Nothing Special   »   [go: up one dir, main page]

EP0796339A1 - Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants - Google Patents

Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants

Info

Publication number
EP0796339A1
EP0796339A1 EP95944069A EP95944069A EP0796339A1 EP 0796339 A1 EP0796339 A1 EP 0796339A1 EP 95944069 A EP95944069 A EP 95944069A EP 95944069 A EP95944069 A EP 95944069A EP 0796339 A1 EP0796339 A1 EP 0796339A1
Authority
EP
European Patent Office
Prior art keywords
aav
cell
packaging
vector
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95944069A
Other languages
German (de)
English (en)
Inventor
James M. Allen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ampliphi Biosciences Corp
Original Assignee
Targeted Genetics Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Targeted Genetics Corp filed Critical Targeted Genetics Corp
Publication of EP0796339A1 publication Critical patent/EP0796339A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • This invention relates to gene therapy, and more specifically to materials and methods used for the generation of high titers of recombinant AAV vectors for use in gene therapy procedures.
  • AAV vectors may have utility for gene therapy but heretofore a significant obstacle has been the inability to generate sufficient quantities of such recombinant vectors in amounts that would be clinically useful for human gene therapy application. This is a particular problem for in vivo applications such as direct delivery to the lung.
  • Adeno-associated virus (AAV) vectors are among a small number of recombinant virus vector systems which have been shown to have utility as in vivo gene transfer agents (reviewed in Carter, 1992, Current Opinion in Biotechnology- 3:533-539; Muzcyzka, 1992, Curr. Top. Microbiol. Immunol. 158:97-129) and thus are potentially of great importance for human gene therapy.
  • AAV vectors are capable of high- frequency stable DNA integration and expression in a variety of cells including cystic fibrosis (CF) bronchial and nasal epithelial cells (see, e.g., Flotte et al., 1992a, Am. J. Respir. Cell Mol. Biol.
  • AAV may not require active cell division for stable expression which would be a clear advantage over retroviruses, especially in tissue such as the human airway epithelium where most cells are terminally differentiated and non-dividing.
  • AAV is a defective parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus (see Fig. 1) .
  • a co-infecting helper virus see Fig. 1 .
  • Examples of co-infecting viruses that provide helper functions for AAV growth and replication are adenoviruses, herpesviruses and in some cases poxviruses such as vaccinia.
  • the nature of the helper function is not entirely known but appears to be some indirect effect of the helper virus which renders the cell permissive for AAV replication. This belief is supported by the observation that in certain cases AAV replication may occur at a low level of efficiency in the absence of helper virus co-infection if the cells are treated with agents that are either genotoxic or that disrupt the cell cycle.
  • AAV may replicate to a limited extent in the absence of helper virus in certain unusual conditions, as noted above, the more general result is that infection of cells with AAV in the absence of helper functions results in integration of AAV into the host cell genome.
  • the integrated AAV genome may be rescued and replicated to yield a burst of infectious progeny AAV particles if cells containing an integrated AAV provirus are superinfected with a helper virus such as adenovirus. Because the integration of AAV appears to be an efficient event, this suggested that AAV would be a useful vector for introducing genes into cells for stable expression for uses such as human gene therapy.
  • AAV has a very broad host range with neither any obvious species nor tissue specificity and will replicate in virtually any cell line of human, simian or rodent origin provided an appropriate helper is present.
  • AAV is ubiquitous and has been isolated from a wide variety of animal species including most mammalian and several avian species.
  • AAV has not been associated with the cause of any disease.
  • AAV is not a transforming or oncogenic virus.
  • AAV integration into chromosomes of human cell lines does not cause any significant alteration in the growth properties or morphological characteristics of the cells.
  • These properties of AAV also recommend it as a potentially useful human gene therapy vector because most of the other viral systems proposed for this application such as retroviruses, adenoviruses, herpesviruses, or poxviruses are disease-causing viruses.
  • AAV particles are comprised of a protein capsid having three capsid proteins, VPl, VP2, and VP3, and enclosing a DNA genome.
  • the AAV DNA genome is a linear single-stranded DNA molecule having a molecular weight of about 1.5 x 10 ⁇ daltons or approximately 4680 nucleotides long. Strands of either complementary sense, "plus” or “minus” strands, are packaged into individual particles but each particle has only one DNA molecule. Equal numbers of AAV particles contain either a plus or minus strand.
  • Either strand is equally infectious and replication occurs by conversion of the parental infecting single strand to a duplex form and subsequent ampli ication of a large pool of duplex molecules from which progeny single strands are displaced and packaged into capsids.
  • Duplex or single-strand copies of AAV genomes inserted into bacterial plasmids or phagemids are infectious when transfected into adenovirus-infected cells, and this has allowed the study of AAV genetics and the development of AAV vectors.
  • the AAV2 genome has two copies of a 145-nucleotide-long
  • ITR inverted terminal repeat
  • the unique region contains three transcription promoters p5, pl9, and p40 (Laughlin et al., 1979, Proc. Natl. Acad. Sci. USA. 76:5567-5571) that are used to express the rep and cap genes.
  • the ITR sequences are required in cis and are sufficient to provide a functional origin of replication (ori ) and also are sufficient to provide signals required for integration into the cell genome as well as for efficient excision and rescue from host cell chromosomes or from recombinant plasmids.
  • the ITR can function directly as a transcription promoter in an AAV vector (Flotte et al., 1993, vide supra) .
  • the rep and cap genes are required in tr ⁇ ms to provide functions for replication and encapsidation of viral genome respectively.
  • the rep gene is expressed from two promoters, p5 and pl9. Transcription from p5 yields an unspliced 4.2 kb mRNA which encodes a protein, Rep78, and a spliced 3.9 kb mRNA which encodes a protein, Rep68. Transcription from pl9 yields an unspliced mRNA which encodes Rep52 and a spliced 3.3 kb mRNA which encodes Rep40.
  • the four Rep proteins all comprise a common internal region sequence but differ with respect to their amino and carboxyl terminal regions.
  • Rep78 and Rep68 are required for AAV duplex DNA replication, but Rep52 and Rep40 appear to be needed for progeny,- single- strand DNA accumulation. Mutations in Rep78 and Rep68 are phenotypically Rep- whereas mutations affecting only Rep52 and Rep40 are Rep+ but Ssd-. Rep68 and Rep78 bind specifically to the hairpin conformation of the AAV ITR and possess several enzyme activities required for resolving replication at the AAV termini. Rep52 and Rep40 have none of these properties.
  • the Rep proteins, primarily Rep78 and Rep68 exhibit several pleiotropic regulatory activities including positive and negative regulation of AAV genes and expression from some heterologous promoters, as well as inhibitory effects on cell growth (Tratschin et al., 1986, Mol. Cell.
  • Virology. 166:154-165) reported a very low level expression of some Rep proteins in certain cell lines after stable integration of AAV genomes.
  • the proteins VPl, VP2, and VP3 all share a common overlapping sequence but differ in that VPl and VP2 contain additional amino terminal sequence. All three are coded from the same cap gene reading frame expressed from a spliced 2.3 kb mRNA transcribed from the p40 promoter. VP2 and VP3 are generated from the same mRNA by use of alternate initiation codons. VPl is coded from a minor mRNA using 3' donor site that is 30 nucleotides upstream from the 3' donor used for the major mRNA that encodes VP2 and VP3. VPl, VP2, and VP3 are all required for capsid production. Mutations which eliminate all three proteins (Cap-) prevent accumulation of single- strand progeny AAV DNA whereas mutations in the VPl amino- terminus (Lip-, Inf-) permit single-strand production but prevent assembly of stable infectious particles.
  • AAV infectious genomes of AAV were constructed by insertion of double-strand molecules of AAV into plasmids by procedures such as GC tailing (Samulski et al. , 1982, Proc. Natl. Acad. Sci. USA. 79:2077-2081), addition of synthetic linkers containing restriction endonuclease (Laughlin et al., 1983, Gene. 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem..
  • AAV vector construction were defined as reviewed recently (Carter, 1992, Current Opinions in Biotechnology. 3:533-539; Muzyczka, 1992, Current Topics in Microbiology and Immunology. 158:97-129).
  • AAV vectors are constructed in AAV recombinant plasmids by substituting portions of the AAV coding sequence with foreign DNA to generate a vector plasmid.
  • the terminal (ITR) portions of the AAV sequence must be retained intact because these regions are required in cis for several functions including excision from the plasmid after transfection, replication of the vector genome and integration and rescue from a host cell genome.
  • the vector can then be packaged into an AAV particle to generate an AAV transducing virus by transfection of the vector plasmid into cells that are infected by an appropriate helper virus such as adenovirus or herpesvirus.
  • an appropriate helper virus such as adenovirus or herpesvirus.
  • the vector plasmid In order to achieve replication and encapsidation of the vector genome into AAV particles, the vector plasmid must be complemented for any AAV functions required in trans , namely rep and cap, that were deleted in construction of the vector plasmid.
  • the transducing vector must be generated at sufficiently high titers that it is practicable as a delivery system. This is especially important for gene therapy stratagems aimed at in vivo delivery of the vector.
  • the required dose of transducing vector may be in excess of 10 10 .
  • the vector preparations must be free of wild-type AAV virus.
  • the attainment of high titers of AAV vectors has been difficult for several reasons including preferential encapsidation of wild-type AAV genomes if they are present or generated by recombination, and the inability to generate sufficient complementing functions such as rep or cap.
  • Useful cell lines expressing such complementing functions have not been generated, in part, because of several inhibitory functions of the rep gene.
  • the first AAV vectors that were described contained foreign reporter genes such as neo or cat or dhfr that were expressed from AAV transcription promoters or an SV40 promoter (Tratschin et al., 1984b, Mol. Cell. Biol. 4:2072-2081; Hermonat & Muzyczka, 1984, Proc. Natl. Acad. Sci. USA. 81:6466-6470; Tratschin et al., 1985, Mol. Cell. Biol. 5:3251- 3260; McLaughlin et al., 1988, J. Virol.. 62:1963-1973; Lebkowski et al., 1988 Mol. Cell. Biol.. 7:349-356).
  • foreign reporter genes such as neo or cat or dhfr that were expressed from AAV transcription promoters or an SV40 promoter
  • AAV rep or cap gene still met with generation of wild-type AAV and still produced very low transduction frequencies on human cell lines.
  • McLaughlin et al., 1988 reported that AAV rep- cap- vectors containing the neo gene packaged with the same packaging plasmid used earlier by Hermonat & Muzyczka
  • Lebkowski et al., 1988 packaged AAV vectors which did not contain either a rep or cap gene and used an ori- packaging plasmid pBalA identical to that used earlier by Tratschin et al., (1984b, 1985) and reported transduction frequencies that were similarly low, in that for several human cell lines not more than 1% of the cells could be transduced to geneticin resistance even with their most concentrated vector stocks.
  • Lebkowski et al. , (1988) did not report the actual vector titers in a meaningful way but the biological assays showing not more than 1% transduction frequency when 5 x 10 6 cells were exposed to three ml of vector preparation indicates that the titer was less than 2 x 10 4 .
  • the pBal packaging plasmid contains overlapping ho ology with the ITR sequence in the vector and leads to generation by recombination of wild-type AAV.
  • Laface et al., (1988) used the same vector as that used by Hermonat & Muzyczka (1984) prepared in the same way and obtained a transduction frequency of 1.5% in murine bone marrow cultures again showing very low titer.
  • Sa ulski et al. (1987, J. Virol.. 61:3096-3101) constructed a plasmid called pSub201 which was an intact AAV genome in a bacterial plasmid but which had a deletion of 13 nucleotides at the extremity of each ITR and thus was rescued and replicated less efficiently than other AAV plasmids that contained the entire AAV genome.
  • Samulski et al. (1989, J ⁇ _ Virol.. 63:3822-3828) constructed AAV vectors based on pSub20l but deleted for rep and cap and containing either a hyg or neo gene expressed from an SV40 early gene promoter.
  • pAAV/Ad packaged these vectors by co-transfection with a packaging plasmid called pAAV/Ad which consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490 enclosed at either end with one copy of the adenovirus ITR.
  • pAAV/Ad a packaging plasmid which consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490 enclosed at either end with one copy of the adenovirus ITR.
  • the AAV rep and cap genes were expressed from the natural AAV promoters p5, pl9 and p40.
  • the function of the adenovirus ITR in pAAV/Ad was thought to be to enhance the expression level of AAV capsid proteins.
  • rep is expressed from its homologous promoter and is negatively regulated and thus its expression is limited.
  • Chatterjee et al., and Wong et al. used a packaging system known to give only low titer and which can lead to generation of wild-type AAV genomes because of the overlapping homology in the vector and packaging sequences.
  • Other reports have described the use of AAV vectors to express genes in human lymphocytes (Muro-Cacho et al., 1992, J. Immunotherapy. 11:231-237) or a human erythroid leukemia cell line (Walsh et al., 1992, Proc. Natl. Acad. Sci. USA. 89:7257-7261) with vectors based on the pSub201 vector plasmid and pAAV/Ad packaging plasmid. Again, titers of vector stocks were not reported and were apparently low because a selective marker gene was used to identify those cells that had been successfully transduced with the vector.
  • AAV vectors may have potential utility as vectors for treatment of human disease by gene therapy.
  • the ability to generate sufficient amounts of AAV vectors has been a severe limitation on the development of human gene therapy using AAV vectors.
  • One aspect of this limitation is that there have been very few studies using AAV vectors in in vivo animal models (see, e.g., Flotte et al., 1993b; and Kaplitt et al., 1994, Nature Genetics 8:148-154). This is generally a reflection of the difficulty associated with generating sufficient amounts of AAV vector stocks having a high enough titer to be useful in analyzing in vivo delivery and gene expression.
  • AAV gene therapy has been the relative inefficiency of the vector packaging systems that have been used. Because of the lack of cell lines expressing the AAV trans complementing functions, such as rep and cap, packaging of AAV vectors has been achieved in adenovirus-infected cells by co-transfection of a packaging plasmid and a vector plasmid. The efficiency of this process may be limited by the efficiency of transfection of each of the plasmid constructs, and by the level of expression of Rep proteins from the packaging plasmids described to date. Each of these problems appears to relate to the biological activities of the AAV Rep proteins. In addition, as noted above, all of the packaging systems described above have the ability to generate wild-type AAV by recombination.
  • Lebkowski et al. introduce rep and cap genes into the cell genome but the method again requires the use of episomal AAV transducing vectors comprising an Epstein-Barr virus nuclear antigen (EBNA) gene and an Epstein-Barr virus latent origin of replication; and, again, the only information relative to titer showed a fairly low titer.
  • EBNA Epstein-Barr virus nuclear antigen
  • AAV vectors can achieve in vivo gene transfer in the respiratory tract, for example, but high titers are critical so as to allow for the delivery of sufficiently high multiplicity of vector in as small a volume as possible.
  • Stable, helper-free AAV packaging cell lines have been elusive, mainly due to the activities of Rep protein, which down-regulates its own expression and can negatively affect the host cell.
  • Rep protein which down-regulates its own expression and can negatively affect the host cell.
  • the approaches described in this invention effectively circumvent these problems and have allowed for substantial improvements in packaging efficiency.
  • a method of producing a mammalian cell capable of high efficiency packaging of a recombinant AAV (rAAV) vector comprising the steps of: (a) providing a mammalian cell which comprises a stably integrated AAV cap gene operably linked to a promoter, and a stably integrated AAV rep gene operably linked to a heterologous promoter; (b) replicating the cell of step (a) to produce a population of cells; (c) introducing a helper virus to the population of cells of step (b) ; and (d) selecting a cell exhibiting helper-virus-inducible rep protein activity.
  • rAAV recombinant AAV
  • step (a) comprises the combined rep and cap genes of AAV in which the p5 promoter has been replaced by a heterologous promoter.
  • heterologous promoter is a mouse metallothionein I (mMT-I) promoter.
  • mMT-I mouse metallothionein I
  • a mammalian cell capable of high efficiency packaging of a recombinant AAV (rAAV) vector, said cell comprising a stably integrated cap gene operably linked to a promoter, and a stably integrated rep gene operably linked to a heterologous promoter; wherein said cell exhibits helper- virus-inducible rep protein activity.
  • rAAV recombinant AAV
  • said heterologous promoter is a mouse metallothionein I (mMT-I) promoter.
  • ITR inverted terminal repeat
  • a method of packaging a recombinant AAV vector comprising the steps of: (a) providing an AAV packaging cell of embodiment 10; (b) introducing a recombinant AAV vector, said vector comprising a polynucleotide sequence of interest located between two AAV inverted terminal repeat (ITR) regions; (c) introducing a helper virus; and (d) incubating the cell under conditions suitable for replication and packaging of AAV.
  • ITR inverted terminal repeat
  • a method of packaging a recombinant AAV vector comprising the steps of: (a) providing an AAV packaging cell of embodiment 15 which comprises a stably integrated rAAV vector; (b) introducing a helper virus; and (c) incubating the cell under conditions suitable for replication and packaging of AAV.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Figure 1 is a diagram of plasmid pMt-rep/cap//pKO-neo, as described in Example 1.
  • Figure 2 is a reproduction of Southern blots demonstrating that packaging cells produced according to the present invention have sufficient rep activity to replicate an incoming rAAV vector, as described in Example 4.
  • Figure 3 is a reproduction of Southern blots demonstrating that packaging cells produced according to the present invention are capable of replicating an AAV genome in the presence of adenovirus, and that this activity can be used to quantify the number of infectious viral particles present in a given sample, as described in Example 5.
  • Figure 4 is a reproduction of Southern blots demonstrating that packaging cells produced according to the present invention express rep protein and are able to replicate recombinant AAV plasmid DNA genomes introduced by transfection, as described in Example 6.
  • Figure 5 is a reproduction of Southern blots demonstrating that the infectious rAAV titering assay described in Example 5 had proceeded for a sufficient amount of time to reach a maximum, as described in Example ' 7.
  • Figures 6 and 7 are reproductions of Southern blots demonstrating that packaging cells produced according to the present invention can replicate and package rAAV vector genomes into infectious virions by either transfection or infection, as described in Examples 9 and 10.
  • Figure 8 is a reproduction of a Southern blot demonstrating that packaging cells produced according to the present invention possess sufficient rep activity to recognize, excise and amplify an integrated rAAV vector, as described in Example 11. DETAILED DESCRIPTION OF THE INVENTION
  • AAV vectors are recombinant constructs of the AAV virus comprising AAV components necessary for replication and encapsidation, along with a heterologous polynucleotide encoding a protein of interest. These recombinant AAV vectors are potentially powerful tools for human gene therapy, particularly for diseases such as cystic fibrosis and sickle cell anemia.
  • a major advantage of AAV vectors over other approaches to gene therapy is that they do not require ongoing replication of the target cell in order to integrate permanently into the cell's genome.
  • the invention described herein provides methods and materials for use in the production of high titers of recombinant AAV vectors for use in gene therapy. It also provides methods and materials for determining the relative infectious titer of rAAV preparations.
  • polypeptide polypeptide
  • peptide protein
  • proteins that are post- translationally modified through reactions that include glycosylation, acetylation and phosphorylation.
  • Polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers only to the primary structure of the molecule. Thus, double- and single-stranded DNA, as well as double- and single- stranded RNA are included. It also includes modified polynucleotides such as methylated or capped polynucleotides.
  • Recombinant as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • Sequence overlap occurs when the nucleotides share a homologous sequence of sufficient length and identity that recombination is facilitated.
  • the level of homology and corresponding frequency of recombination increase with increasing length of the homologous sequences and with their level of shared identity.
  • the level of homology that will pose a concern in a given system can be determined theoretically and confirmed experimentally, as is known in the art.
  • recombination can be substantially reduced or eliminated if the overlapping sequence is less than about a 25 nucleotide sequence if it is at least 80% identical over its entire length, or less than about a 50 nucleotide sequence if it is at least 70% identical over its entire length.
  • a "vector” refers to a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.
  • the polynucleotide to be delivered sometimes referred to as a "target polynucleotide” may comprise a coding sequence of interest in gene therapy.
  • a “recombinant AAV vector” refers to vector comprising one or more polynucleotides of interest tha are flanked by AAV inverted terminal repeat sequences (ITRs) .
  • ITRs AAV inverted terminal repeat sequences
  • Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus and is expressing the AAV rep and cap genes.
  • AAV "rep” and “cap” genes (encoding replication and encapsidation proteins, respectively) have been found in all AAV serotypes examined, and are described above and in the references cited therein. Typically, the rep and cap genes are found adjacent to each other in the AAV genome, and they are generally conserved among AAV serotypes.
  • helper virus for AAV refers to a second virus that allows wild-type AAV (which is a "defective" parvovirus) to b replicated and packaged by a host cell.
  • helper viruses include adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • Packaging refers to a series of subcellular events that results in the assembly and ' encapsidation of an rAAV vector. Thus, when a suitable vector plasmid is introduced into a packaging cell line under appropriate conditions, it will be assembled into a vector viral particle.
  • Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide) .
  • a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter.
  • Promoter refers to a geno ic region that enhances the transcription of a gene or coding sequence to which it is operably linked.
  • operably linked refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter promotes transcription of the coding sequence.
  • An operably linked promoter is usually in cis configuration with the coding sequence, but is not necessarily contiguous with it.
  • “Host cells”, “cell lines”, “cell cultures”, and other such terms denote higher eukaryotic cells, most preferably mammalian cells, which can be used as recipients for recombinant vectors or other transfer polynucleotides, and include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.
  • “Stable integration" of a polynucleotide into a cell means that the polynucleotide has been introduced into a chromosome or mini-chromosome of the cell and, therefore, becomes a relatively permanent part of the cellular genome.
  • “episomes” such as plasmids can sometimes be maintained for many generations (particularly if kept under selective pressure)
  • genetic material carried episomally is generally more susceptible to loss than chromosomally- integrated material.
  • chromatin structure of eukaryotic chromosomes can influence the level of expression of an integrated polynucleotide; and we believe that such effects can sometimes prove beneficial in situations such as those described herein (in which the level of expression of the AAV rep gene can have negative effects upon cellular metabolism) .
  • the selection of stable cell lines having properties that are particularly desirable in the context of the present invention, are described in the Detailed Description and Examples below. 6/17947 PC17US95/15892
  • Efficiency when used in describing a cell line refers to the useful properties of the line; in particular, the growth rate, and (for packaging cell lines) the number of virus particles produced per cell. "High efficiency packaging” indicates production of at least 100 viral particles per cell.
  • the method for producing high titers of recombinant•AAV vectors comprises several steps.
  • the general strategy involves preparation of mammalian packaging cell lines that comprise a stably integrated AAV cap gene operably linked to a promoter, and a stably integrated AAV rep gene operably linked to a heterologous promoter.
  • Packaging cells are then infected or transfected with a plasmid comprising the AAV ITR regions and the target polynucleotide.
  • suitable conditions including suitable growth conditions and infection with a competent helper virus
  • expression of the rep and cap genes of the packaging cell results in the synthesis of rep and cap proteins which mediate replication and encapsidation of the
  • AAV vector Providing a polynucleotide of interest (also referred to as a "target polynucleotide”) in- between the AAV ITR sequences of the rAAV vector, thus results in packaging of the target polynucleotide into an infectious rAAV particle which can be used to deliver the polynucleotide to a desired host cell.
  • a polynucleotide of interest also referred to as a "target polynucleotide”
  • the proportion of wild-type AAV i.e., particles not containing the target polynucleotide
  • the presence of contaminating wild-type AAV limits the therapeutic potential of rAAV vector preparations.
  • the p5 promoter region is replaced with a different promoter.
  • the packaging cell lines of the present invention enable the efficient production of rAAV preparations that are of high titer and are substantially free of any contaminating wild- type AAV; attributes that are especially useful in the context of AAV-mediated gene therapy.
  • the degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to ITRs.
  • the similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.
  • the parental lines from which packaging cells are generated may be obtained from any cell line that is susceptible to AAV infection, and amenable to culture in vitro.
  • AAV has a very broad host range and has been isolated from a variety of mammalian cell types, including simian, human and rodent cells.
  • human cell lines in which appropriate helper functions can be expressed are typically preferred.
  • Such human cell lines from which the packaging cell lines may be derived include, for example, Hela, A549, 293, KB, Detroit, and WI38 cells. We initially selected both Hela cells and A549 cells for demonstrations of the present invention. As described in the Examples below, we were readily able to generate packaging cells from both parental lines tested.
  • the rep gene is under regulation of the p5 promoter, which is itself strongly down-regulated by rep expression.
  • the cells are provided with a stably integrated AAV cap gene operably linked to a promoter, and a stably integrated AAV rep gene operably linked to a heterologous promoter; as described and illustrated herein. Any heterologous promoter that is not strongly down-regulated by rep gene expression is suitable; but inducible promoters are preferred because constitutive expression of the rep gene can have a negative impact on the host cell.
  • inducible promoters are known in the art; including, by way of illustration, heavy metal ion inducible promoters (such as metallothionein promoters) ; steroid hormone inducible promoters (such as the MMTV promoter or growth hormone promoters) ; and promoters such as those from T7 phage which are active in the presence of T7 RNA polymerase.
  • inducible promoters are those that are induced by the helper virus that is used to complement the replication and packaging of the rAAV vector.
  • helper-virus-inducible promoters include for example, the adenovirus early gene promoter which is inducible by adenovirus E1A protein; the adenovirus major late promoter; the herpesvirus promoter which is inducible by herpesvirus proteins such as VP16 or 1CP ; as well as vaccinia or poxvirus inducible promoters.
  • the Examples below illustrate a generally applicable method that can be used to test putative promoters to readily determine whether or not they are helper-virus-inducible and whether or not they will be useful in the generation of high efficiency packaging cells.
  • the method involves replacing the p5 promoter of the AAV rep gene with the putative helper-virus-inducible promoter (either known in the art or identified using well-known techniques such as linkage to promoter-less "reporter" genes) .
  • the AAV rep-cap genes (with p5 replaced) , preferably linked to a positive selectable marker such as an antibiotic resistance gene, are then stably integrated into a suitable host cell (such as the Hela or A549 cells exemplified below) .
  • Cells that are able to grow relatively well under selection conditions are then tested for their ability to express the rep and cap genes upon addition of a helper virus.
  • As an initial test for rep and/or cap expression cells can be readily screened using immunofluorescence to detect rep and/or cap proteins (as illustrated in the Examples below) . Confirmation of packaging capabilities and efficiencies can then be determined by functional tests for replication and packaging of incoming rAAV vectors (also illustrated below) .
  • the p5 promoter with a helper-virus-inducible promoter derived from the mouse etallothionein gene, and used the resulting constructs to generate packaging cell lines capable of producing high titers of rAAV particles.
  • the AAV cap gene is also stably integrated into the packaging cell line.
  • the rep and cap genes are introduced into the parental line together, by using a plasmid that contains them both (essentially as they are arranged in the AAV genome, except for replacement of the sequences upstream of rep, i.e. the p5 promoter region) .
  • plasmid designated pMt-rep/cap//pKO-neo shown in Figure 1 .
  • the plasmid contains a heterologous promoter linked to a region containing the rep-cap genes. The rest of the rep-cap region, including the pl9 promoter and the p40 promoter are retained.
  • the plasmid also contains an AAV polyadenylation signal.
  • the components of native AAV that are not present in the plasmid include the p5 promoter region (which has been substituted by the heterologous promoter) and the ITRs (which are present in the vector plasmid to be introduced separately) .
  • Cells transfected with rep and cap genes as described above are then selected from untransfected cells according to methods that are routine in the art. Most conveniently, selection is accomplished by linking the rep and cap genes to one or more selectable markers (such as antibiotic resistance genes) .
  • selectable markers such as antibiotic resistance genes
  • the neo-resistance gene was included next to the rep-cap sequences.
  • selectable markers are driven by constitutive promoters; and preferably, such markers are introduced in an opposite orientation relative to the AAV rep-cap genes since that tends to reduce the potential for the promoter driving the selectable marker to effect expression of the rep gene (which can be detrimental to the host cell) .
  • the cell lines are exposed to the antibiotic for which resistance has been provided (geneticin was used in the case of the constructs referred to above) .
  • the selectable marker is included on the same plasmid as the rep-cap sequences; and both are stably integrated into the host genome.
  • the plasmid Mt-rep/cap//pKOneo for example, geneticin- resistant cells would be expected to possess an integrated copy of the neo gene as well as pMt-rep/cap. Since the rep sequences cannot readily be lost in our system, the prior art would predict that the recipient cells would exhibit reduced growth rates.
  • our constructs were introduced into exemplary mammalian host cells (Hela and A549) , the rate of proliferation of the geneticin-resistant clones was not significantly affected in either of the cell lines.
  • packaging cells that are capable of replicating at least one half as rapidly as the parental cells, and capable of producing more than 100 rAAV particles/cell.
  • the cells grow at least two-thirds as rapidly as the parental line, and produce more than 250 rAAV particles/cell.
  • packaging cells that replicate substantially as rapidly as the parent cells (at least about 80% of the rate), and that produce more ' than about 500 rAAV particles per cell.
  • the packaging cell line is supplied with a recombinant AAV vector comprising AAV inverted terminal repeat (ITR) regions surrounding one or more polynucleotides of interest (or "target" polynucleotides) .
  • ITR inverted terminal repeat
  • the target polynucleotide is operably linked to a promoter, either its own or a heterologous promoter.
  • a promoter either its own or a heterologous promoter.
  • suitable promoters are known in the art, the choice of which depends on the desired level of expression of the target polynucleotide; whether one wants constitutive expression, inducible expression, cell-specific or tissue-specific expression, etc.
  • the rAAV vector will also contain a positive selectable marker in order to allow for selection of cells that have been infected by the rAAV vector.
  • Negative selectable markers can also be included; as a means of selecting against those same cells should that become necessary or desirable.
  • those constructs involve direct translational fusions between a dominant positive selectable marker a negative selectable marker.
  • Preferred positive selectable markers are derived from genes selected from the group consisting of hph, neo, and gpt
  • preferred negative selectable markers are derived from genes selected from the group consisting of cytosine dea inase, HSV-I TK, VZV TK, HPRT, APRT and gpt
  • Especially preferred markers are bifunctional selectable fusion genes wherein the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene.
  • CFTR operably linked to a promoter.
  • CFTR polypeptides that are capable of reconstructing CFTR functional deficiencies in cells derived from cystic fibrosis patients.
  • Rich et al. (1991, Science, 253: 205-207) described a CFTR derivative missing amino acid residues 708-835, that was capable of transporting chloride and capable of correcting a naturally occurring CFTR defect.
  • Egan et al. (1993) described another CFTR derivative (comprising about 25 amino acids from an unrelated protein followed by the sequence of native CFTR beginning at residue 119) that was also capable of restoring electrophysiological characteristics of normal CFTR.
  • polynucleotides include, but are not limited to: (i) polynucleotides encoding proteins useful in other forms of gene therapy to relieve deficiencies caused by missing, defective or sub-optimal levels of a structural protein or enzyme; (ii) polynucleotides that are transcribed into anti-sense molecules; (iii) polynucleotides that are transcribed into decoys that bind transcription or translation factors; (iv) polynucleotides that encode cellular modulators such as cytokines; (v) polynucleotides that can make recipient cells susceptible to specific drugs, such as the herpes virus thymidine kinase gene; and (vi) polynucleotides for cancer therapy, such as the wild-type p53 tumor suppressor cDNA for replacement of the missing or damaged p53 gene associated with some lung and breast cancer
  • the same packaging cell line can be used for any of these applications.
  • the plasmid comprising the specific target polynucleotide is introduced into the packaging cell for production of the AAV vector by one of several possible methods; including, for example, electroporation.
  • Helper virus can be introduced before, during or after introduction of the rAAV vector. As illustrated in Example 10, the plasmid can be co-infected into the culture along with the helper virus.
  • the cells are then cultured for a suitable period, typically 2-5 days, in conditions suitable for replication and packaging as known in the art (see references above and examples below) . Lysates are prepared, and the recombinant AAV vector particles are purified by techniques known in the art.
  • the recombinant AAV vector is itself stably integrated into a clone of the packaging cell line.
  • a stable, vector-containing packaging line can be grown and stored until ready for use.
  • the user simply infects the cells with helper virus and cultures the cells under conditions suitable for replication and packaging of AAV (as described below) .
  • the amount of helper virus and the incubation time influence the amount of rep activity, they can be readily optimized and kept constant, as illustrated below.
  • To conduct the assay aliquots of the packaging cell line are introduced with a standard amount of helper virus and serial dilutions of the rAAV preparation to be tested.
  • the relative infectious titer of the AAV is indicated by the amount of replicated AAV present in each aliquot after suitable incubation; and can be compared to a preparation of known titer.
  • the examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.
  • Example l Construction of a plasmid encoding the rep-cap sequences operably linked to a heterologous promoter
  • a plasmid containing the wild type rep and cap genes from deoxyribonucleotide 311 to 4493 of the AAV genome
  • mMt-I mouse metallothionein I
  • This construction effectively removes both ITR's and substitutes the mMt-I promoter for the p5 promoter while maintaining all of the AAV reading frames, the pl9 and p40 promoters and the polyadenylation signal.
  • pKOneo contains the neo gene ⁇ providing resistance to neomycin and gentamicin) under control of the SV40 early promoter; as well as SV40 small t intron and SV40 polyadenylation signal oriented in the opposite transcriptional direction relative to pMt-rep/cap (Ito et al. 1994 Cancer Lett. 76:33-39).
  • pMt-rep/cap//pKO-neo The resulting plasmid, designated pMt-rep/cap//pKO-neo, is shown in Figure 1.
  • Example 2 Integration of the rep-cap genes into mammalian cell lines
  • DMEM Dulbecco's modified Eagle's medium
  • the cells were plated at low density in the presence of l mg/ml active component geneticin (Gibco-BRL) . Individual colonies were ring cloned, expanded and maintained in l mg/ml geneticin.
  • Gabco-BRL active component geneticin
  • the selectable marker is included on the same plasmid as the rep-cap sequences; and both are stably integrated into the host genome.
  • the plasmid Mt-rep/cap//pKOneo for example, geneticin- resistant cells would be expected to possess an integrated copy of the neo gene as well as pMt-rep/cap. Since the rep sequences cannot readily be lost in our system, the prior art would predict that the recipient cells would exhibit reduced growth rates.
  • PBS phosphate buffered saline
  • the cells were then washed three additional times with PBS and incubated overnight with "WT" medium (1% nonfat dry milk, 0.5 mg/ml bovine serum albumin, 150 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% Tween 20 and 1 mM NaN 3 ) .
  • WT "WT” medium
  • Anti-rep antibody (rabbit anti-Rep78.93; Trempe et al. 1987 Virology 161:18-28) was diluted 1:250 in WT and 100 ⁇ l added to each well for 1 hour at room temperature (RT) .
  • the cells were washed five times with WT and then incubated with 100 ⁇ l of a 1:100 dilution of anti-rabbit IgG FITC conjugate secondary antibody (Sigma Chemical Corp.) in the dark for 1 hour at RT.
  • the cells were then washed three times with WT and two times with PBS in the dark and examined with an Axioskop H fluorescence microscope (Zeiss, Germany) .
  • rep protein was detectable in a number of the cells examined (8 out of 23 A549 clones and 3 out of 28 Hela clones) .
  • the addition of heavy metals did not significantly affect the observed rep expression under any conditions.
  • helper-virus-inducible promoter is a general one that can be readily applied to any promoter of potential interest by simply swapping it into rep constructs and screening for colonies as we describe herein.
  • exemplary clones (of Hela and A549 origin) were tested for their ability to replicate recombinant AAV genomes after infection, as described below.
  • Replication activity of IF+ cells We examined whether the pMt-rep/cap//pKO-neo transfected cell lines exhibited functional replication activity.
  • MOI 25 pfu/cell
  • the culture medium from each well was removed to a labeled tube and any cells still attached to the culture dish were trypsinized and pooled with cells present in the media.
  • the cell suspension was centrifuged at 3000 rpm for 5 min. , after which the supernatant was removed and total nucleic acid was prepared from the cell pellet (according to Ausubel et al. (ed.) 1987 Current Protocols in Molecular Biology Greene Publishing Associates, Brooklyn, N.Y.) .
  • Negative controls for the experiment included the incubation of 1.2 x 10 8 AAVCFTR particles on either cell line without adenovirus. Fifteen micrograms of nucleic acid for each sample, as well as untreated Hela clone 37 DNA +/- 20 pg of AAVCFTR plasmid (positive control for Southern) , was digested with EcoRI, subjected to gel electrophoresis, transferred to nitrocellulose and probed with a 1.488 kb EcoRI fragment from within the CFTR cDNA. Lanes 15-18 (Fig.
  • a hybridization signal migrating at 1.488 kb is present in DNA isolated from both the Hela clone 37 and A549 clone 20 cell lines after infection by AAVCFTR virus and adenovirus (Fig. 2, lanes 1, 2 and 8).
  • Example 5 rAAV infectious titer assays Additional rep activity assays were performed in order to determine whether there was a linear relationship between incoming AAVCFTR virus and replicated AAVCFTR sequences (which could be exploited as the basis of an rAAV infectious titer assay) .
  • Three log dilutions from 1.2 x 10 9 to 1.2 x 10 7 AAVCFTR particles, as determined by slot blot hybridization, were cultured in 2.5 ml media on 2.5 x 10 5 Hela clone 37 cells plus adenovirus (MOI 25 pfu/cell) for 48 hours in a 6 well culture dish.
  • MOI 25 pfu/cell
  • packaging cells produced according to the present invention are capable of replicating an AAV genome in the presence of adenovirus, and that this activity can be used to quantify the number of infectious viral particles present in a given sample.
  • the particle number was determined by slot blot hybridization of the AAVCFTR virus preparation and may reflect the contribution of infectious and defective AAVCFTR particles; whereas the infectious assay described above should only detect infectious particles.
  • AAV recombinant AAV
  • Current methods for the production of recombinant AAV (rAAV) virus include the transient transfection of plasmid vectors containing the rAAV sequences. One or more steps are undertaken to remove the plasmid DNA from a rAAV preparation.
  • AAVCFTR plasmid DNA was incubated directly onto packaging cells (Hela clone 37) +/- adenovirus to determine whether the above-described infection assay would detect non-viral DNA.
  • AAVCFTR plasmid (10 ⁇ g) was electroporated as previously described into 4 x 10 6 Hela clone 37 cells and then transferred to a 100 mm culture dish.
  • FIG. 4 shows the hybridization pattern of the endogenous CFTR gene and the migration of CFTR cDNA (20 pg) spiked into human genomic DNA when digested with EcoRI and probed with the 1.488 kb CFTR cDNA fragment. Electroporation of the AAVCFTR plasmid into Hela clone 37 cells resulted in a signal migrating at 1.488 k (Fig.4, lane 1) and represents the amount of AAVCFTR plasmid present in these cells 24 hours after transfection.
  • Lanes 5-8 show the results of incubating 1 ⁇ g, 100 ng, 10 ng and 1 ng AAVCFTR plasmid, respectively, on Hela clone 37 cell in the presence of adenovirus.
  • Example 8 Adenovirus titration of rAAV infectious titer assay
  • MOI 2.5 ml culture media +/- adenovirus
  • a slight signal migrating at 1.488 kb can be detected in DNA isolated from Hela clone 37 cells incubated with supernatant derived from the minus adenovirus control and reflects a small amount of contaminating input AAVCFTR plasmid from the electroporation (Fig. 7, lane 1).
  • Supernatant derived from a duplicate well cultured with adenovirus and titered on Hela clone 37 cells revealed significantly more hybridization migrating at 1.488 kb relative to control conditions (Fig. 7, compare lanes 3 and 1) .
  • Example 11 Rescue and amplification of an integrated rAAV 'vector from packaging cells
  • Packaging cells derived from Hela clone 37
  • a recombinant AAV vector designated rAAV-CMV-Hygro
  • hygromycin resistance gene operably linked to the CMV enhancer/promoter
  • a stable, polyclonal line was derived by selection in 300 ⁇ g/ml hygromycin.
  • the polyclonal, hygro-resistant Hela clone 37 line (2.5 x 10 5 cells/well) was seeded onto a 6 well dish for infection with adenovirus
  • Lane 1 represents DNA isolated from the parental Hela clone 37 cell line, and hence does not contain the hygro-resistance gene.
  • Lane 2 contains DNA from the polyclonal, hygro-resistant Hela clone 37 line which at this exposure time does not show the presence of the resistance gene which is present at an average of about 1 copy/well (data not shown) .
  • DNA isolated from a duplicate well containing the hygro-resistant Hela clone 37 cells treated with adenovirus was run in lane 3.
  • the hybridization present at 1048 bp represents material derived from the excision and amplification of the integrated AAVCMVHygro vector integrated in the Hela clone cells.
  • the addition of wild-type AAV to the adenovirus infection gave similar results (lane 4) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Pulmonology (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Data Exchanges In Wide-Area Networks (AREA)

Abstract

Cette invention concerne les vecteurs AAV (virus associé à un adénovirus), lesquels peuvent être utiles en thérapie génétique. Jusqu'à présent, toutefois, l'obstacle principal a été l'incapacité de produire de tels vecteurs recombinants en quantité suffisante pour qu'ils soient utiles, d'un point de vue clinique, lors d'applications de la thérapie génétique chez l'homme. Les lignées cellulaires stables d'encapsidation d'AAV étaient difficiles à isoler, notamment à cause de l'activité de la protéine rep, qui, par la rétro-régulation de sa propre expression, peut avoir des effets négatifs sur la cellule hôte. Cette invention propose des systèmes d'encapsidation, ainsi que des procédés d'encapsidation de vecteurs AAV, permettant d'éviter ces problèmes et d'accroître sensiblement l'efficacité d'encapsidation.
EP95944069A 1994-12-06 1995-12-06 Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants Withdrawn EP0796339A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US35021994A 1994-12-06 1994-12-06
US350219 1994-12-06
US48057595A 1995-06-07 1995-06-07
US480575 1995-06-07
PCT/US1995/015892 WO1996017947A1 (fr) 1994-12-06 1995-12-06 Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants

Publications (1)

Publication Number Publication Date
EP0796339A1 true EP0796339A1 (fr) 1997-09-24

Family

ID=26996529

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95944069A Withdrawn EP0796339A1 (fr) 1994-12-06 1995-12-06 Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants

Country Status (5)

Country Link
EP (1) EP0796339A1 (fr)
JP (1) JPH10511264A (fr)
AU (1) AU707866B2 (fr)
CA (1) CA2207927A1 (fr)
WO (1) WO1996017947A1 (fr)

Families Citing this family (277)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6326356B1 (en) 1996-10-18 2001-12-04 Board Of Regents, The University Of Texas System Suppression of neu overexpression using a mini-E1A gene
US6924128B2 (en) 1994-12-06 2005-08-02 Targeted Genetics Corporation Packaging cell lines for generation of high titers of recombinant AAV vectors
US5843742A (en) * 1994-12-16 1998-12-01 Avigen Incorporated Adeno-associated derived vector systems for gene delivery and integration into target cells
US6306830B1 (en) 1996-09-05 2001-10-23 The Regents Of The University Of California Gene therapy for congestive heart failure
US6752987B1 (en) 1995-02-28 2004-06-22 The Regents Of The University Of California Adenovirus encoding human adenylylcyclase (AC) VI
US20020103147A1 (en) 1997-09-05 2002-08-01 Hammond H. Kirk Gene therapy for congestive heart failure
CN1136920C (zh) 1995-02-28 2004-02-04 加利福尼亚大学董事会 基因转移介导的血管形成疗法
US6294370B1 (en) 1997-06-24 2001-09-25 Medigene Ag System for the production of AAV vectors
DE19625188A1 (de) * 1996-06-24 1998-01-08 Medigene Gmbh System zur Herstellung von AAV-Vektoren
US6541012B2 (en) 1996-06-24 2003-04-01 Christoph Bogedain System for the production of AAV vectors
IL128780A0 (en) * 1996-09-06 2000-01-31 Univ Pennsylvania An inducible method for production of recombinant adeno-associated viruses utilizing T7 polymerase
US5866552A (en) * 1996-09-06 1999-02-02 The Trustees Of The University Of Pennsylvania Method for expressing a gene in the absence of an immune response
WO1998010086A1 (fr) * 1996-09-06 1998-03-12 Trustees Of The University Of Pennsylvania Procede d'utilisation de cre-lox pour la production de virus adeno-associes de recombinaison
JP2001506133A (ja) 1996-12-18 2001-05-15 ターゲティッド ジェネティクス コーポレイション 組換えａａｖベクターの産生における使用のための、ａａｖスプリット−パッケージング遺伝子およびこのような遺伝子を含む細胞株
AU5603998A (en) * 1996-12-18 1998-07-15 Targeted Genetics Corporation Recombinase-activatable AAV packaging cassettes for use in the production of AV vectors
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
JP2001514845A (ja) 1997-09-05 2001-09-18 ターゲティッド ジェネティクス コーポレイション 組換えａａｖベクターの高力価ヘルパーなし調製物を生成するための方法
US6995006B2 (en) 1997-09-05 2006-02-07 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
CA2304168A1 (fr) 1997-09-19 1999-04-01 The Trustees Of The University Of Pennsylvania Procedes et lignee cellulaire utiles pour la production de virus adeno-associes recombines
AU9319198A (en) 1997-09-19 1999-04-05 Trustees Of The University Of Pennsylvania, The Methods and vector constructs useful for production of recombinant aav
US6346415B1 (en) 1997-10-21 2002-02-12 Targeted Genetics Corporation Transcriptionally-activated AAV inverted terminal repeats (ITRS) for use with recombinant AAV vectors
US6642051B1 (en) 1997-10-21 2003-11-04 Targeted Genetics Corporation Amplifiable adeno-associated virus(AAV) packaging cassettes for the production of recombinant AAV vectors
IT1297074B1 (it) * 1997-11-21 1999-08-03 Angeletti P Ist Richerche Bio Forme ormone-dipendenti delle proteine rep del virus adeno-associato (aav-2), sequenze di dna codificanti per esse, vettori che le
GB9811172D0 (en) * 1998-05-22 1998-07-22 Royal Free Hosp School Med Virus production
EP1930418B1 (fr) 1998-09-04 2015-04-08 Genzyme Corporation Procédés pour produire des préparations de vecteurs AAV recombinants de forte teneur dépourvues de virus assistants
DE19905501B4 (de) 1999-02-10 2005-05-19 MediGene AG, Gesellschaft für molekularbiologische Kardiologie und Onkologie Verfahren zur Herstellung eines rekombinanten Adeno-assoziierten Virus, geeignete Mittel hierzu sowie Verwendung zur Herstellung eines Arzneimittels
US6893865B1 (en) 1999-04-28 2005-05-17 Targeted Genetics Corporation Methods, compositions, and cells for encapsidating recombinant vectors in AAV particles
US6793926B1 (en) 1999-05-27 2004-09-21 Genovo, Inc. Methods for production of a recombinant adeno-associated virus
WO2000073481A1 (fr) 1999-05-28 2000-12-07 Targeted Genetics Corporation Techniques et compositions permettant d'abaisser le niveau de facteur de necrose tumorale (tnf) dans les troubles associes au tnf
EP1939300A1 (fr) 1999-05-28 2008-07-02 Targeted Genetics Corporation Procédés et compositions pour diminuer le niveau de facteur de nécrose de tumeur (TNF) pour des troubles associés au TNF
EP1916258B1 (fr) 1999-08-09 2014-04-23 Targeted Genetics Corporation Améliorations de l'expression d'une séquence de nucléotides hétérologues à brin unique à partir de vecteurs viraux recombinants par la désignation de la séquence de manière à ce qu'elle forme des paires de bases intrabrins
EP2369002A1 (fr) 1999-08-09 2011-09-28 Targeted Genetics Corporation Améliorations de l'expression d'une séquence de nucléotides hétérologues à brin unique à partir de vecteurs viraux recombinants par la désignation de la séquence de manière à ce qu'elle forme des paires de bases intrabrins
US7115391B1 (en) 1999-10-01 2006-10-03 Genovo, Inc. Production of recombinant AAV using adenovirus comprising AAV rep/cap genes
EP1572893B1 (fr) 2001-11-09 2009-01-07 THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by THE SECRETARY of the DEPARTMENT OF HEALTH AND HUMAN SERVICES Production d'un virus adeno-associe dans des cellules d'insectes
US6723551B2 (en) 2001-11-09 2004-04-20 The United States Of America As Represented By The Department Of Health And Human Services Production of adeno-associated virus in insect cells
EP1587923B1 (fr) 2003-01-22 2011-08-24 Duke University Constructions ameliorees permettant d'exprimer des polypeptides lysosomiaux
CA2555675A1 (fr) 2004-02-10 2005-08-25 Trustees Of Dartmouth College Compositions de nicotinamide riboside kinase et procedes d'utilisation
DK1945779T3 (da) 2005-10-20 2013-06-03 Uniqure Ip Bv Forbedrede AAV vektorer frembragt i insektceller
US8512981B2 (en) 2006-06-21 2013-08-20 Amsterdam Molecular Therapeutics B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
EP2061891B1 (fr) 2006-08-24 2012-04-11 Virovek, Inc. Expression dans des cellules d'insecte de gènes ayant des cadres ouverts de lecture chevauchants, procédés et compositions pour cela
WO2008067480A2 (fr) 2006-11-29 2008-06-05 Nationwide Children's Hospital Inhibition de la myostatine destinée à améliorer le muscle et/ou à améliorer la fonction musculaire
US9415121B2 (en) 2008-12-19 2016-08-16 Nationwide Children's Hospital Delivery of MECP2 polynucleotide using recombinant AAV9
US11219696B2 (en) 2008-12-19 2022-01-11 Nationwide Children's Hospital Delivery of polynucleotides using recombinant AAV9
CN102448501A (zh) 2009-03-27 2012-05-09 西马生物医学计划公司 治疗肝硬化和肝纤维化的方法和组合物
SG10202109219SA (en) 2009-05-02 2021-10-28 Genzyme Corp Gene therapy for neurodegenerative disorders
US20120225933A1 (en) 2009-11-05 2012-09-06 Gonzalez Aseguinolaza Gloria Regulated expression systems
US20140114279A1 (en) 2010-11-23 2014-04-24 Richard Klinghoffer Modulation neural pathways
US20140155469A1 (en) 2011-04-19 2014-06-05 The Research Foundation Of State University Of New York Adeno-associated-virus rep sequences, vectors and viruses
US10196636B2 (en) 2011-04-21 2019-02-05 Nationwide Children's Hospital, Inc. Recombinant virus products and methods for inhibition of expression of myotilin
WO2012145597A2 (fr) 2011-04-21 2012-10-26 Nationwide Children's Hospital, Inc. Produits de virus recombinant et procédés pour l'inhibition de l'expression de la myotiline
US20130039888A1 (en) 2011-06-08 2013-02-14 Nationwide Children's Hospital Inc. Products and methods for delivery of polynucleotides by adeno-associated virus for lysosomal storage disorders
AU2012286994B2 (en) 2011-07-25 2017-08-10 Nationwide Children's Hospital, Inc. Recombinant virus products and methods for inhibition of expression of DUX4
US9434928B2 (en) 2011-11-23 2016-09-06 Nationwide Children's Hospital, Inc. Recombinant adeno-associated virus delivery of alpha-sarcoglycan polynucleotides
US9163259B2 (en) 2012-05-04 2015-10-20 Novartis Ag Viral vectors for the treatment of retinal dystrophy
CA2880653C (fr) 2012-08-01 2022-05-17 Nationwide Children's Hospital Administration intrathecale du virus de type 9 adeno-associe recombinant
CA2885216A1 (fr) 2012-09-17 2014-03-20 The Research Institute At Nationwide Children's Hospital Compositions et procedes de traitement de la sclerose laterale amyotrophique
ES2747833T3 (es) 2013-04-04 2020-03-11 Dartmouth College Composiciones y procedimientos para la escisión in vivo del ADN proviral del vih-1
US9828587B2 (en) 2013-04-08 2017-11-28 University Of Iowa Research Foundation Chimeric adeno-associated virus/ bocavirus parvovirus vector
AU2014253730B2 (en) 2013-04-20 2018-09-13 Research Institute At Nationwide Children's Hospital Recombinant adeno-associated virus delivery of exon 2-targeted U7snRNA polynucleotide constructs
JP2016523835A (ja) 2013-05-15 2016-08-12 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ 中枢神経系へのアデノ随伴ウイルスを介した遺伝子導入
WO2015031392A1 (fr) 2013-08-27 2015-03-05 Research Institute At Nationwide Children's Hospital Produits et méthodes de traitement de la sclérose latérale amyotrophique
CA2929669A1 (fr) 2013-11-05 2015-05-14 The Research Institute At Nationwide Children's Hospital Compositions et procedes d'inhibition de nf-kb et sod-1 afin de traiter la sclerose laterale amyotrophique
WO2015142984A1 (fr) 2014-03-18 2015-09-24 Washington University Procédés et compositions de substitution du chromophore décalée vers le rouge pour des applications optogénétiques
WO2015191508A1 (fr) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Capsides chimériques
AU2015301978C1 (en) 2014-08-09 2022-06-09 Research Institute At Nationwide Children's Hospital Methods and materials for activating an internal ribosome entry site in exon 5 of the DMD gene
WO2016057975A2 (fr) 2014-10-10 2016-04-14 Research Institute At Nationwide Children's Hospital Injections guidées pour transfert de gène par vaa au muscle
EP3215602B1 (fr) 2014-11-05 2019-12-25 The Research Institute at Nationwide Children's Hospital Procédés et matériels pour la production de virus recombinés dans des microalgues eucaryotes
RU2719192C2 (ru) 2014-11-14 2020-04-17 Вояджер Терапьютикс, Инк. Модулирующие полинуклеотиды
EP3230441A4 (fr) 2014-12-12 2018-10-03 Voyager Therapeutics, Inc. Compositions et méthodes pour la production de scaav
JP2018506530A (ja) 2015-01-30 2018-03-08 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 脊柱軟膜下遺伝子送達システム
MA41451A (fr) 2015-02-04 2017-12-12 Univ Washington Constructions anti-tau
SG11201706766WA (en) 2015-02-23 2017-09-28 Crispr Therapeutics Ag Materials and methods for treatment of hemoglobinopathies
WO2016187017A1 (fr) 2015-05-15 2016-11-24 Mcivor R Scott Virus adéno-associé pour une administration thérapeutique au système nerveux central
US10017832B2 (en) 2015-08-25 2018-07-10 Washington University Compositions and methods for site specific recombination at asymmetric sites
CA2998636A1 (fr) 2015-09-17 2017-03-23 Research Institute At Nationwide Children's Hospital Procedes et materiaux pour therapie genique par galgt2
US20190048340A1 (en) 2015-09-24 2019-02-14 Crispr Therapeutics Ag Novel family of rna-programmable endonucleases and their uses in genome editing and other applications
EP4279084A1 (fr) 2015-10-28 2023-11-22 Vertex Pharmaceuticals Inc. Matériaux et méthodes pour traiter la dystrophie musculaire de duchenne
US20180230489A1 (en) 2015-10-28 2018-08-16 Voyager Therapeutics, Inc. Regulatable expression using adeno-associated virus (aav)
MX2018005332A (es) 2015-11-06 2018-11-09 Crispr Therapeutics Ag Materiales y metodos para tratamiento de la enfermedad de almacenamiento de glucogeno tipo 1a.
EA201891192A1 (ru) 2015-11-16 2019-01-31 Рисёрч Инститьют Эт Нейшнвайд Чилдрен'С Хоспитал Средства и способы лечения миопатий, связанных с титином, и других титинопатий
JP6932698B2 (ja) 2015-12-01 2021-09-08 クリスパー・セラピューティクス・アクチェンゲゼルシャフトＣＲＩＳＰＲ Ｔｈｅｒａｐｅｕｔｉｃｓ ＡＧ アルファ１アンチトリプシン欠乏症の治療のための材料および方法
MX2018007840A (es) 2015-12-23 2019-05-02 Crispr Therapeutics Ag Materiales y metodos para el tratamiento de la esclerosis lateral amiotrofica y/o la degeneracion lobar frontotemporal.
WO2017120589A1 (fr) 2016-01-08 2017-07-13 Washington University Compositions comprenant de la chémérine et méthodes pour les utiliser
US20190038771A1 (en) 2016-02-02 2019-02-07 Crispr Therapeutics Ag Materials and methods for treatment of severe combined immunodeficiency (scid) or omenn syndrome
US11702672B2 (en) 2016-02-08 2023-07-18 University Of Iowa Research Foundation Methods to produce chimeric adeno-associated virus/bocavirus parvovirus
EP3416689B1 (fr) 2016-02-18 2023-01-18 CRISPR Therapeutics AG Matériel et procédés de traitement d'une immunodéficience combinée grave (scid) ou d'un syndrome d'omenn
US11066456B2 (en) 2016-02-25 2021-07-20 Washington University Compositions comprising TREM2 and methods of use thereof
WO2017147467A1 (fr) 2016-02-26 2017-08-31 Research Institute At Nationwide Children's Hospital Produits viraux recombinants et procédés d'induction d'un saut d'exon dux4
WO2017158422A1 (fr) 2016-03-16 2017-09-21 Crispr Therapeutics Ag Matières et méthodes pour le traitement d'hémochromatoses héréditaires
KR102489437B1 (ko) 2016-03-28 2023-01-16 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 뉴런 과흥분성 치료를 위한 방법 및 조성물
US11013774B2 (en) 2016-03-28 2021-05-25 Ultragenyx Pharmaceutical Inc. Methods of heat inactivation of adenovirus
MA44528A (fr) 2016-04-02 2019-02-06 Res Inst Nationwide Childrens Hospital Système promoteur u6 modifié pour l'expression spécifique d'un tissu
MA45477A (fr) 2016-04-15 2019-02-20 Res Inst Nationwide Childrens Hospital Administration à vecteurs de virus adéno-associé de microarn-29 et micro-dystrophine pour traiter la dystrophie musculaire
FI3442600T3 (fi) 2016-04-15 2024-05-14 Res Inst Nationwide Childrens Hospital Adeno-assosioidulla virusvektorilla b-sarkoglykaanin ja mikro-rna-29:n toimittaminen ja lihasdystrofian hoito
IL262416B2 (en) 2016-04-18 2024-02-01 Crispr Therapeutics Ag Materials and methods for the treatment of hemoglobin diseases
WO2017189964A2 (fr) 2016-04-29 2017-11-02 Voyager Therapeutics, Inc. Compositions pour le traitement de maladies
WO2017189959A1 (fr) 2016-04-29 2017-11-02 Voyager Therapeutics, Inc. Compositions pour le traitement de maladies
WO2017191503A1 (fr) 2016-05-05 2017-11-09 Crispr Therapeutics Ag Substances et méthodes pour le traitement d'hémoglobinopathies
CN109831916B (zh) 2016-05-18 2023-07-21 沃雅戈治疗公司 治疗亨廷顿氏舞蹈病的组合物和方法
CA3024448A1 (fr) 2016-05-18 2017-11-23 Voyager Therapeutics, Inc. Polynucleotides modulateurs
WO2018002762A1 (fr) 2016-06-29 2018-01-04 Crispr Therapeutics Ag Matériaux et méthodes pour traiter la sclérose latérale amyotrophique (als) et d'autres troubles associés
WO2018002783A1 (fr) 2016-06-29 2018-01-04 Crispr Therapeutics Ag Matériels et méthodes de traitement de l'ataxie de friedreich et d'autres troubles associés
EP3478829A1 (fr) 2016-06-29 2019-05-08 Crispr Therapeutics AG Matériels et méthodes de traitement de la dystrophie myotonique de type 1 (dm1) et d'autres troubles associés
JP7548696B2 (ja) 2016-07-06 2024-09-10 バーテックス ファーマシューティカルズ インコーポレイテッド 疼痛関連障害を処置するための物質及び方法
EP3481856A1 (fr) 2016-07-06 2019-05-15 Crispr Therapeutics AG Matériaux et procédés de traitement de troubles liés à la douleur
WO2018007871A1 (fr) 2016-07-08 2018-01-11 Crispr Therapeutics Ag Matériels et méthodes pour le traitement de l'amyloïdose de la transthyrétine
WO2018020323A2 (fr) 2016-07-25 2018-02-01 Crispr Therapeutics Ag Matériels et méthodes pour le traitement de troubles liés aux acides gras
US11795473B2 (en) 2016-10-14 2023-10-24 Ultragenyx Pharmaceutical Inc. Use of tonicifying agents to enhance recombinant adeno-associated virus yield
WO2018093925A1 (fr) 2016-11-15 2018-05-24 Laoharawee Kanut Procédé d'amélioration de la fonction neurologique dans la mpsi et la mpsii et d'autres troubles neurologiques
WO2018094251A1 (fr) 2016-11-17 2018-05-24 Kaspar Brian K Administration intrathécale de virus adéno-associé recombinant codant pour la protéine 2 de liaison méthyl-cpg
EP3555296A4 (fr) 2016-12-13 2020-07-29 Seattle Children's Hospital (DBA Seattle Children's Research Institute) Méthodes d'activation de médicament exogène de complexes de signalisation induits par agents chimiques exprimés dans des cellules modifiées in vitro et in vivo
WO2018132747A1 (fr) 2017-01-13 2018-07-19 University Of Iowa Research Foundation Petit arn non codant pour bocaparvovirus et ses utilisations
EP3585900B1 (fr) 2017-02-22 2022-12-21 CRISPR Therapeutics AG Matériels et méthodes de traitement d'ataxia spinocerebellaire de type 2 (sca2) et d'autres maladies ou de troubles liés au gene du protéine ataxia spinocerebellaire de type 2 (sca2)
EP3585895A1 (fr) 2017-02-22 2020-01-01 CRISPR Therapeutics AG Compositions et méthodes pour l'édition génétique
US11407997B2 (en) 2017-02-22 2022-08-09 Crispr Therapeutics Ag Materials and methods for treatment of primary hyperoxaluria type 1 (PH1) and other alanine-glyoxylate aminotransferase (AGXT) gene related conditions or disorders
EP3585898A1 (fr) 2017-02-22 2020-01-01 CRISPR Therapeutics AG Matériaux et procédés pour le traitement de l'ataxie spinocérébelleuse de type 1 (sca1) et d'autres états ou troubles liés au gène de l'ataxie spinocérébelleuse de type 1 (atxn1)
WO2018154418A1 (fr) 2017-02-22 2018-08-30 Crispr Therapeutics Ag Matériaux et procédés pour le traitement de la maladie de parkinson à début précoce (park1) et d'autres états pathologiques ou troubles associés au gène alpha (snca)
JP7162021B2 (ja) 2017-03-17 2022-10-27 ニューカッスル ユニバーシティ 筋ジストロフィーを治療するためのマイクロジストロフィン断片のアデノ随伴ウイルスベクター送達
SG11201908575SA (en) 2017-03-17 2019-10-30 Res Inst Nationwide Childrens Hospital Adeno-associated virus vector delivery of muscle specific micro-dystrophin to treat muscular dystrophy
EP3601581A4 (fr) 2017-03-22 2021-05-19 Ultragenyx Pharmaceutical Inc. Procédés de culture cellulaire faisant intervenir des inhibiteurs de hdac ou des protéines rep
AU2018261790B2 (en) 2017-05-05 2024-10-03 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
JP2020518259A (ja) 2017-05-05 2020-06-25 ボイジャー セラピューティクス インコーポレイテッドＶｏｙａｇｅｒ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． ハンチントン病治療組成物および方法
WO2019097305A2 (fr) 2017-05-12 2019-05-23 Crispr Therapeutics Ag Matériaux et procédés de génie cellulaire et leurs utilisations en immuno-oncologie
CA3068906A1 (fr) 2017-07-08 2019-01-17 Genethon Traitement de l'amyotrophie spinale
US11497576B2 (en) 2017-07-17 2022-11-15 Voyager Therapeutics, Inc. Trajectory array guide system
EP3662060A2 (fr) 2017-08-03 2020-06-10 Voyager Therapeutics, Inc. Compositions et procedes de délivrance d'aav
US20200231986A1 (en) 2017-09-29 2020-07-23 Massachusetts Eye And Ear Infirmary Production of adeno-associated viruses in insect cells
CN111479924B (zh) 2017-10-16 2024-06-14 沃雅戈治疗公司 肌萎缩性侧索硬化症(als)的治疗
US20200237799A1 (en) 2017-10-16 2020-07-30 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (als)
US20190247517A1 (en) 2017-10-17 2019-08-15 Casebia Therapeutics Limited Liability Partnership Compositions and methods for gene editing for hemophilia a
EP3697915A4 (fr) 2017-10-18 2021-12-08 Research Institute at Nationwide Children's Hospital Administration par vecteur à virus adéno-associé de micro-dystrophine spécifique de muscles pour traiter la dystrophie musculaire
SG11202003285PA (en) 2017-10-20 2020-05-28 Res Inst Nationwide Childrens Hospital Methods and materials for nt-3 gene therapy
MA50849A (fr) 2017-10-26 2020-09-02 Vertex Pharma Substances et procédés pour le traitement d'hémoglobinopathies
SG11202004406VA (en) 2017-11-08 2020-06-29 Avexis Inc Means and method for preparing viral vectors and uses of same
EP3707155A2 (fr) 2017-11-09 2020-09-16 Vertex Pharmaceuticals Incorporated Systèmes crispr/cas pour le traitement de dmd
CN111727251B (zh) 2017-11-21 2024-09-20 克里斯珀医疗股份公司 用于治疗常染色体显性色素性视网膜炎的材料和方法
CN111801417B (zh) 2017-12-14 2024-10-29 克里斯珀医疗股份公司 新的rna-可编程的内切核酸酶系统及其在基因组编辑和其他应用中的用途
JP7402163B2 (ja) 2017-12-21 2023-12-20 クリスパー セラピューティクス アーゲー ２ａ型アッシャー症候群の処置のための材料および方法
EP3728595A1 (fr) 2017-12-21 2020-10-28 CRISPR Therapeutics AG Substances et méthodes pour le traitement du syndrome d'usher de type 2a et/ou de la rétinite pigmentaire autosomique récessive (arrp) non syndromique
WO2019140330A1 (fr) 2018-01-12 2019-07-18 Casebia Therapeutics Limited Liability Partnership Compositions et méthodes pour l'édition génique par ciblage de la transferrine
US11566236B2 (en) 2018-02-05 2023-01-31 Vertex Pharmaceuticals Incorporated Materials and methods for treatment of hemoglobinopathies
WO2019150203A1 (fr) 2018-02-05 2019-08-08 Crispr Therapeutics Ag Substances et méthodes pour traiter des hémoglobinopathies
EP3752616A1 (fr) 2018-02-16 2020-12-23 CRISPR Therapeutics AG Compositions et méthodes pour l'édition génique par ciblage du fibrinogène-alpha
JP7550648B2 (ja) 2018-03-19 2024-09-13 クリスパー セラピューティクス アーゲー 新規ｒｎａプログラム可能エンドヌクレアーゼ系およびその使用
US12037707B2 (en) 2018-04-05 2024-07-16 Massachusetts Eye And Ear Infirmary Methods of making and using combinatorial barcoded nucleic acid libraries having defined variation
WO2019204668A1 (fr) 2018-04-18 2019-10-24 Casebia Therapeutics Limited Liability Partnership Compositions et procédés d'inactivation de l'apo (a) par édition génique pour le traitement d'une maladie cardiovasculaire
CN112313331A (zh) 2018-04-27 2021-02-02 沃雅戈治疗公司 用于测量aadc病毒载体效力的方法
EP3784259A4 (fr) 2018-04-27 2021-12-29 Seattle Children's Hospital (DBA Seattle Children's Research Institute) Cellules résistantes à la rapamycine
KR20210019996A (ko) 2018-05-15 2021-02-23 보이저 테라퓨틱스, 인크. 파킨슨병의 치료를 위한 조성물 및 방법
EP3793616A1 (fr) 2018-05-15 2021-03-24 Voyager Therapeutics, Inc. Compositions et procédés pour l'administration de vaa
WO2019222444A2 (fr) 2018-05-16 2019-11-21 Voyager Therapeutics, Inc. Évolution dirigée
WO2019222441A1 (fr) 2018-05-16 2019-11-21 Voyager Therapeutics, Inc. Sérotypes de vaa pour l'administration de charge utile spécifique au cerveau
CN112601557A (zh) 2018-06-08 2021-04-02 诺华股份有限公司 用于测量药物产品效力的基于细胞的测定
GB201809588D0 (en) 2018-06-12 2018-07-25 Univ Bristol Materials and methods for modulating intraocular and intracranial pressure
US12060567B2 (en) 2018-06-13 2024-08-13 Voyager Therapeutics, Inc. Engineered untranslated regions (UTR) for AAV production
AU2019290544A1 (en) 2018-06-18 2021-01-28 Research Institute At Nationwide Children's Hospital Adeno-associated virus vector delivery of muscle specific micro-dystrophin to treat muscular dystrophy
AU2019288291B2 (en) 2018-06-18 2024-09-26 Research Institute At Nationwide Children's Hospital Recombinant adeno-associated virus products and methods for treating dystroglycanopathies and laminin-deficient muscular dystrophies
WO2020006458A1 (fr) 2018-06-29 2020-01-02 Research Institute At Nationwide Children's Hospital Produits de virus adéno-associés recombinants et méthodes de traitement de la dystrophie des ceintures 2a
US20210254103A1 (en) 2018-07-02 2021-08-19 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis and disorders associated with the spinal cord
SG11202100704PA (en) 2018-07-24 2021-02-25 Voyager Therapeutics Inc Systems and methods for producing gene therapy formulations
WO2020047268A1 (fr) 2018-08-29 2020-03-05 Research Institute At Nationwide Children's Hospital Compositions et procédés pour l'inhibition de l'expression de la protéine gars mutante
US20210395776A1 (en) 2018-09-28 2021-12-23 Voyager Therapeutics, Inc. Frataxin expression constructs having engineered promoters and methods of use thereof
TW202035689A (zh) 2018-10-04 2020-10-01 美商航海家醫療公司 測量病毒載體粒子的效價及強度之方法
CA3115248A1 (fr) 2018-10-05 2020-04-09 Voyager Therapeutics, Inc. Constructions d'acides nucleiques modifies codant pour des proteines de production d'aav
WO2020077165A1 (fr) 2018-10-12 2020-04-16 Voyager Therapeutics, Inc. Compositions et procédés pour l'administration d'aav
CA3116701A1 (fr) 2018-10-15 2020-04-23 Voyager Therapeutics, Inc. Vecteurs d'expression pour la production a grande echelle de raav dans le systeme baculovirus/sf9
CA3116885A1 (fr) 2018-10-17 2020-04-23 Crispr Therapeutics Ag Compositions et methodes d'administration de transgene
US20220001028A1 (en) 2018-11-30 2022-01-06 Novartis Ag Aav viral vectors and uses thereof
CA3122319A1 (fr) 2018-12-21 2020-06-25 Genethon Cassettes d'expression pour vecteurs de therapie genique
CA3124963A1 (fr) 2018-12-31 2020-07-09 Research Institute At Nationwide Children's Hospital Extinction d'arn dux4 a l'aide d'arn ciblant crispr-cas13b
MX2021008542A (es) 2019-01-18 2021-11-12 Voyager Therapeutics Inc Métodos y sistemas para la producción de partículas de aav.
AU2020216480A1 (en) * 2019-01-31 2021-09-09 Oregon Health & Science University Methods for using transcription-dependent directed evolution of AAV capsids
MX2021009404A (es) 2019-02-04 2021-11-12 Res Inst Nationwide Childrens Hospital Administracion del virus adenoasociado de polinucleotido de cln6.
JP2022519596A (ja) 2019-02-04 2022-03-24 リサーチ・インスティチュート・アット・ネーションワイド・チルドレンズ・ホスピタル Ｃｌｎ３ポリヌクレオチドのアデノ随伴ウイルス送達
WO2020168362A1 (fr) 2019-02-15 2020-08-20 Crispr Therapeutics Ag Édition de gène pour l'hémophilie a avec une expression de facteur viii améliorée
JP2022521025A (ja) 2019-02-25 2022-04-04 ノバルティス アーゲー Ｂｉｅｔｔｉクリスタリン網膜症を治療するための組成物及び方法
JP2022520875A (ja) 2019-02-25 2022-04-01 ノバルティス アーゲー Ｂｉｅｔｔｉクリスタリン網膜症を治療するための組成物及び方法
BR112021016570A2 (pt) 2019-02-26 2021-11-03 Res Inst Nationwide Childrens Hospital Entrega de vetor de vírus adenoassociado de beta-sarcoglicano e tratamento de distrofia muscular
SG11202109741VA (en) 2019-03-12 2021-10-28 Crispr Therapeutics Ag Novel high fidelity rna-programmable endonuclease systems and uses thereof
SG11202110904PA (en) 2019-04-12 2021-10-28 Ultragenyx Pharmaceutical Inc Engineered producer cell lines and methods of making and using the same
WO2020214672A1 (fr) 2019-04-15 2020-10-22 University Of Iowa Research Foundation Procédés et compositions pour expression de transgène
EP3955969A1 (fr) 2019-04-15 2022-02-23 Sanford Research Thérapie génique pour le traitement ou la prévention d'effets visuels dans une maladie de batten
EP3962536A1 (fr) 2019-04-29 2022-03-09 Voyager Therapeutics, Inc. Système et procédé pour la production de cellules d'insectes infectées par baculovirus (ceib) dans les bioréacteurs
EP3966227A1 (fr) 2019-05-07 2022-03-16 Voyager Therapeutics, Inc. Compositions et méthodes d'augmentation vectorisée de la destruction, de l'expression et/ou de la régulation de protéines
EP3966327A1 (fr) 2019-05-08 2022-03-16 Vertex Pharmaceuticals Incorporated Systèmes de vecteurs crispr/cas en deux parties pour le traitement de dmd
KR20220009427A (ko) 2019-05-17 2022-01-24 더 리서치 인스티튜트 앳 네이션와이드 칠드런스 하스피탈 글리코시드 가수분해효소를 사용하는 망막 세포에 대한 유전자 치료 벡터의 개선된 전달
US20200407729A1 (en) 2019-06-28 2020-12-31 Crispr Therapeutics Ag Materials and methods for controlling gene editing
KR20220035937A (ko) 2019-07-25 2022-03-22 노파르티스 아게 조절 가능한 발현 시스템
US20220290182A1 (en) 2019-08-09 2022-09-15 Voyager Therapeutics, Inc. Cell culture medium for use in producing gene therapy products in bioreactors
CN114945583A (zh) 2019-08-21 2022-08-26 全国儿童医院研究所 Α-肌聚糖的腺相关病毒载体递送和肌营养不良症的治疗
EP4022070A1 (fr) 2019-08-26 2022-07-06 Voyager Therapeutics, Inc. Expression contrôlée de protéines virales
WO2021046155A1 (fr) 2019-09-03 2021-03-11 Voyager Therapeutics, Inc. Édition vectorisée d'acides nucléiques pour corriger des mutations manifestes
US20230416776A1 (en) 2019-10-08 2023-12-28 Regents Of The University Of Minnesota Crispr-mediated human genome editing with vectors
US20220403438A1 (en) 2019-10-08 2022-12-22 Trustees Of Boston College Proteins containing multiple, different unnatural amino acids and methods of making and using such proteins
EP4045092A1 (fr) 2019-10-18 2022-08-24 Research Institute at Nationwide Children's Hospital Thérapie génique ciblant des cellules cochléaires
CA3158131A1 (fr) 2019-10-18 2021-04-22 Nicolas Sebastien Wein Materiaux et procedes pour le traitement de troubles associes au gene irf2bpl
WO2021102435A1 (fr) 2019-11-22 2021-05-27 Research Institute At Nationwide Children's Hospital Matériaux et méthodes pour le traitement de troubles associés au gène cargo
IL294072A (en) 2019-12-20 2022-08-01 Res Inst Nationwide Childrens Hospital Gene therapy is efficient for targeting muscle in muscle diseases
CA3171959A1 (fr) 2020-02-18 2021-08-26 Research Institute At Nationwide Children's Hospital Ciblage d'arnmi medie par aav dans le traitement de troubles lies a l'x
EP4114449A2 (fr) 2020-03-05 2023-01-11 Neotx Therapeutics Ltd. Méthodes et compositions pour le traitement du cancer à l'aide de cellules immunitaires
KR20220155334A (ko) 2020-03-16 2022-11-22 울트라제닉스 파마수티컬 인코포레이티드 재조합 아데노-연관 바이러스 수율을 증진시키는 방법
CN115379863A (zh) 2020-04-14 2022-11-22 吉尼松公司 用于治疗酸性神经酰胺酶缺乏症的载体
BR112022020753A2 (pt) 2020-04-15 2022-12-20 Voyager Therapeutics Inc Compostos de ligação a tau
WO2021247995A2 (fr) 2020-06-04 2021-12-09 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de la douleur neuropathique
US20210393801A1 (en) 2020-06-15 2021-12-23 Research Institute At Nationwide Children's Hospital Adeno-Associated Virus Vector Delivery for Muscular Dystrophies
US20230374483A1 (en) 2020-07-08 2023-11-23 Regents Of The University Of Minnesota Modified hexosaminidase and uses thereof
WO2022018638A1 (fr) 2020-07-21 2022-01-27 Crispr Therapeutics Ag Méthodes et compositions d'édition génomique pour moduler la faah pour le traitement de troubles neurologiques
WO2022026410A2 (fr) 2020-07-27 2022-02-03 Voyager Therapeutics, Inc Compositions et méthodes pour le traitement de la maladie de niemann-pick de type c1
CN117120619A (zh) 2020-07-27 2023-11-24 沃雅戈治疗公司 用于治疗与葡萄糖神经酰胺酶β缺陷相关的神经病症的组合物和方法
EP4192514A1 (fr) 2020-08-06 2023-06-14 Voyager Therapeutics, Inc. Milieu de culture cellulaire destiné à être utilisé dans la production de produits de thérapie génique dans des bioréacteurs
US20230414787A1 (en) 2020-08-27 2023-12-28 University Of Iowa Research Foundation Gene knock-out for treatment of glaucoma
TW202227634A (zh) 2020-09-08 2022-07-16 美商薩羅塔治療公司 表現γ—肌聚醣之腺相關病毒載體之全身性遞送及肌肉失養症之治療
US20230357795A1 (en) 2020-09-15 2023-11-09 Research Institute At Nationwide Children's Hospital Aav-mediated homology-independent targeted integration gene editing for correction of diverse dmd mutations in patients with muscular dystrophy
US20240261434A1 (en) 2020-09-28 2024-08-08 Research Institute At Nationwide Children's Hospital Products and methods for treating muscular dystrophy
EP4222264A1 (fr) 2020-09-30 2023-08-09 CRISPR Therapeutics AG Matériaux et méthodes de traitement de la sclérose latérale amyotrophique
IL302128A (en) 2020-11-02 2023-06-01 Biomarin Pharm Inc A process for the enrichment of adeno-associated virus
JP2023551279A (ja) 2020-11-30 2023-12-07 リサーチ インスティチュート アット ネイションワイド チルドレンズ ホスピタル 顔面肩甲上腕筋ジストロフィー（ｆｓｈｄ）を治療するための組成物及び方法
EP4263829A1 (fr) 2020-12-17 2023-10-25 Vertex Pharmaceuticals Incorporated Compositions et procédés pour l'édition de bêta-globine pour le traitement d'hémoglobinopathies
WO2022164860A1 (fr) 2021-01-27 2022-08-04 Research Institute At Nationwide Children's Hospital Matériels et méthodes pour le traitement d'un déficit en lipase acide lysosomale (dlal)
US20240093191A1 (en) 2021-02-03 2024-03-21 Research Institute At Nationwide Children's Hospital Compositions and methods for treating disease associated with dux4 overexpression
WO2022170082A1 (fr) 2021-02-05 2022-08-11 Regents Of The University Of Minnesota Méthodes de prévention de défauts cardiaques ou squelettiques dans des maladies comprenant des mucopolysaccharidoses
WO2022170038A1 (fr) 2021-02-05 2022-08-11 Amicus Therapeutics, Inc. Administration de virus adéno-associé de polynucléotide cln3
CA3212108A1 (fr) 2021-03-04 2022-09-09 Research Institute At Nationwide Children's Hospital Produits et procedes de traitement de myopathies a base de dystrophine utilisant crispr-cas9 pour corriger les duplications d'exon dmd
CN115427561B (zh) 2021-03-09 2024-06-04 辉大(上海)生物科技有限公司 工程化CRISPR/Cas13系统及其用途
AU2022256458A1 (en) 2021-04-13 2023-11-16 Research Institute At Nationwide Children's Hospital Recombinant adeno-associated virus encoding methyl-cpg binding protein 2 for treating pitt hopkins syndrome via intrathecal delivery
US20240189447A1 (en) 2021-04-16 2024-06-13 Asklepios Biopharmaceutical, Inc. Rational polyploid aav virions that cross the blood brain barrier and elicit reduced humoral response
AU2022262420A1 (en) 2021-04-23 2023-11-16 Research Institute At Nationwide Children's Hospital Products and methods for treating muscular dystrophy
EP4334447A1 (fr) 2021-05-07 2024-03-13 UCL Business Ltd Édition du génome abca4
IL308431A (en) 2021-05-17 2024-01-01 Sarepta Therapeutics Inc Production of recombinant AAV vectors for the treatment of muscular dystrophy
EP4108263A3 (fr) 2021-06-02 2023-03-22 Research Institute at Nationwide Children's Hospital Produits de virus adéno-associés recombinants et méthodes de traitement de la dystrophie musculaire des ceintures 2a
JP2024526062A (ja) 2021-06-11 2024-07-17 バイエル・アクチエンゲゼルシヤフト Ｖ型ｒｎａプログラム可能エンドヌクレアーゼ系
EP4101928A1 (fr) 2021-06-11 2022-12-14 Bayer AG Systèmes d'endonucléase programmables à arn de type v
WO2023283962A1 (fr) 2021-07-16 2023-01-19 Huigene Therapeutics Co., Ltd. Capside aav modifiée pour thérapie génique et méthodes associées
CA3228365A1 (fr) 2021-08-11 2023-02-16 Solid Biosciences Inc. Traitement de la dystrophie musculaire
JP2024533174A (ja) 2021-09-03 2024-09-12 ビオマリン プハルマセウトイカル インコーポレイテッド Ａａｖカプシド組成物及び送達方法
WO2023034990A1 (fr) 2021-09-03 2023-03-09 Biomarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
WO2023034989A1 (fr) 2021-09-03 2023-03-09 Biomarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
EP4396202A1 (fr) 2021-09-03 2024-07-10 BioMarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
EP4396201A1 (fr) 2021-09-03 2024-07-10 BioMarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
WO2023034980A1 (fr) 2021-09-03 2023-03-09 Bomarin Pharmaceutical Inc. Compositions capsidiques de vaa et méthodes d'administration
EP4144841A1 (fr) 2021-09-07 2023-03-08 Bayer AG Nouveaux systèmes d'endonucléase programmables à petit arn à spécificité pam améliorée et leurs utilisations
CN117980484A (zh) 2021-09-16 2024-05-03 诺华股份有限公司 新颖的转录因子
EP4405396A2 (fr) 2021-09-20 2024-07-31 Voyager Therapeutics, Inc. Compositions et procédés pour le traitement du cancer positif her2
WO2023060215A1 (fr) 2021-10-07 2023-04-13 Research Institute At Nationwide Children's Hospital Produits et procédés pour la désactivation de la protéine zéro de la myéline et le traitement de la maladie cmt1b
WO2023060233A1 (fr) 2021-10-08 2023-04-13 Amicus Therapeutics, Inc. Biomarqueurs de maladies du surcharge lysosomale
JP2023059858A (ja) 2021-10-15 2023-04-27 リサーチ インスティチュート アット ネイションワイド チルドレンズ ホスピタル 自己相補的アデノ随伴ウイルスベクター及び筋ジストロフィーの治療におけるその使用
WO2023077078A1 (fr) 2021-10-29 2023-05-04 Ultragenyx Pharmaceutical Inc. Lignées cellulaires modifiées pour une production accrue de virus adéno-associé recombiné (raav)
WO2023091949A2 (fr) 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de troubles neurologiques liés à un déficit en bêta glucosylcéramidase
US20230279431A1 (en) 2021-11-30 2023-09-07 Research Institute At Nationwide Children's Hospital Self-Complementary Adeno-Associated Virus Vector and its Use in Treatment of Muscular Dystrophy
EP4198134A1 (fr) 2021-12-16 2023-06-21 Genethon Augmentation de transfert de gène de gamma-sarcoglycane utilisant des séquences itr modifiées
EP4198047A1 (fr) 2021-12-16 2023-06-21 Genethon Augmentation du transfert de gènes de la protéine apparentée à la fukutine à l'aide de séquences itr modifiées
EP4198048A1 (fr) 2021-12-16 2023-06-21 Genethon Augmentation du transfert du gène calpaïne-3 à l'aide de séquences itr modifiées
EP4198046A1 (fr) 2021-12-16 2023-06-21 Genethon Augmentation de transfert de gène d'alpha-sarcoglycane utilisant des séquences itr modifiées
EP4453013A1 (fr) 2021-12-21 2024-10-30 Research Institute at Nationwide Children's Hospital Matériaux et méthodes pour le traitement de la dystrophie musculaire des ceintures
WO2023118068A1 (fr) 2021-12-23 2023-06-29 Bayer Aktiengesellschaft Nouveaux petits systèmes programmables d'endonucléases à arn de type v
AU2023227443A1 (en) 2022-03-01 2024-10-10 Crispr Therapeutics Ag Methods and compositions for treating angiopoietin-like 3 (angptl3) related conditions
AU2023227586A1 (en) 2022-03-03 2024-09-12 Research Institute At Nationwide Children's Hospital Materials and methods for the treatment of eif2b5 mutations and diseases resulting therefrom
WO2023178067A1 (fr) * 2022-03-16 2023-09-21 Rutgers, The State University Of New Jersey Distribution contrôlée de gène spécifique à un muscle
WO2023196818A1 (fr) 2022-04-04 2023-10-12 The Regents Of The University Of California Compositions et procédés de complémentation génétique
WO2023214346A1 (fr) 2022-05-06 2023-11-09 Novartis Ag Nouveaux polypeptides de fusion vp2 d'aav recombinants
WO2023240177A1 (fr) 2022-06-08 2023-12-14 Research Instiitute At Nationwide Children's Hospital Produits et méthodes pour le traitement de maladies ou de pathologies associées à l'expression mutante ou pathogène de kcnq3
WO2023237587A1 (fr) 2022-06-10 2023-12-14 Bayer Aktiengesellschaft Nouveaux petits systèmes programmables d'endonucléases à arn de type v
WO2023240236A1 (fr) 2022-06-10 2023-12-14 Voyager Therapeutics, Inc. Compositions et procédés pour le traitement de troubles liés à l'amyotrophie spinale
WO2024011115A1 (fr) 2022-07-06 2024-01-11 Research Institute At Nationwide Children's Hospital Administration de polynucléotide cln1 par un virus adéno-associé
WO2024035782A1 (fr) 2022-08-10 2024-02-15 Aav Gene Therapeutics, Inc. Administration intramusculaire d'insuline médiée par vaa
TW202428602A (zh) 2022-09-15 2024-07-16 美商航海家醫療公司 Ｔａｕ結合化合物
WO2024064913A1 (fr) 2022-09-23 2024-03-28 Sarepta Therapeutics, Inc. Vecteurs aav recombinants pour le traitement de la dystrophie musculaire
WO2024081706A1 (fr) 2022-10-11 2024-04-18 Research Institute At Nationwide Children's Hospital Administration de virus adéno-associé pour traiter l'atrophie musculaire spinale avec détresse respiratoire de type 1 (smard1) et charcot-marie-tooth de type 2s (cmt2s)
WO2024092171A1 (fr) 2022-10-26 2024-05-02 University Of Iowa Research Foundation Procédé d'administration de grands gènes à l'aide d'un virus et d'un système de recombinaison d'adn
WO2024092126A1 (fr) 2022-10-27 2024-05-02 Cargo Therapeutics, Inc. Compositions et méthodes pour immunothérapies améliorées
WO2024129743A2 (fr) 2022-12-13 2024-06-20 Bluerock Therapeutics Lp Endonucléases de type v modifiées programmables par arn et leurs utilisations
WO2024145474A2 (fr) 2022-12-29 2024-07-04 Voyager Therapeutics, Inc. Compositions et procédés de régulation de mapt
WO2024151982A1 (fr) 2023-01-13 2024-07-18 Amicus Therapeutics, Inc. Constructions de thérapie génique pour le traitement de la maladie de pompe
WO2024163560A1 (fr) 2023-02-01 2024-08-08 Sarepta Therapeutics, Inc. Procédés de production de raav
WO2024163012A1 (fr) 2023-02-02 2024-08-08 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de troubles neurologiques liés à un déficit en bêta glucosylcéramidase
WO2024168276A2 (fr) 2023-02-09 2024-08-15 Cargo Therapeutics, Inc. Compositions et méthodes pour immunothérapies
WO2024220592A2 (fr) 2023-04-18 2024-10-24 Research Institute At Nationwide Children's Hospital, Inc. Thérapie génique pour le traitement de la dystrophie musculaire des ceintures r9 et de la dystrophie musculaire congénitale 1c

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5173414A (en) * 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
US5478745A (en) * 1992-12-04 1995-12-26 University Of Pittsburgh Recombinant viral vector system
DE69433592T2 (de) * 1993-11-09 2005-02-10 Targeted Genetics Corp., Seattle Die erzielung hoher titer des rekombinanten aav-vektors
JP3952312B2 (ja) * 1993-11-09 2007-08-01 メディカル カレッジ オブ オハイオ アデノ関連ウイルス複製遺伝子を発現可能な安定な細胞株

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9617947A1 *

Also Published As

Publication number Publication date
WO1996017947A1 (fr) 1996-06-13
JPH10511264A (ja) 1998-11-04
AU4596396A (en) 1996-06-26
AU707866B2 (en) 1999-07-22
CA2207927A1 (fr) 1996-06-13

Similar Documents

Publication Publication Date Title
AU707866B2 (en) Packaging cell lines for generation of high titers of recombinant AAV vectors
US6924128B2 (en) Packaging cell lines for generation of high titers of recombinant AAV vectors
WO1996017947A9 (fr) Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants
US6541258B2 (en) AAV split-packaging genes and cell lines comprising such genes for use in the production of recombinant AAV vectors
AU688428B2 (en) Generation of high titers of recombinant AAV vectors
US6936466B2 (en) Transcriptionally-activated AAV inverted terminal repeats (ITRs) for use with recombinant AAV vectors
US6642051B1 (en) Amplifiable adeno-associated virus(AAV) packaging cassettes for the production of recombinant AAV vectors
EP0728214B1 (fr) Lignees cellulaires stables aptes a exprimer le gene de replication du virus adeno-associe
WO1998027204A9 (fr) Genes d'encapsidation fractionnes de virus adeno-associe (aav) et lignees cellulaires comprenant ces genes utilises pour la production de vecteurs d'aav de recombinaison
CA2269661A1 (fr) Cassettes d'encapsidation d'aav activables par la recombinase servant a la production de vecteurs d'aav
CA2304801C (fr) Sequences terminales repetees inverses (itr) d'aav a action transcriptionnelle, a utiliser avec des vecteurs d'aav recombines
AU758541B2 (en) Amplifiable adeno-associated virus (AAV) packaging cassettes for the production of recombinant AAV vectors
AU2003203790B2 (en) Transcriptionally-activated AAV inverted terminal repeats (ITRs) for use with recombinant AAV vectors

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19970701

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20011128

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060727