: 12, and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product, wherein the step of administering comprises pulmonary, endobronchial, intranasal, intratracheal and/or intrabronchial administration. 2. The method according to claim 1, wherein the lung cell is selected from an airway epithelial cell, a smooth muscle cell, and an endothelial cell. 3. The method of claim 2, wherein the airway epithelial cell is a basal cell, a goblet cell, or a ciliated cell. 4. The method of claim 3, wherein the airway epithelial cell is an alveolar epithelial cell of the lung type 1 (AECI), an alveolar epithelial cell of the lung type 2 (AECII), a bronchial epithelial cell, or a tracheal epithelial cell. 5. The method according to any one of paragraphs 1-4, wherein the composition is formulated as an aerosol. 6. The method according to claim 5, comprising introducing the composition by means of a sprayer. 7. The method according to any one of claims 1-6, wherein the composition contains from 10 ¹¹ to 10 ¹⁴ vector genomes (vg) of rAAV per 1 ml. 8. The method according to any one of claims 1-7, wherein the nucleotide sequence encoding the gene product is operably linked to a promoter. 9. The method according to claim 8, wherein the promoter is a constitutive promoter. 10. The method according to claim 8, wherein the promoter is a tissue-specific promoter. 11. The method according to any of paragraphs. 1-10, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a gene product selected from the human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active fragment thereof, SFTPA1 (surfactant A1), caveolin-1, alpha-1-antitrypsin, alpha-1-antichymotrypsin, alpha-1-macroglobulin, matrix metalloproteinase-1 (MMP1), matrix metalloproteinase-12 (MMP12), microsomal epoxide hydrolyase, CYP1A1, glutathione S-transferase, heme oxygenase-1, TGF-beta-1, TNF-alpha, IL-1 complex, IL-8, IL-13, human leukocyte antigen (HLA-B7 and Bw16), vitamin D binding protein, and beta-2-adrenergic receptor. 12. The method according to claim 11, wherein the gene product is a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein that lacks amino acids 708-759 of the human CFTR protein sequence. 13. The method of claim 12, wherein the gene product is a biologically truncated CFTR protein that lacks amino acids 708-759 of the human CFTR protein sequence. 14. The method according to claim 13, wherein the heterologous nucleic acid comprises the nucleotide sequence presented in SEQ ID NO: 43, or a sequence at least 80% identical thereto. 15. A pharmaceutical composition for the treatment of cystic fibrosis, comprising an rAAV vector, wherein said rAAV vector comprises (i) a capsid comprising a capsid protein of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising Thr at amino acid 469 and Ala at amino acid 598, based on the amino acid numbering specified in SEQ ID NO: 12, and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding CFTR or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence, wherein said nucleotide sequence is operably linked to an expression control sequence. 16. The pharmaceutical composition according to claim 15, wherein the nucleotide sequence encoding the CFTR protein is at least 80% identical to the nucleotide sequence presented as SEQ ID NO: 43. 17. The pharmaceutical composition according to claim 16, wherein the nucleotide sequence encoding CFTR comprises the nucleotide sequence presented as SEQ ID NO: 43. 18. The pharmaceutical composition according to any one of claims 15-17, wherein the expression control sequence comprises a universal promoter, preferably wherein the universal promoter is CMV173. 19. A pharmaceutical composition according to any one of claims 15-18, wherein the composition is formulated for inhalation or is suitable for inhalation. 20. A pharmaceutical composition according to any one of claims 15-19, comprising from about 20 to about 50 mM citrate, from about 85 to about 125 mM NaCl and about 0.005% Pluronic F68 and having a pH of about 6.0. 21. A pharmaceutical composition according to claim 20, comprising about 20 mM citrate, about 125 mM NaCl and about 0.005% Pluronic F68 and having a pH of about 6.0. 22. A method for treating cystic fibrosis in a subject, comprising administering to the subject a pharmaceutical composition according to any one of claims 15-21, wherein the step of administering comprises pulmonary, endobronchial, intranasal, intratracheal and/or intrabronchial administration. 23. Use of a pharmaceutical composition according to any one of claims 15-21 in the preparation of a medicinal product for the treatment of cystic fibrosis, where the medicinal product is intended for pulmonary, endobronchial, intranasal, intratracheal or intrabronchial administration. 24. A composition comprising a recombinant adeno-associated virus (rAAV) vector for treating a lung disease by delivering a heterologous nucleic acid to a lung cell in a subject, wherein said rAAV vector comprises (i) a capsid comprising a capsid protein comprising the amino acid sequence set forth as SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 and comprising Thr at amino acid 469 and Ala at amino acid 598, based on the amino acid numbering set forth in SEQ ID NO: 12, and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product, wherein the composition is administered by pulmonary, endobronchial, intranasal, intratracheal, or intrabronchial administration. 25. The composition of claim 24, wherein the composition is used in the treatment of cystic fibrosis, a pulmonary disease or a related pulmonary disease. 26. The composition according to claim 25, wherein the composition is used in the treatment of cystic fibrosis. 27. The method according to claim 1, wherein the capsid protein comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 12. 28. The method according to claim 1, wherein the capsid protein comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 12. 29. The pharmaceutical composition according to claim 15, wherein the capsid protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 12. 30. The pharmaceutical composition according to claim 15, wherein the capsid protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 12.

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