RU2773691C2 - TREATMENT OF DISEASES BY MEANS OF EXPRESSION OF ENZYME WITH DEOXYRIBONUCLEASE (DNase) ACTIVITY IN LIVER - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the treatment of a disease or a condition in a subject accompanied by the accumulation of extracellular DNA (hereinafter – ecDNA) in the port-sinusoidal blood flow of the liver. A method for the treatment of the above-mentioned disease or condition in a subject is proposed, including the administration to the subject of therapeutically effective amount of a recombinant expression vector of an adeno-associated virus (rAAV), containing (i) capsid protein and (ii) nucleic acid containing a liver-specific promoter functionally connected to a nucleotide sequence encoding an enzyme with deoxyribonuclease (hereinafter – DNase) activity. A method for the treatment of the above-mentioned disease or condition in a subject is also proposed, including the administration to the subject of an expression vector containing nucleic acid containing a promoter functionally connected to a sequence encoding an enzyme with DNase activity, where the promoter is a liver-specific promoter and/or where the vector includes one or more molecules targeting nucleic acid into liver cells.

EFFECT: invention provides an increase in the clearance of ecDNA accumulated in the port-sinusoidal blood flow of the liver.

24 cl, 21 dwg, 13 tbl, 15 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No. 62/617,879, filed January 16, 2018, the contents of which are hereby incorporated by reference in their entirety.

SEQUENCE LIST

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on January 10, 2019, is named 252732.000003_ST25.txt and has a size of 52177 bytes.

FIELD OF TECHNOLOGY

[0003] The present invention relates to the liver-specific delivery and/or expression of an enzyme that has deoxyribonuclease (DNase) activity for increased clearance of extracellular DNA (cfDNA) accumulated in the portosinusoidal circulation of the liver, and the use of such liver-specific delivery and/or expression for the treatment of various diseases and conditions, including cancer and neurodegeneration.

BACKGROUND OF THE INVENTION

[0004] Cancer patients have elevated plasma and serum levels of circulating extracellular DNA (cfDNA) compared to healthy individuals (Fleischhacker, 2007). In cancer patients, circulating cfDNA originates both from dying non-tumor cells and from tumor cells and neutrophils. Tumors trigger the release of extracellular DNA traps that contribute to the establishment of a prothrombotic state, cachexia, and organ failure in cancer patients (Demmers, 2012). Tumor-derived cfDNA may contribute to the development of metastases and resistance to chemotherapy (García-Olmo, 2013). The amount of circulating cfDNA increases with tumor progression (Sawyers, 2008), reaching maximum levels in patients with advanced disease and metastatic disease (Butt, 2008). Higher amounts of circulating cfDNA have been shown to be highly correlated with poor patient survival (Schwarzenbach, 2008).

[0005] Neurodegeneration is a distinct clinical pathological condition with progressive loss of neuronal structure and/or function, including neuronal death. The molecular pathways leading to neurodegeneration are highly disease-specific (eg, accumulation of abnormally folded beta-amyloid and tau bey in the brain in patients with Alzheimer's disease; accumulation of alpha-synuclein in Parkinson's disease; accumulation of mutant huntingtin in Huntington's disease; accumulation protein aggregates TDP-43 and FUS in amyotrophic lateral sclerosis (ALS); accumulation of mitochondrial DNA mutations and disruption of the mechanics of mitochondrial division during aging) and lead to the death of nerve cells in the late stages of disease progression. Programmed cell death, including apoptosis, seems to play a key role in the progression of neurodegeneration at an advanced stage of the disease, as shown by studies in animal models and cell lines (Radi E., et al., J Alzheimers Dis. 2014; 42).

[0006] Patients with neurodegenerative diseases have elevated levels of circulating cfDNA, including cfDNA of microbial origin, which greatly contributes to the progression of neurodegeneration (see, for example, international application WO2016/190780) through various mechanisms, including transmission across the blood-brain barrier ( BBB) and direct damage to neurons, release of intracerebral neutrophil DNA traps (Zenaro, 2015) and initiation of cerebral thrombotic arteriopathy.

[0007] The present inventors have previously demonstrated that the systemic administration of high doses of DNase protein into a patient's bloodstream may be useful in the treatment of a number of diseases and conditions associated with elevated blood levels of cfDNA, including cancer (e.g., carcinoma, sarcoma, lymphoma, melanoma; see, for example, US Pat. US Patent Application No. US20170100463), neurodegenerative diseases (see, for example, International Publication No. WO2016/190780), infections (see, for example, US Patent Nos. 8431123 and 9072733), diabetes (see, for example, US Patent No. 8388951), atherosclerosis (see, for example, US patent No. 8388951), stroke (see, for example, US patent No. 8796004), angina pectoris (see, for example, US patent No. 8796004), ischemia (see, for example, patent US #8796004), damaged kidney failure (see, for example, US Pat. No. 9,770,492), delayed-type hypersensitivity reactions such as, for example, graft-versus-host disease [GVHD]) (see, for example, US Pat. No. 8,535,663), decreased fertility (see ., for example, US patent No. 8916151), age-related impairment of sperm motility (see, for example, US patent No. 8871200) and aging (see, for example, US patent application publication No. US20150110769). All of these patents and applications are hereby incorporated by reference in their entirety.

[0008] Others later demonstrated similar effects. (Wen, 2013; Cederval, 2015; Tohme, 2016; Patutina, 2011; Li, 2015). rhDNase I also suppressed the development of metastatic disease by 60-90% at a daily dose of 0.02-2.3 mg/kg in a model of metastatic disease of lung carcinoma and hepatoma (Patutina, 2011). Li (Li, 2015) reported the successful use of DNase I to treat GVHD in mice.

[0009] Although systemic administration of DNase protein appears to be suitable for the treatment of diseases and conditions associated with increased amounts of circulating cfDNA, systemic treatment with DNase protein has shown limited effects in clinical settings (see, for example, International Publication No. WO2014/020564 ). Thus, there is a need for more effective methods for reducing circulating cfDNA levels in order to improve the treatment of diseases and conditions associated with elevated cfDNA levels in the blood, such as, for example, cancer, the development of somatic mosaicism, side effects associated with chemotherapy or radiation therapy. , neurodegenerative diseases, infections, delayed-type hypersensitivity reactions, diabetes, atherosclerosis, ischemia, stroke, angina pectoris, reduced fertility, age-related impairment of sperm motility, aging, etc.

BRIEF DESCRIPTION OF THE INVENTION

[0010] As discussed in the Background section above, there is a need for more effective methods for reducing circulating cfDNA levels in order to improve the treatment of diseases and conditions associated with elevated cfDNA levels in the blood. The present invention addresses these and other needs by providing methods and compositions for the liver-specific delivery and/or expression of enzymes having DNase activity.

[0011] In one aspect, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid containing a promoter operably linked to a nucleotide sequence encoding an enzyme that has a deoxyribonuclease (DNase) activity, where the promoter is a liver-specific promoter. In one embodiment, the liver-specific promoter mediates a significantly increased expression of the enzyme in the liver compared to other tissues and organs.

[0012] In another aspect, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid containing a promoter operably linked to a nucleotide sequence encoding an enzyme having a deoxyribonuclease (DNase) activity where the capsid protein provides efficient and/or preferential targeting of the vector to the liver when administered in vivo . In one embodiment, the capsid protein is VP3. In one embodiment, the capsid protein includes one or more mutations that improve the efficiency and/or specificity of vector delivery to the liver compared to the corresponding wild-type capsid protein. In one particular embodiment, the improved efficiency and/or specificity of vector delivery to the liver results in a significant increase in liver expression of the enzyme compared to other tissues and organs.

[0013] Non-limiting examples of enzymes having DNase activity that can be used in the vectors of the present invention include, for example, DNase I, DNase X, DNase γ, DNase 1L1, DNase 1L2, DNase 1L3, DNase II, DNase IIα, DNase IIβ , caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, or mutants or derivatives thereof. In one embodiment, the enzyme having DNase activity is DNase I (eg, human DNase I) or a mutant or derivative thereof. In one embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In one embodiment, the DNase I mutant comprises one or more mutations at the actin binding site (e.g., Gln-9, Glu-13, Thr-14 , His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combination thereof; provisions are in relation to the mature protein sequence lacking the secretory signal sequence) . In one embodiment, one of the mutations in the actin-binding site is a mutation in Ala-114. In one embodiment, the DNase I mutant comprises one or more DNase activity-enhancing mutations (e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combination thereof; the positions are in relation to the sequence of the mature protein lacking secretory signal sequence). In one embodiment, the DNase I mutant includes one or more mutations selected from the group consisting of Q9R, E13R, N74K, and A114F, and any combinations thereof. In one embodiment, the DNase I mutant includes a Q9R mutation. In one embodiment, the DNase I mutant comprises an E13R mutation. In one embodiment, the DNase I mutant includes the N74K mutation. In one embodiment, the DNase I mutant comprises the A114F mutation.

[0014] In one embodiment, the DNase I mutant includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of SEQ ID NO: 5. In one embodiment, the DNase I mutant includes mutations Q9R, E13R, N74K and A114F. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 5.

[0015] In one embodiment, the DNase I mutant includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of SEQ ID NO: 2. In one embodiment, the DNase I mutant includes mutations Q9R, E13R, N74K and A114F. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 2.

[0016] In one embodiment, the DNase I mutant consists of the sequence of SEQ ID NO: 2 or SEQ ID NO: 5.

[0017] In one embodiment, the DNase I mutant contains one or more mutations selected from the group consisting of H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H44A:D53R:Y65A, H44A:Y65A:E69R, H64N: V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T and any combination thereof. In one embodiment, the nucleotide sequence encodes a DNase I comprising the sequence of SEQ ID NO: 4. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises a nucleotide sequence a sequence that is at least 90% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 23. In one embodiment, the nucleotide sequence encodes a DNase I comprising the sequence of SEQ ID NO: 1. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 22. In one embodiment, realization nucleic acid includes nucleotide sequence that is at least 90% identical to SEQ ID NO: 22. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 22. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 22. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 32. In one embodiment, the nucleotide sequence encodes DNase I mutant containing the sequence of SEQ ID NO: 2 4. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 29. In one embodiment, the nucleotide sequence encodes a DNase I mutant, containing the sequence of SEQ ID NO: 26. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 90% identical SEQ ID NO: 28. In one embodiment, the nucle The nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 28. In one embodiment, the nucleotide sequence encodes a DNase I mutant comprising the sequence of SEQ ID NO: 5. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 21. In one embodiment, the nucleotide sequence encodes a DNase mutant I containing follower SEQ ID NO: 2. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 19.

[0018] In one embodiment, the enzyme having DNase activity is a fusion protein comprising (i) a DNase enzyme or fragment thereof linked to (ii) an albumin or Fc polypeptide or fragment thereof. In one embodiment, the sequence encoding an enzyme having DNase activity includes a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates efficient secretion of the enzyme into the hepatic sinusoidal system when the vector is expressed in the liver. Non-limiting examples of suitable secretory signal sequences include, for example, a DNase I secretory signal sequence, an IL2 secretory signal sequence, an albumin secretory signal sequence, a β-glucuronidase secretory signal sequence, an alkaline protease secretory signal sequence, and a fibronectin secretory signal sequence. In one particular embodiment, the secretory signal sequence includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identical to MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one particular embodiment, the secretory signal sequence includes the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one particular embodiment, the secretory signal sequence includes the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one particular embodiment, the secretory signal sequence includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the MYRMQLLSCIALSLALVTNS sequence (SEQ ID NO: 7). In one particular embodiment, the secretory signal sequence includes the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one particular embodiment, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one specific embodiment, a sequence encoding a secretory signal sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 20. In one specific embodiment, a sequence encoding a secretory signal sequence includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence includes a nucleotide sequence that is at least 95% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence sequence, includes the nucleotide sequence of SEQ ID NO: 20.

[0019] In one particular embodiment, the secretory signal sequence includes the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one particular embodiment, the secretory signal sequence consists of the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one particular embodiment, the secretory signal sequence contains a sequence that is at least 80% identical to the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one specific embodiment, a sequence encoding a secretory signal sequence includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 27. In one specific embodiment, a sequence encoding a secretory signal sequence includes a nucleotide sequence that is at least 90% identical to SEQ ID NO: 27. In one specific embodiment, the sequence encoding the secretory signal sequence includes a nucleotide sequence that is at least 95% identical to SEQ ID NO: 27. In one specific embodiment, the sequence encoding the secretory signal sequence sequence, includes the nucleotide sequence of SEQ ID NO: 27.

[0020] Non-limiting examples of promoters that can be used in the vectors of the present invention include, for example, albumin promoter, α1-antitrypsin (AAT) promoter, thyroid hormone-binding globulin promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, factor VIII promoter ( FVIII), HBV basic promoter (BCP), HBV PreS2 promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, thyroxine-binding globulin (TBG) promoter, Hepatic Control Region (HCR)-ApoCII hybrid promoter, HCR-hAAT hybrid promoter , apolipoprotein E (ApoE) promoter, low-density lipoprotein promoter, pyruvate kinase promoter, phospholpyruvate carboxykinase promoter, phenylalanine hydroxylase promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, apolipoprotein A-II (APOA2) promoter, transferrin promoter , transthyretin promoter, α-fibrinogen promoter, β-fibrinogen promoter, alpha promoter α-1-antichymotrypsin, α2-HS glycoprotein promoter, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, complement protein promoter, α1-acid glycoprotein promoter, LSP1 promoter, serpin peptidase inhibitor promoter, clade member 1 A (SERPINA1) (hAAT) promoter , cytochrome P450 family 3 subfamily A polypeptide 4 promoter (CYP3A4), microRNA 122 (miR-122) promoter, IGF-II liver-specific P1 promoter, transthyretin (MTTR) promoter, and α-fetoprotein (AFP) promoter.

[0021] In one embodiment, the promoter is an albumin promoter. In one specific embodiment, the albumin promoter comprises the sequence SEQ ID NO: 8. In one specific embodiment, the albumin promoter consists of the sequence SEQ ID NO: 8. See also Frain et al., Mol. Cell Biol., 1990, 10(3):991-999.

[0022] In one embodiment, the promoter is the α1-antitrypsin (AAT) promoter. In one embodiment, the promoter is the human α1-antitrypsin (hAAT) promoter. In one specific embodiment, the antitrypsin promoter includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 15. In one embodiment, the AAT promoter includes the sequence of SEQ ID NO: 15. In one in a particular embodiment, the AAT promoter consists of the sequence of SEQ ID NO: 15.

[0023] In one embodiment, the capsid protein comprises one or more mutations selected from the group consisting of S279A, S671A, K137R, T252A, and any combinations thereof. In one embodiment, one or more mutations in the capsid protein include the K137R mutation. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:3 [Anc80]. In one embodiment, the implementation of the capsid protein consists of the sequence of SEQ ID NO:3 [Anc80]. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:9 [Anc80]. In one embodiment, the implementation of the capsid protein consists of the sequence of SEQ ID NO:9 [Anc80]. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:34 [Anc80L65]. In one embodiment, the implementation of the capsid protein consists of the sequence of SEQ ID NO:34 [Anc80L65]. In one embodiment, the capsid protein comprises the sequence of SEQ ID NO:35 [Anc80L65 variant]. In one embodiment, the implementation of the capsid protein consists of the sequence of SEQ ID NO:35 [Anc80L65 variant]. In one embodiment, the capsid protein is an AAV8 capsid protein mutant such as, for example, AAV3G1, AAVT20, or AAVTR1, or another capsid protein mutant as described in International Patent Application Publication No. WO2017/180854 (e.g., containing VP3 mutations at amino acids 263 -267 [e.g. 263NGTSG267->SGTH or 263NGTSG267->SDTH] and/or amino acids 457-459 [e.g. 457TAN459->SRP] and/or amino acids 455-459 [e.g. 455GGTAN459 ->DGSGL] and/or amino acids 583-597).

[0024] Non-limiting examples of AAVs that can be used in the vectors of the present invention include, for example, serotype 1 (AAV1), AAV2, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVLK03, AAVhu37, AAVrh64R1, and Anc 80. In one particular embodiment, AAV belongs to serotype 8 or Anc 80.

[0025] In one embodiment, the AAV vector nucleic acid further comprises two AAV inverted terminal repeats (ITRs), the ITRs flanking a nucleotide sequence encoding an enzyme having DNase activity.

[0026] In one embodiment, the implementation of the AAV vector nucleic acid further includes one or more enhancers located upstream or downstream of the promoter. In one embodiment, the enhancer may be located directly upstream of the promoter, for example, where the 3' end of the enhancer sequence is fused directly to the 5' end of the promoter sequence. Non-limiting examples of suitable enhancers include, for example, apolipoprotein E (ApoE) enhancer (e.g., liver control region-1 (HCR-1) ApoE enhancer or HCR-2 ApoE enhancer), alpha-fetoprotein enhancer, TTR enhancer, LSP enhancer, α1 enhancer -microglobulin/bicunin, albumin gene enhancer (Ealb) and any combination thereof. In one particular embodiment, the enhancer is an ApoE enhancer. In one particular embodiment, the enhancer is an ApoE enhancer upstream of a promoter. In one particular embodiment, the enhancer is an ApoE enhancer fused to the 5' end of the promoter. In one particular embodiment, the ApoE enhancer is a liver control region (HCR) enhancer. In one particular embodiment, the enhancer includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 17. In one particular embodiment, the enhancer comprises the sequence of SEQ ID NO: 17. In one particular embodiment, implementation of the enhancer consists of the sequence SEQ ID NO: 17.

[0027] In one embodiment, the AAV vector nucleic acid further comprises a polyadenylation signal operably linked to a nucleotide sequence encoding an enzyme having DNase activity.

[0028] In one embodiment, the nucleic acid further comprises a Kozak sequence. In one particular embodiment, the Kozak sequence includes the sequence 5'-GCCGCCACC-3' (SEQ ID NO: 33). In one embodiment, the nucleic acid comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 30. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 30. In one embodiment, in an embodiment, the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 30. In one embodiment, the nucleic acid includes a nucleotide sequence that is at least 95% identical to SEQ ID NO: 30. In one embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 30.

[0029] In one embodiment, the nucleic acid further comprises a post-transcriptional regulatory element. In one embodiment, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In one particular embodiment, WPRE does not encode a functional X protein. In one embodiment, the post-transcriptional regulatory element includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In one particular embodiment, the post-transcriptional regulatory element comprises the sequence of SEQ ID NO: 16. B In one particular embodiment, the post-transcriptional regulatory element consists of the sequence SEQ ID NO: 16.

[0030] In one embodiment, the rAAV vector nucleic acid of the present invention comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the sequence of SEQ ID NO: 19. In one particular embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 19. In one specific embodiment, the nucleic acid consists of the sequence of SEQ ID NO: 19. In one specific embodiment, the nucleic acid has a map shown in Figure 10.

[0031] In one embodiment, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence of SEQ ID NO: 34, and (ii) a nucleic acid comprising a nucleotide sequence encoding the enzyme deoxyribonuclease ( DNase) containing the sequence of SEQ ID NO: 4, functionally linked to the promoter of albumin or promoter α1-antitrypsin (AAT).

[0032] In one embodiment, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence of SEQ ID NO: 34, and (ii) a nucleic acid comprising a nucleotide sequence encoding the enzyme deoxyribonuclease ( DNase) containing the sequence SEQ ID NO: 5 operably linked to an albumin promoter or an α1-antitrypsin (AAT) promoter.

[0033] In one embodiment, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence of SEQ ID NO: 34, and (ii) a nucleic acid comprising a nucleotide sequence encoding a deoxyribonuclease enzyme ( DNase) containing the sequence of SEQ ID NO: 1, functionally linked to the promoter of albumin or promoter α1-antitrypsin (AAT).

[0034] In one embodiment, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence of SEQ ID NO: 34, and (ii) a nucleic acid comprising a nucleotide sequence encoding a deoxyribonuclease enzyme ( DNase) containing the sequence SEQ ID NO: 2 operably linked to an albumin promoter or an α1-antitrypsin (AAT) promoter.

[0035] In one embodiment, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence of SEQ ID NO: 34, and (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 30 .

[0036] In one embodiment, the present invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence of SEQ ID NO: 34, and (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 31 .

[0037] In a related aspect, the present invention provides pharmaceutical compositions and dosage forms comprising any of the rAAV vectors of the present invention and a pharmaceutically acceptable carrier and/or excipient.

[0038] In a related aspect, the present invention provides a method for delivering an enzyme having deoxyribonuclease (DNase) activity to the liver of a subject in need thereof, comprising administering to the subject any of the rAAV vectors or pharmaceutical compositions described above.

[0039] In another aspect, the present invention provides a method of treating a disease or condition (e.g., cancer, neurodegenerative disease, atherosclerosis, or a delayed-type hypersensitivity reaction) in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of extracellular DNA ( cfDNA) in the portosinusoidal circulation of the subject's liver, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above.

[0040] In a further aspect, the present invention provides a method of treating cancer in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above. In one embodiment, the cancer originates and/or metastasizes in organs, tissues, and/or structures from which it drains into the portal vein. In one embodiment, the method is effective in inhibiting metastasis. Non-limiting examples of cancers treatable by the methods of the present invention include, for example, peritoneal carcinomatosis, lymphoma, gastric cancer, colon cancer, intestinal cancer, colorectal cancer, pancreatic cancer, liver cancer, bile duct cancer, gallbladder cancer, sarcoma, and metastatic liver disease of any origin.

[0041] In another aspect, the present invention provides a method of treating a neurodegenerative disease in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above. Non-limiting examples of neurodegenerative diseases treatable by the methods of the present invention include, for example, Alzheimer's disease, mild cognitive impairment (MCI), CADASIL syndrome, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granules (AGD), Pick's disease (PiD), Huntington's disease (HD), and frontotemporal dementia with parkinsonism-17 (FTDP-17). In one embodiment, the neurodegenerative disease is associated with the formation of a misfolded protein due to the presence of DNA. In one particular embodiment, the DNA is human DNA, microbial DNA, extracellular DNA, or intracellular DNA.

[0042] In one embodiment, the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, amyloidosis, asthma, or prion disease. In one embodiment, the amyloidosis is amyloidosis with hereditary cerebral hemorrhage, primary systemic amyloidosis, secondary systemic amyloidosis, serum amyloidosis, senile systemic amyloidosis, hemodialysis associated amyloidosis, Finnish-type hereditary systemic amyloidosis, atrial amyloidosis, lysozyme systemic amyloidosis. Lysozyme systemic amyloidosis), amyloidosis associated with insulin, or amyloidosis associated with the a-chain of fibrinogen.

[0043] In another aspect, the present invention provides a method of treating a delayed type hypersensitivity reaction in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above. In one embodiment, the delayed-type hypersensitivity reaction is graft-versus-host disease (GVHD).

[0044] In a further aspect, the present invention provides a method for treating atherosclerosis in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above.

[0045] In another aspect, the present invention provides a method for preventing or eliminating one or more side effects associated with chemotherapy or radiation therapy in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above. In one embodiment, said one or more side effects of chemotherapy are selected from the group consisting of weight loss, bone marrow toxicity, catabolic changes in blood chemistry, cardiotoxicity (e.g., myocardial necrosis), gastrointestinal toxicity, suppression immunity and neutropenia. In one embodiment, chemotherapy comprises administering one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule targeting agents, topoisomerase inhibitors, alkaloids, and targeted therapeutic agents. In one embodiment, chemotherapy comprises administering one or more compounds selected from the group consisting of anthracycline, doxorubicin, 5-fluorouracil (5-FU), etoposide, taxane, and cyclophosphamide. In one embodiment, said one or more side effects of radiation therapy are selected from the group consisting of weight loss, skin irritation, skin damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, and secondary cancer. In one embodiment, the radiation therapy is external beam radiation therapy or systemic radioisotope therapy. In one embodiment, the rAAV vector or vector composition is administered during a cycle of chemotherapy or radiation therapy. In another embodiment, the rAAV vector or vector composition is administered after a cycle of chemotherapy or radiation therapy. In one embodiment, chemotherapy or radiation therapy is used to treat a cancer selected from the group consisting of peritoneal carcinomatosis, lymphoma, stomach cancer, colon cancer, bowel cancer, colorectal cancer, pancreatic cancer, liver cancer, bile duct cancer, bile duct cancer. bladder, sarcoma and metastatic liver disease of any origin.

[0046] In one embodiment of any of the above methods according to the present invention, the introduction of the rAAV vector or vector composition results in the expression of an enzyme that has DNase activity and its secretion into the portosinusoidal circulation of the liver of the subject.

[0047] In one embodiment of any of the above methods according to the present invention, the rAAV vector or vector composition is administered at a dose and regimen that is sufficient to reduce the level of extracellular DNA (ecDNA) in the portosinusoidal bloodstream of the specified subject.

[0048] In one embodiment of any of the above methods according to the present invention, the subject is a human.

[0049] In another aspect, the present invention provides a method for delivering an enzyme having deoxyribonuclease (DNase) activity to the liver of a subject in need thereof, comprising administering to the subject an expression vector containing a nucleic acid containing a promoter operably linked to a sequence encoding said enzyme. where the promoter is a liver-specific promoter.

[0050] In a further aspect, the present invention provides a method for delivering an enzyme that has deoxyribonuclease (DNase) activity to the liver of a subject in need thereof, comprising administering to the subject an expression vector containing a nucleic acid containing a promoter operably linked to a sequence encoding said an enzyme, wherein the vector includes one or more molecules capable of targeting the nucleic acid to liver cells when administered in vivo.

[0051] In another aspect, the present invention provides a method of treating a disease or condition in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of extracellular DNA (cfDNA) in the portosinusoidal circulation of the liver of the subject, said method comprising administering to the subject a vector expression containing a nucleic acid containing a promoter functionally linked to a sequence encoding an enzyme with deoxyribonuclease (DNase) activity, while the promoter is a liver-specific promoter. In one embodiment, the disease is selected from the group consisting of cancer (including metastatic liver disease), neurodegenerative disease, atherosclerosis, and delayed type hypersensitivity reaction.

[0052] In another aspect, the present invention provides a method of treating a disease or condition in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of extracellular DNA (cfDNA) in the portosinusoidal circulation of the liver of the subject, said method comprising administering to the subject a vector an expression containing a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme having deoxyribonuclease (DNase) activity, wherein the vector includes one or more molecules capable of targeting the nucleic acid to liver cells. In one embodiment, the disease is selected from the group consisting of cancer (including metastatic liver disease), neurodegenerative disease, atherosclerosis, and delayed type hypersensitivity reaction.

[0053] In a further aspect, the present invention provides a method for treating cancer in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme that has a deoxyribonuclease (DNase) ) activity, where the promoter is a liver-specific promoter. In one embodiment, the cancer originates and/or metastasizes in organs, tissues, and/or structures from which it drains into the portal vein.

[0054] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme having a deoxyribonuclease (DNase) activity, wherein the vector includes one or more molecules capable of targeting the nucleic acid to liver cells. In one embodiment, the cancer originates and/or metastasizes in organs, tissues, and/or structures from which it drains into the portal vein.

[0055] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, said method comprising administering to the subject an expression vector containing a nucleic acid encoding an enzyme having deoxyribonuclease (DNase) activity, said expression vector providing synthesis of the enzyme DNase in the liver of the specified subject, and in this case, the cancer arises and/or metastasizes in organs, tissues and/or structures from which the outflow occurs in the portal vein.

[0056] Non-limiting examples of cancers to be treated with any of the above cancer treatments include, for example, peritoneal carcinomatosis, lymphoma, gastric cancer, colon cancer, intestinal cancer, colorectal cancer, pancreatic cancer, liver cancer, bile duct cancer, cancer gallbladder, sarcoma and metastatic liver disease of any origin.

[0057] In one embodiment of any of the above cancer treatment methods, the method is effective in inhibiting metastasis.

[0058] In another aspect, the present invention provides a method for treating a neurodegenerative disease in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme that has a deoxyribonuclease ( DNase) activity, where the promoter is a liver-specific promoter.

[0059] In another aspect, the present invention provides a method for treating a neurodegenerative disease in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme having a deoxyribonuclease (DNase) ) activity, wherein the vector includes one or more molecules capable of targeting the nucleic acid to liver cells.

[0060] In another aspect, the present invention provides a method for treating a neurodegenerative disease in a subject in need thereof, said method comprising administering to the subject an expression vector containing a nucleic acid encoding an enzyme having deoxyribonuclease (DNase) activity, said expression vector provides for the synthesis of the DNase enzyme in the liver of the specified subject, and at the same time, microbial extracellular DNA (cfDNA) of intestinal origin is detected in the blood of the specified patient.

[0061] Non-limiting examples of neurodegenerative diseases treatable by any of the above neurodegenerative disease treatments include, for example, Alzheimer's disease, mild cognitive impairment (MCI), CADASIL syndrome, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granule disease (AGD), Pick's disease (PiD), Huntington's disease (HD), and frontotemporal dementia with parkinsonism-17 (FTDP-17). In one embodiment, the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, amyloidosis, asthma, or prion disease. In one embodiment, the amyloidosis is amyloidosis with hereditary cerebral hemorrhage, primary systemic amyloidosis, secondary systemic amyloidosis, serum amyloidosis, senile systemic amyloidosis, hemodialysis associated amyloidosis, Finnish-type hereditary systemic amyloidosis, atrial amyloidosis, lysozyme systemic amyloidosis. Lysozyme systemic amyloidosis), amyloidosis associated with insulin, or amyloidosis associated with the a-chain of fibrinogen.

[0062] In another aspect, the present invention provides a method for treating a delayed-type hypersensitivity reaction in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme that has deoxyribonuclease (DNase) activity, where the promoter is a liver-specific promoter. In one embodiment, the delayed-type hypersensitivity reaction is graft-versus-host disease (GVHD).

[0063] In another aspect, the present invention provides a method for treating a delayed-type hypersensitivity reaction in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme that has deoxyribonuclease (DNase) activity, wherein the vector includes one or more molecules capable of targeting the nucleic acid to liver cells. In one embodiment, the delayed-type hypersensitivity reaction is graft-versus-host disease (GVHD).

[0064] In a further aspect, the present invention provides a method for treating atherosclerosis in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme that has a deoxyribonuclease (DNase) ) activity, where the promoter is a liver-specific promoter.

[0065] In another aspect, the present invention provides a method for treating atherosclerosis in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter operably linked to a sequence encoding an enzyme having a deoxyribonuclease (DNase) activity, wherein the vector includes one or more molecules capable of targeting the nucleic acid to liver cells.

[0066] In another aspect, the present invention provides a method for preventing or eliminating one or more side effects associated with chemotherapy or radiation therapy in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter, operably linked to a sequence encoding an enzyme that has deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.

[0067] In another aspect, the present invention provides a method for preventing or eliminating one or more side effects associated with chemotherapy or radiation therapy in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid containing a promoter, operably linked to a sequence encoding an enzyme having deoxyribonuclease (DNase) activity, wherein the vector includes one or more molecules capable of targeting the nucleic acid to liver cells.

[0068] In one embodiment of any of the above methods for preventing or eliminating one or more side effects associated with chemotherapy, said one or more side effects of chemotherapy are selected from the group consisting of weight loss, bone marrow toxicity, catabolic changes in blood biochemistry, cardiotoxicity (eg, myocardial necrosis), gastrointestinal toxicity, immune suppression, and neutropenia.

[0069] In one embodiment of any of the above methods for preventing or eliminating one or more side effects associated with chemotherapy, the chemotherapy comprises administering one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, agents targeting microtubules, topoisomerase inhibitors, alkaloids and targeted therapeutic agents.

[0070] In one embodiment of any of the above methods for preventing or eliminating one or more side effects associated with chemotherapy, the chemotherapy comprises administering one or more compounds selected from the group consisting of anthracycline, doxorubicin, 5-fluorouracil (5-FU) , etoposide, taxane and cyclophosphamide.

[0071] In one embodiment of any of the above methods for preventing or eliminating one or more side effects associated with radiation therapy, said one or more side effects of radiation therapy are selected from the group consisting of weight loss, skin irritation, skin damage, fatigue , nausea, vomiting, fibrosis, intestinal damage, memory loss, infertility and secondary cancer.

[0072] In one embodiment of any of the above methods for preventing or eliminating one or more side effects associated with radiation therapy, the radiation therapy is external beam radiation therapy or systemic radioisotope therapy.

[0073] In one embodiment of any of the above methods for preventing or eliminating one or more side effects associated with chemotherapy or radiation therapy, the vector is administered during a cycle of chemotherapy or radiation therapy.

[0074] In one embodiment of any of the above methods for preventing or eliminating one or more side effects associated with chemotherapy or radiation therapy, the vector is administered after a cycle of chemotherapy or radiation therapy.

[0075] In one embodiment of any of the methods of the present invention, the vector further comprises one or more molecules capable of targeting the nucleic acid to liver cells.

[0076] In one embodiment of any of the methods of the present invention, the promoter is a liver-specific promoter.

[0077] In one embodiment of any of the methods according to the present invention, the vector is placed in a liposome or nanoparticle.

[0078] In one embodiment of any of the methods of the present invention, the vector is used in the form of naked DNA.

[0079] In one embodiment of any of the methods of the present invention, the vector is a viral vector. Non-limiting examples of suitable viral vectors include, for example, adeno-associated viral vectors, adenovirus vectors, retroviral vectors (eg, lentiviral vectors), and hepatotropic viral vectors (eg, hepatitis B virus (HBV) vectors).

[0080] In one embodiment of any of the methods of the present invention, the enzyme having DNase activity is selected from the group consisting of DNase I, DNase X, DNase γ, DNase 1L1, DNase 1L2, DNase 1L3, DNase II, DNase IIα, DNase IIβ, caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, or mutants or derivatives thereof. In one embodiment, the enzyme, DNase is DNase I (eg, human DNase I) or a mutant or derivative thereof. In one embodiment, the DNase I mutant includes one or more mutations at the actin binding site (e.g., Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67 , Glu-69, Asn-74, Ala-114, and any combination thereof; provisions are in relation to the mature protein sequence lacking the secretory signal sequence). In one embodiment, one of the mutations in the actin-binding site is a mutation in Ala-114. In one embodiment, the DNase I mutant comprises one or more DNase activity-enhancing mutations (e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combination thereof; the positions are in relation to the sequence of the mature protein lacking secretory signal sequence). In one embodiment, the DNase I mutant includes one or more mutations selected from the group consisting of Q9R, E13R, N74K, and A114F, and any combinations thereof. In one embodiment, the DNase I mutant includes a Q9R mutation. In one embodiment, the DNase I mutant comprises an E13R mutation. In one embodiment, the DNase I mutant includes the N74K mutation. In one embodiment, the DNase I mutant comprises the A114F mutation.

[0081] In one embodiment, the DNase I mutant contains one or more mutations selected from the group consisting of H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H44A:D53R:Y65A, H44A:Y65A:E69R, H64N: V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T and any combination thereof. In one embodiment, the DNase I mutant is a long acting form of DNase. In one embodiment, DNase I comprises the sequence of SEQ ID NO: 4. In one embodiment, DNase I comprises the sequence of SEQ ID NO: 1. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 5. In one embodiment, the DNase I mutant includes the sequence SEQ ID NO: 2.

[0082] In one embodiment of any of the methods of the present invention, the sequence encoding the enzyme having DNase activity includes a secretory signal sequence, said secretory signal sequence mediating efficient secretion of the enzyme into the portosinusoidal circulation of the liver upon administration of the vector to a subject. In one embodiment, the secretory signal sequence is selected from the group consisting of a DNase I secretory signal sequence, an IL2 secretory signal sequence, an albumin secretory signal sequence, a β-glucuronidase secretory signal sequence, an alkaline protease secretory signal sequence, and a fibronectin secretory signal sequence. In one particular embodiment, the secretory signal sequence includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identical to MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one embodiment, the secretory signal sequence includes the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one embodiment, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one particular embodiment, the secretory signal sequence includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the MYRMQLLSCIALSLALVTNS sequence (SEQ ID NO: 7). In one embodiment, the secretory signal sequence includes the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one particular embodiment, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).

[0083] In one embodiment of any of the methods according to the present invention, the liver-specific promoter mediates a significantly increased expression of the enzyme in the liver compared to other tissues and organs. Non-limiting examples of liver-specific promoters that can be used in the methods and vectors of the present invention include, for example, albumin promoter, α1-antitrypsin (AAT) promoter, thyroid hormone-binding globulin promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, factor promoter VIII (FVIII), HBV basic promoter (BCP), HBV PreS2 promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, thyroxine-binding globulin (TBG) promoter, Hepatic Control Region (HCR)-ApoCII hybrid promoter, HCR hybrid promoter -hAAT, Apolipoprotein E (ApoE) promoter, Low density lipoprotein promoter, Pyruvate kinase promoter, Phosphenol pyruvate carboxykinase promoter, Phenylalanine hydroxylase promoter, Lecithin cholesterol acyltransferase (LCAT) promoter, Apolipoprotein H (ApoH) promoter, Apolipoprotein A-II (APOA2) promoter, transferrin promoter, transthyretin promoter, α-fibrinogen promoter, β-fib promoter rhinogen, alpha-1-antichymotrypsin promoter, α2-HS glycoprotein promoter, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, complement protein promoter, α1-acid glycoprotein promoter, LSP1 promoter, serpin peptidase inhibitor promoter, clade member 1 A (SERPINA1) promoter (hAAT), cytochrome P450 family 3 subfamily A (CYP3A4) polypeptide 4 promoter, microRNA 122 (miR-122) promoter, IGF-II liver-specific P1 promoter, transthyretin (MTTR) promoter, and α-fetoprotein (AFP) promoter. In one embodiment, the liver-specific promoter is an albumin promoter. In one specific embodiment, the albumin promoter comprises the sequence SEQ ID NO: 8. In one embodiment, the albumin promoter consists of the sequence SEQ ID NO: 8. See also Frain et al., Mol. Cell Biol., 1990, 10(3):991-999.

[0084] In one specific embodiment, the liver-specific promoter is the α1-antitrypsin (AAT) promoter. In one particular embodiment, the antitrypsin promoter is the human α1-antitrypsin (AAT) promoter. In one particular embodiment, the AAT promoter comprises the sequence SEQ ID NO: 15. In one particular embodiment, the AAT promoter comprises the sequence SEQ ID NO: 15. In one particular embodiment, the AAT promoter consists of the sequence SEQ ID NO: 15. In various embodiments, any of the methods according to the present invention, the vector contains an enhancer. The enhancer may be a liver control site enhancer such as HCR-1 or HCR-2 linked to an ApoE gene, eg the human ApoE gene. In one specific embodiment, the enhancer comprises the sequence SEQ ID NO: 17. In one specific embodiment, the enhancer consists of the sequence SEQ ID NO: 17.

[0085] In various embodiments of any of the methods of the present invention, the vector contains a Kozak sequence upstream of the DNase coding sequence. The Kozak sequence may have the sequence 5'-GCCGCCACC-3' (SEQ ID NO: 33).

[0086] In various embodiments of any of the methods of the present invention, the vector comprises a post-transcriptional regulatory element, such as WPRE, that does not encode a functional protein X. In one particular embodiment, the post-transcriptional regulatory element comprises the sequence of SEQ ID NO: 16. In one particular embodiment, implementation, the post-transcriptional regulatory element consists of the sequence SEQ ID NO: 16.

[0087] In various embodiments of any of the methods of the present invention, the vector includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the sequence of SEQ ID NO: 19. In one particular embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 19. In one specific embodiment, the nucleic acid consists of the sequence of SEQ ID NO: 19. In one specific embodiment, the nucleic acid has a map shown in Figure 10.

[0088] In one embodiment of any of the methods of the present invention, administration of the vector results in the expression of the enzyme and its secretion into the liver portosinusoidal circulation of the subject.

[0089] In one embodiment of any of the methods according to the present invention, the vector is administered at a dose and regimen that is sufficient to reduce the level of extracellular DNA (cfDNA) in the portosinusoidal bloodstream of the specified subject.

[0090] In one embodiment of any of the methods of the present invention, the subject is a human.

[0091] In one embodiment of any of the methods of the present invention, the method further comprises selecting a subject with an increased level of extracellular DNA (cfDNA) in the bloodstream compared to the level of cfDNA in the bloodstream of a normal healthy subject.

[0092] In one embodiment of any of the methods of the present invention, the method further comprises administering to the subject a deoxyribonuclease (DNase) enzyme (e.g., DNase I, DNase X, DNase γ, DNase 1L1, DNase 1L2, DNase 1L3, DNase II (including DNase IIα and DNase IIβ), caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, or mutants or derivatives thereof). In one particular embodiment, the DNase is DNase I, or a mutant or derivative thereof.

[0093] These and other aspects of the present invention will be apparent to those skilled in the art from the following description, claims, and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The figure 1 shows the organs and tissues, the outflow of which occurs in the portal vein of the liver. For example, the portal vein drains blood from the lower esophagus, stomach, pancreas, gallbladder, spleen, small intestine, colon, upper bladder, and liver.

[0095] Figures 2A-2I show pooled patient data and individual blood electrophoretic data for nine patients with advanced metastatic disease treated with intravenous bovine DNase I. (A) rectal carcinoma, (B) lung carcinoma, ( C) lung carcinoma, (D) melanoma, (E) breast cancer, (F) adenocarcinoma, (G) kidney cancer, (H) rectal cancer, (I) breast cancer.

[0096] Figure 3 shows a gamma immunoscintigraphic image of a tumor-bearing mouse taken on day 15, one hour after injection of 99mTc labeled anti-DNA antibodies, with significantly more radioactivity in the liver area (b) in the center/right than in the tumor zone (a) at the bottom left.

[0097] Figure 4 shows IHC micrographs of liver tissue from the various groups listed in Table 3 and treated as described in Example 4. Large amounts of cfDNA were found in the liver of a gemcitabine-treated mouse (group 7). Relatively little cfDNA was found in the liver of a mouse treated with mAFP/Alb AAV8 DNase I, an AAV8 vector targeting the DNase I gene in liver cells for expression with an albumin promoter (group 2).

[0098] Figure 5A shows a map of the mAFP/Alb AAV8 4658_pAAV-AFP-Alb-huDNaseI_mut vector. The vector contains (i) elements for reproduction in bacteria, ie. an AmpR marker, origins of replication colE1, F1 and M13, and (ii) an hDNAseI_mut sequence (SEQ ID NO: 2; hyperactive actin-resistant DNase I mutant) operably linked to the mouse albumin minimal promoter, mouse α-fetoprotein (AFP) enhancer II ) (with L-ITR), hGH polyA signal flanked by AAV8 L-ITR and R-ITR, M13 origin of replication, and F1 origin of replication. Figure 5B shows the sequence alignment of the Anc80, AAV2 and AAV8 VP3 proteins (adapted from Zinn et al., Cell Rep., 2015, 12 (67): 1056-1068). Figure 5C shows a map of the ADV-207186 vector based on human adenovirus type 5 (dE1/E3) with human DNase I expressed under the control of the CMV promoter.

[0099] Figure 6 shows bioluminescence data reflecting tumor size. The tumors of both MIA PaCa-2 orthotopic tumor implant mice and non-orthotopic MIA PaCa-2 tumor implant mice decreased in size when treated with hDNaseI_mut (SEQ ID NO: 5; hyperactive DNase I actin-resistant mutant) mAFP/Alb AAV8.

[00100] Figure 7 presents liver histology data showing that animals treated with DNase I mAFP/Alb AAV8 have a much lower inflammatory cell density, no necrosis, and much less nucleophilic material on the vascular surface when treated with the DNase I mAFP/Alb expression vector. Alb AAV8 instead of DNase I protein.

[00101] Figure 8 shows a significant reduction in the total number of donor-derived CD4+ and CD8+ donor-derived T cells in peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches in mice treated with mAFP/Alb AAV8 DNase I, by compared with mice treated with recombinant DNase I.

[00102] Figure 9 shows a restriction map of the pLV2-IL2ss-DNaseI mut lentiviral vector used to deliver DNase I in Example 7. The vector includes the AmpR marker and replication origins for F1 and SV40. The vector includes the EF-1a promoter upstream of the IL-2 leader upstream of DNase I.

[0100] Figure 10A shows the complete sequence of the ApoEHCR enhancer construct: hAAT>DNaseI promoter (hyperactive) correct leader-WPRE Xinact or ApoE-HCR enhancer: hAAT promoter>hyperactive human DNase I variant with correct leader sequence (secretory sequence): post-transcriptional regulatory Woodchuck Hepatitis Virus Element (WPRE) that does not encode a functional X protein. Figure 10B shows a vector map of the ApoEHCR enhancer:hAAT>hDNaseI promoter (hyperactive) correct leader-WPRE Xinact. Figure 10C shows a vector map of the ApoEHCR enhancer:hAAT>hDNaseI promoter (wild-type)-WPRE Xinact.

[0101] Figure 11 shows a map of the pAAV-MCS expression vector without a promoter used to generate the ApoEHCR enhancer:hAAT>hDNaseI promoter (hyperactive) correct Xinact WPRE leader.

[0102] Figure 12 shows the results of SDS-PAGE analysis of the purified AAV vector Anc80 VR-18013AD. Three bands of 60, 72 and 90 kDa were observed in a ratio of approximately 1:1:10, which corresponds to VP1-3 proteins.

[0103] Figures 13A and 13B illustrate data in which Xinact ApoEHCR enhancer-hAAT-hDNaseI promoter (hyperactive) correct leader-WPRE and Xinact ApoEHCR promoter-hAAT-hDNaseI (wild type)-WPRE vectors efficiently transduce cells of hepatocyte origin only and cause high level expression and secretion of a DNase I enzyme or a wild-type DNase I enzyme with biologically increased expression from transduced G2 cells.

[0104] Figure 14 shows the results of the Y Maze Behavior Test.

[0105] Figure 15 shows the results of a situational fear state test.

[0106] Figure 16 shows that microglial activation was significantly reduced in VR-18013AD treated 3xTg-AD mice compared to 3xTg-AD control mice, especially in the cerebral cortex.

[0107] Figure 17 shows data indicating a significant reduction in microglial activation in the cerebral cortex of VR-18013AD treated 3xTg-AD mice compared to 3xTg-AD control mice.

[0108] Figure 18 shows data indicating that treatment with VR-18013AD significantly reduces amyloid deposition in VR-18013AD D treated 3xTg-AD mice compared to 3xTg-AD control mice.

[0109] Figure 19 shows that treatment with VR-18013AD significantly reduces the deposition of hyperphosphorylated tau protein in VR-18013AD treated 3xTg-AD mice compared to 3xTg-AD control mice.

[0110] Figure 20 shows data indicating a significant reduction in the reduction of p-tau in the hippocampus after treatment with VR-18013AD.

[0111] Figure 21 shows a typical morphometric image of mice treated with control (single intravenous injection of PBS) and VR-18014AD (single intravenous injection of VR-18014AD at a dose of 1.0 x 10 ¹¹ GC/mouse).

DETAILED DESCRIPTION OF THE INVENTION

[0112] The present invention is based on the unexpected discovery that during diseases and conditions characterized by increased levels of circulating cfDNA (e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.), a significant amount of cfDNA accumulates in the liver , predominantly in the periendothelial space and space of Disse. Even large amounts of systemically administered DNase protein show limited efficacy in cleaving liver cfDNA. This cfDNA continuously flows from the liver depot into the portal vein of the liver and then into the systemic circulation. As shown in the Examples section below, transgenic expression of DNase I in the liver (eg, using viral expression vectors) results in almost complete removal of cfDNA accumulated in the liver and in the portosinusoidal circulation when the DNase is secreted into the sinusoidal space of the liver. Such transgenic expression of DNase in the liver leads to a significant antitumor effect (especially in tumors of organs and tissues, outflow from which occurs into the portal vein), a decrease in the toxicity of antitumor chemotherapy, a slowdown in the progression of autoimmune and neurodegenerative diseases, etc. This transgenic expression of DNase in the liver, when the DNase is secreted into the sinusoidal space, provides even more benefits when the outflow from the tumor occurs in the portal vein system and the cfDNA formed in the tumor site undergoes a "first pass" through the portosinusoidal blood flow. (Figure 1 shows organs and systems that drain into the portal vein.) The same applies to neurodegenerative and autoimmune conditions, which are promoted by increased intestinal permeability of microbial DNA entering the systemic circulation, with a corresponding "first pass" through the portosinusoidal circulation .

[0113] The present invention provides various vectors for delivering DNase to the liver. Specific non-limiting examples of such vectors include AAV vectors, adenoviral vectors, retroviral vectors (e.g., lentiviral vectors), hepatotropic viral vectors (e.g., hepatitis B virus (HBV) vectors), nanoparticles (e.g., phosphoramidite nanoparticles), liposomes (e.g., cationic liposomes). such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)), lactoferrin, poly L-lysine, polyethyleneimine, chitosan, etc.).

[0114] An enzyme having DNase activity may be expressed under the control of a liver-specific promoter and/or other liver-specific control element (eg, enhancer). Specific non-limiting examples of liver-specific promoters and controls include, for example, the albumin promoter; human antitrypsin alpha-1 promoter (hAAT); TBG (thyroxine-binding globulin); apolipoprotein E hepatic control region; apolipoprotein A-II (APOA2) promoter; serpin peptidase inhibitor, promoter of clade A, member 1 (SERPINA1) (hAAT); cytochrome P450 subfamily A subfamily 4 (CYP3A4) promoter; miRNA promoter 122 (MIR122); liver-specific P1 promoter of IGF-II; mouse transthyretin promoter (MTTR); and an alpha-fetoprotein (AFP) promoter. The promoter may be located upstream or downstream of the enhancer. The promoter sequence can be directly fused to the enhancer sequence.

[0115] Specific non-limiting examples of enzymes having DNase activity that can be used in the compositions and methods of the present invention include DNase I, DNase X, DNase γ, DNase 1L1, DNase 1L2, DNase 1L3, DNase II (e.g., DNase IIα, DNase IIβ), caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, or mutants or derivatives thereof.

[0116] If the enzyme having DNase activity is DNase I, various actin-binding mutants can be used. Specific non-limiting examples of residues in wild-type recombinant human DNase I (SEQ ID NO: 4) that can be mutated include, for example, Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr- 65, Val-66, Val-67, Glu-69, Asn-74 and Ala-114. In various embodiments, the Ala-114 mutation is used. For example, in a hyperactive human DNase I mutant containing the sequence of SEQ ID NO: 5, the Ala-114 residue is mutated. Complementary residues in other DNases can also be mutated. Specific non-limiting examples of mutations in wild-type recombinant human DNase I include H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H44A:D53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T.

[0117] Various DNase mutants can be used to increase DNase activity. Specific non-limiting examples of mutations in wild-type recombinant human DNase I include, for example, Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69 , Asn-74 and Ala-114. Specific non-limiting examples of mutations to increase wild-type recombinant DNase I activity include Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and A114F. For example, a combination of Q9R, E13R, N74K and A114F mutations can be used, such a combination being found in at least a hyperactive DNase I mutant containing the sequence of SEQ ID NO: 5.

[0118] When AAV vectors are used for DNase expression, they can be derived from any serotype, e.g. serotype 1 (AAV1), AAV2, AAV3 (e.g. AAV3A, AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 , AAV10, AAV11, AAV12, AAVrh10 (as described, for example, in US Patent No. 9790472, International Patent Application Publication Nos. Ther., 2015, 23(12):1877-1887), AAVhu37 (as described, for example, in International Patent Application Publication No. WO2017180857), AAVrh64R1 (as described, for example, in International Patent Application Publication No. WO2017180857) or Anc80 (Zinn et al., Cell Rep., 2015, 12(67): 1056-1068).

[0119] Point mutations can be introduced into the capsid protein (eg, VP3) to increase the efficiency and/or specificity of liver-specific delivery. Specific non-limiting examples of such point mutations in the AAV8 VP3 capsid protein include, for example, S279A, S671A, K137R, and T252A, as well as the AAV8 capsid mutations described in International Patent Application Publication No. WO2017/180854 (for example, AAV3G1, AAVT20 or AAVTR1, VP3 mutations in amino acids 263-267 [e.g. 263NGTSG267->SGTH or 263NGTSG267->SDTH] and/or amino acids 457-459 [e.g. 457TAN459->SRP] and/or amino acids 455-459 [e.g. 455GGTAN459->DGSGL ] and/or amino acids 583-597).

[0120] Vectors and compositions according to the present invention can be targeted to the liver in various ways. Specific non-limiting examples of routes of administration to the liver include intrahepatic injection, intravenous injection, and intra-arterial injection.

Sequences

SEQ ID NO: 1 - wild type human DNase I (WT), precursor; Registration number in Genbank NP_005214.2; the secretory signal sequence is underlined:

MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

SEQ ID NO: 2 - human DNase I mutant, precursor; mutant residues compared to SEQ ID NO: 1 are in bold and underlined; the secretory signal sequence is underlined:

MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

SEQ ID NO: 3 - Anc80 VP1 capsid protein:

AADGYLPDWLEDNLSEGIREWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAX ¹ KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMX ² AGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTALPTYNNHLYKQISSQSGX ³ STNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX ⁴ LNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX ⁵ FSYTFEDVPFHSSYAHSQSLDRLNPLIDQYLYYLSRTQTTSGTAGNRX ⁶ LQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTX ⁷ NQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITX ⁸ EEEIKTTNPVATEX ⁹ YGTVATNLQSX ¹⁰ NTAPATGTVNSQGALPGMVWQX ¹¹ RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

X ¹ = K or R; X ² = A or S; X ³ = A or G; X ⁴ = R or K; X ⁵ = E or Q; X ⁶ = T or E; X ⁷ = A or T; X ⁸ = S or N; X ⁹ = Q or E; X ¹⁰ = S or A and X ¹¹ = N or D.

SEQ ID NO: 4 - Mature human wild-type (WT) DNase I (no secretory signal sequence; Genbank accession no. 4AWN_A:

LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

SEQ ID NO: 5 - mature human DNase I mutant (no secretory signal sequence); mutated residues compared to SEQ ID NO: 4 are in bold and underlined:

LKIAAFNIRTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

SEQ ID NO: 6 - human DNase I secretory signal sequence:

MRGMKLLGALLALAALLQGAVS

SEQ ID NO: 7 - IL2 secretory signal sequence:

MYRMQLLSCIALSLALVTNS

SEQ ID NO: 8 - human albumin promoter:

ACTAGTTCCAGATGGTAAATATACACAAGGGATTTAGTCAAACAATTTTTTGGCAAGAATATTATGAATTTTGTAATCGGTTGGCAGCCAATGAAATACAAAGATGAGTCTAGTTAATAATCTACAATTATTGGTTAAAGAAGTATATTAGTGCTAATTTCCCTCCGTTTGTCCTAGCTTTTCTCTTCTGTCAACCCCACACGCCTTTGGCACC

SEQ ID NO: 9 - Anc80 VP1 capsid protein:

AADGYLPDWLEDNLSEGIREWDLKPGAPKPKANQQKQDDGRGLVLPGYYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAX ¹ KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMX ² AGGGAPADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTALPTYNNHLYKQISSQSGX ³ STNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX ⁴ LNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX ⁵ FSYTFEDVPFHSSYAHSQSLDRLNPLIDQYLYYLSRTQTTSGTAGNRX ⁶ LQFSQAGPSSANQAKNWLPGPCYRQQRVSKTX ⁷ NQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVITX ⁸ EEEIKTTNPVATEX ⁹ YGTVATNLQSX ¹⁰ NTAPATGTVNSQGALPGVWQX ¹¹ RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

X ¹ = K or R; X ² = A or S; X ³ = A or G; X ⁴ = R or K; X ⁵ = E or Q; X ⁶ = T or E; X ⁷ = A or T; X ⁸ = S or N; X ⁹ = Q or E; X ¹⁰ = S or A and X ¹¹ = N or D.

SEQ ID NO: 10 - human beta-globulin primer:

CAACTTCATCCACGTTCACC

SEQ ID NO: 11 - NLRP3 Forward Primer:

GTCTGAGCTCCAACCATTCT

SEQ ID NO: 12 - NLRP3 reverse primer:

CACTGTGGGTCCTTCATCTTT

SEQ ID NO: 13 - Forward primer of the 16S gene of the universal bacterial RNA:

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG

SEQ ID NO: 14 - universal bacterial RNA 16S gene reverse primer:

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC

SEQ ID NO: 15 - human antitrypsin promoter sequence:

gatcttgctaccagtggaacagccactaaggattctgcagtgagagcagagggccagctaagtggtactctcccagagactgtctgactcacgccaccccctccaccttggacacaggacgctgtggtttctgagccaggtacaatgactcctttcggtaagtgcagtggaagctgtacactgcccaggcaaagcgtccgggcagcgtaggcgggcgactcagatcccagccagtggacttagcccctgtttgctcctccgataactggggtgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacggacgaggacagggccctgtctcctcagcttcaggcaccaccactgacctgggacagtgaat

SEQ ID NO: 16 - Groundhog hepatitis virus post-transcriptional regulatory element not encoding functional X protein:

agtggcggccgctcgagctagcggccgctctagaagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcggactag

SEQ ID NO: 17 - Apolipoprotein E (ApoE) Enhancer, Liver Control Region (HCR):

aggctcagaggcacacaggagtttctgggctcaccctgcccccttccaacccctcagttcccatcctccagcagctgtttgtgtgctgcctctgaagtccacactgaacaaacttcagcctactcatgtccctaaaatgggcaaacattgcaagcagcaaacagcaaacacacagccctccctgcctgctgaccttggagctggggcagaggtcagagacctctctgggcccatgccacctccaacatccactcgaccccttggaatttcggtggagaggagcagaggttgtcctggcgtggtttaggtagtgtgagagg

SEQ ID NO: 18 - Polynucleotide encoding hyperactive human DNase I variant SEQ ID NO: 5:

atgaagctgctgggggcgctgctggcactggcggccctactgcagggggccgtgtccctgaagatcgcagccttcaacatcaggacatttgggaggaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaagagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtga

SEQ ID NO: 19 - polynucleotide encoding human DNase I mutant precursor SEQ ID NO: 2 (secretory signal sequence underlined):

Atgaggggcatgaagctgctgggggcgctgctggcactggcggccctactgcagggggccgtgtccctgaagatcgcagccttcaacatcaggacatttgggaggaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaagagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtga

SEQ ID NO: 20 - polynucleotide encoding the secretory signal sequence (SEQ ID NO: 6) of human DNase I:

atgaggggcatgaagctgctggggcgctgctggcactggcggccctactgcaggggggccgtgtcc

SEQ ID NO: 21 - polynucleotide encoding mature human DNase I mutant (without secretory signal sequence) SEQ ID NO: 5:

ctgaagatcgcagccttcaacatcaggacatttgggaggaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaagagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtga

SEQ ID NO: 22 - polynucleotide encoding human DNase I, wild-type (WT), precursor of SEQ ID NO: 1:

atgaggggcatgaagctgctgggggcgctgctggcactggcggccctactgcagggggccgtgtccctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaag

SEQ ID NO: 23 - polynucleotide encoding human wild-type mature DNase I (WT) SEQ ID NO: 4:

ctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaag

SEQ ID NO: 24 - Mus musculus wild-type DNase I, precursor; Registration number in Genbank No. NP_034191.3; the secretory signal sequence is underlined:

MRYTGLMGTLLTLVNLLQLAGTLRIAAFNIRTFGETKMSNATLSVYFVKILSRYDIAVIQEVRDSHLVAVGKLLDELNRDKPDTYRYVVSEPLGRKSYKEQYLFVYRPDQVSILDSYQYDDGCEPCGNDTFSREPAIVKFFSPYTEVQEFAIVPLHAAPTEAVSEIDALYDVYLDVWQKWGLEDIMFMGDFNAGCSYVTSSQWSSIRLRTSPIFQWLIPDSADTTVTSTHCAYDRIVVAGALLQAAVVPNSAVPFDFQAEYGLSNQLAEAISDHYPVEVTLRKI

SEQ ID NO: 25 - Mus musculus wild-type DNase I secretory signal sequence

MRYTGLMGTLLTLVNLLQLAGT

SEQ ID NO: 26 - Wild-type (WT) Mus musculus DNase I

LRIAAFNIRTFGETKMSNATLSVYFVKILSRYDIAVIQEVRDSHLVAVGKLLDELNRDKPDTYRYVVSEPLGRKSYKEQYLFVYRPDQVSILDSYQYDDGCEPCGNDTFSREPAIVKFFSPYTEVQEFAIVPLHAAPTEAVSEIDALYDVYLDVWQKWGLEDIMFMGDFNAGCSYVTSSQWSSIRLRTSPIFQWLIPDSADTTVTSTHCAYDRIVVAGALLQAAVVPNSAVPFDFQAEYGLSNQLAEAISDHYPVEVTLRKI

SEQ ID NO: 27 - Polynucleotide encoding Mus musculus wild-type DNase I secretory signal sequence

atgcggtacacagggctaatgggaacactgctcaccttggtcaacctgctgcagctggctgggact

SEQ ID NO: 28 - Polynucleotide encoding mature Mus musculus wild-type (WT) DNase I

ctgagaattgcagccttcaacattcggacttttggggagactaagatgtccaatgctaccctctctgtatactttgtgaaaatcctgagtcgctatgacatcgctgttatccaagaggtcagagactcccacctggttgctgttgggaagctcctggatgaactcaatcgggacaaacctgacacctaccgctatgtagtcagtgagccgctgggccgcaaaagctacaaggaacagtacctttttgtgtacaggcctgaccaggtgtctattctggacagctatcaatatgatgatggctgtgaaccctgtggaaatgacaccttcagcagagagccagccattgttaagttcttttccccatacactgaggtccaagaatttgcgatcgtgcccttgcatgcagccccaacagaagctgtgagtgagatcgacgccctctacgatgtttacctagatgtctggcaaaagtggggcctggaggacatcatgttcatgggagacttcaatgctggctgcagctacgtcacttcctcccagtggtcctccattcgccttcggacaagccccatcttccagtggctgatccctgacagtgcggacaccacagtcacatcaacacactgtgcttatgacaggattgtggttgctggagctctgctccaggctgctgttgttcccaactcggctgttccttttgatttccaagcagaatacggactttccaaccagctggctgaagccatcagtgaccattacccagtggaggtgacactcagaaaaatctga

SEQ ID NO: 29 - Polynucleotide encoding wild-type Mus musculus DNase I, precursor

atgcggtacacagggctaatgggaacactgctcaccttggtcaacctgctgcagctggctgggactctgagaattgcagccttcaacattcggacttttggggagactaagatgtccaatgctaccctctctgtatactttgtgaaaatcctgagtcgctatgacatcgctgttatccaagaggtcagagactcccacctggttgctgttgggaagctcctggatgaactcaatcgggacaaacctgacacctaccgctatgtagtcagtgagccgctgggccgcaaaagctacaaggaacagtacctttttgtgtacaggcctgaccaggtgtctattctggacagctatcaatatgatgatggctgtgaaccctgtggaaatgacaccttcagcagagagccagccattgttaagttcttttccccatacactgaggtccaagaatttgcgatcgtgcccttgcatgcagccccaacagaagctgtgagtgagatcgacgccctctacgatgtttacctagatgtctggcaaaagtggggcctggaggacatcatgttcatgggagacttcaatgctggctgcagctacgtcacttcctcccagtggtcctccattcgccttcggacaagccccatcttccagtggctgatccctgacagtgcggacaccacagtcacatcaacacactgtgcttatgacaggattgtggttgctggagctctgctccaggctgctgttgttcccaactcggctgttccttttgatttccaagcagaatacggactttccaaccagctggctgaagccatcagtgaccattacccagtggaggtgacactcagaaaaatctga

SEQ ID NO: 30 - Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNaseI (hyperactive)correct leader-WPRE Xinact (VR-18013AD)

aggctcagaggcacacaggagtttctgggctcaccctgcccccttccaacccctcagttcccatcctccagcagctgtttgtgtgctgcctctgaagtccacactgaacaaacttcagcctactcatgtccctaaaatgggcaaacattgcaagcagcaaacagcaaacacacagccctccctgcctgctgaccttggagctggggcagaggtcagagacctctctgggcccatgccacctccaacatccactcgaccccttggaatttcggtggagaggagcagaggttgtcctggcgtggtttaggtagtgtgagagggatcttgctaccagtggaacagccactaaggattctgcagtgagagcagagggccagctaagtggtactctcccagagactgtctgactcacgccaccccctccaccttggacacaggacgctgtggtttctgagccaggtacaatgactcctttcggtaagtgcagtggaagctgtacactgcccaggcaaagcgtccgggcagcgtaggcgggcgactcagatcccagccagtggacttagcccctgtttgctcctccgataactggggtgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacggacgaggacagggccctgtctcctcagcttcaggcaccaccactgacctgggacagtgaatgccgccaccAtgaggggcatgaagctgctgggggcgctgctggcactggcggccctactgcagggggccgtgtccctgaagatcgcagccttcaacatcaggacatttgggaggaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagc cactgggacggaagagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgaagtggcggccgctcgagctagcggccgctctagaagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttg tcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgacctcagacgagtcggcccccc

[0121] (APOE HCR enhancer: bases 1-320; human alpha-1 antitrypsin promoter: bases 321-717; Kozak sequence: bases 718-726; hyperactive human DNase I variant with natural fully correct leader sequence: bases 727-1575 ; protein WPRE X inactivated: bases 1576-2212)

SEQ ID NO: 31 - Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNAase I wild-type-WPRE Xinact (VR-18014AD)

aggctcagaggcacacaggagtttctgggctcaccctgcccccttccaacccctcagttcccatcctccagcagctgtttgtgtgctgcctctgaagtccacactgaacaaacttcagcctactcatgtccctaaaatgggcaaacattgcaagcagcaaacagcaaacacacagccctccctgcctgctgaccttggagctggggcagaggtcagagacctctctgggcccatgccacctccaacatccactcgaccccttggaatttcggtggagaggagcagaggttgtcctggcgtggtttaggtagtgtgagagggatcttgctaccagtggaacagccactaaggattctgcagtgagagcagagggccagctaagtggtactctcccagagactgtctgactcacgccaccccctccaccttggacacaggacgctgtggtttctgagccaggtacaatgactcctttcggtaagtgcagtggaagctgtacactgcccaggcaaagcgtccgggcagcgtaggcgggcgactcagatcccagccagtggacttagcccctgtttgctcctccgataactggggtgaccttggttaatattcaccagcagcctcccccgttgcccctctggatccactgcttaaatacggacgaggacagggccctgtctcctcagcttcaggcaccaccactgacctgggacagtgaatgccgccaccatgaggggcatgaagctgctgggggcgctgctggcactggcggccctactgcagggggccgtgtccctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagc cactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgaagtggcggccgctcgagctagcggccgctctagaagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttg tcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgacctcagacgagtcggcccccc

[0122] (APOE HCR enhancer: bases 1-320; human alpha-1 antitrypsin promoter: bases 321-717; Kozak sequence: bases 718-726; wild-type human DNase I with natural fully correct leader sequence: bases 727-1575 ; protein WPRE X inactivated: bases 1576-2212)

SEQ ID NO: 32 - polynucleotide encoding human DNase I, wild-type (WT), precursor of SEQ ID NO: 1 with stop codon:

atgaggggcatgaagctgctgggggcgctgctggcactggcggccctactgcagggggccgtgtccctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagTGA

SEQ ID NO: 33 - Kozak sequence

5'-GCCCGCCACC-3'

SEQ ID NO: 34 - Anc80L65 VP1 capsid protein

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATEEYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

SEQ ID NO: Anc80L65 VP1 capsid protein variant 35

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVITNEEEIKTTNPVATEEYGTVATNLQSANTAPATGTVNSQGALPGVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL

Definitions

[0123] The term "enzyme having deoxyribonuclease (DNase) activity" as used herein refers to an enzyme capable of hydrolytically cleaving phosphodiester bonds in the DNA backbone.

[0124] As used herein, the terms "deoxyribonuclease" and "DNase" refer to any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone. A large number of deoxyribonucleases are known that can be used in the methods of the present invention. Non-limiting examples of DNases useful in the methods of the present invention include, for example, DNase I (for example, recombinant human DNase I (rhDNase I) or bovine pancreatic DNase I), DNase I analogues (such as, for example, DNase X, DNase gamma and DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, and acetylcholinesterase. The present invention also encompasses DNase enzymes that have an extended half-life (e.g., albumin and/or Fc fused, or protected from binding to actin by modification of the actin binding site; see, for example, Gibson et al., (1992) J. Immunol Methods, 155, 249-256). The actin-binding site of DNase I can be mutated, for example, at the following residues: Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu- 69, Asn-74, Ala-114 recombinant human DNase (SEQ ID NO: 4). For example, in a hyperactive variant of human DNase I containing the sequence of SEQ ID NO: 5, the Ala-114 residue is mutated. Example mutations include H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C H44A:D53R:Y65A , V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T (in the order of SEQ ID NO: 4). Mutations in DNase I with increased DNase I activity are also covered. Non-limiting examples of such mutations are, for example, Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and A114F of recombinant human DNase I (SEQ ID NO: 4). For example, a combination of mutations Q9R, E13R, N74K, and A114F is found in hyperactive DNase I containing the sequence of SEQ ID NO: 5. DNase I cleaves DNA preferentially at the phosphodiester bonds adjacent to the pyrimidine nucleotide, producing polynucleotides with terminal 5'-phosphate terminal groups with a free hydroxyl group in position 3', on average forming tetranucleotides. DNase I acts on single-stranded DNA, double-stranded DNA and chromatin.

[0125] The term "about" denotes the limits of an acceptable error range for a particular value, as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system. For example, "about" may mean within an acceptable standard deviation in accordance with the practice accepted in the art. Alternatively, "about" may mean a range of no more than ±20%, no more than ±10%, no more than ±5%, and no more than ±1% of the set value. Alternatively, especially in relation to biological systems or processes, the term may mean within an order of magnitude, for example, within 2-fold value. When specific values are described in the application and claims, unless otherwise indicated, the term "about" is implied and in this context means within the allowable error range for a particular value.

[0126] The terms "extracellular DNA" (English extracellular DNA), "extracellular DNA" (English cell-free DNA) and "cfDNA" are used interchangeably to refer to extracellular DNA (for example, eukaryotic, archaeal or prokaryotic origin) found in blood, cerebrospinal fluid (CSF), or intestines of the patient.

[0127] The terms "treat" or "treatment" of a disease, disorder, or condition include: (1) preventing, delaying, or reducing the frequency and/or likelihood of at least one clinical or subclinical symptom of a disease, disorder, or condition developing in a subject, which may be affected by or predisposed to a disease, disorder or condition, but is not yet experiencing or showing clinical or subclinical symptoms of the disease, disorder or condition; or (2) suppression of the disease, disorder, or condition, ie, arrest, reduction, or delay in the development of the disease or its recurrence, or at least one clinical or subclinical symptom thereof; or (3) alleviate the disease, i. e. providing regression of the disease, disorder or condition, or at least one of its clinical or subclinical symptoms. The beneficial effect for the subject to be treated is either statistically significant or at least perceptible to the patient or physician.

[0128] The terms "individual", "subject", "animal", "patient", and "mammal" are used interchangeably to refer to mammals, including humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, and etc.) and experimental animal models of diseases (for example, mice, rats).

[0129] The term "effective", when applied to a dose or amount, refers to that amount of a compound or pharmaceutical composition that is sufficient to achieve the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, an effective amount of the combination may or may not include amounts of each ingredient that would be effective when administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs used, the route of administration, and the like.

[0130] As used herein, the term "therapeutically effective" when applied to a dose or amount, refers to that amount of a compound or pharmaceutical composition that is sufficient to achieve the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients (eg, a combination of DNase and another compound) is administered, an effective amount of the combination may or may not include amounts of each ingredient that would be effective when administered alone.

[0131] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream of the transcription direction of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a DNA-dependent binding site RNA polymerase, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art to have a direct or indirect effect on regulation of the level of transcription from the promoter. A "tissue-specific" promoter may preferably be active in certain tissue or cell types.

[0132] As used herein, the term "liver-specific expression" refers to predominant or exclusive expression in the liver, i. expression to a much greater extent than in other tissues and organs.

[0133] The term "liver-specific promoter" as used herein refers to a promoter that is predominantly or exclusively active in a liver cell (eg, hepatocyte) and directs/initiates transcription in the liver to a much greater extent than in other tissues and organs. As used herein, the term "predominantly" means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (e.g., 100% of said promoter-driven expression) occurs in liver cells. The ratio of liver to non-liver expression can vary between different liver-specific promoters. In some embodiments, a liver-specific promoter may preferentially direct/initiate transcription in a particular liver cell type (eg, hepatocytes, Kupffer cells, endothelial cells, etc.). Some liver-specific promoters used in the expression cassettes of the present invention include at least one, usually more than one, liver nuclear factor binding site. The liver-specific promoters used in the expression cassettes of the present invention may be constitutive or inducible promoters. Some non-limiting examples of hepatic promoters useful in the expression cassettes of the present invention include: albumin promoter (Alb), human antitrypsin alpha-1 promoter (hAAT), thyroxine-binding globulin (TBG), apolipoprotein E-hepatic control region promoter, apolipoprotein promoter A-II (APOA2), serpin peptidase inhibitor, clade A member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3 subfamily A (CYP3A4) polypeptide 4 promoter, microRNA 122 (miR-122) promoter, liver-specific promoter IGF-II P1, mouse transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, and mouse prealbumin gene promoter.

[0134] Non-limiting examples of liver-specific promoters include, for example, albumin (Alb) promoter, human antitrypsin alpha-1 promoter (hAAT), thyroxine-binding globulin (TBG) promoter, apolipoprotein E-hepatic control region promoter, apolipoprotein A promoter -II (APOA2), serpin peptidase inhibitor, clade A member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3 subfamily A (CYP3A4) polypeptide 4 promoter, microRNA 122 (miR-122) promoter, liver-specific IGF promoter -II P1, mouse transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, thyroid hormone-binding globulin promoter, alcohol dehydrogenase promoter, factor VIII (FVIII) promoter, HBV basic promoter (BCP) and PreS2 promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter ), hybrid liver control region (HCR)-ApoCII promoter, AAT promoter in combination with mouse albumin gene enhancer element (Ealb), low-density lipoprotein promoter, pyruvate promoter kinases, phosphenolpyruvate carboxykinase promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, transferrin promoter, transthyretin promoter, alpha-fibrinogen and beta-fibrinogen promoters, alpha-1-antichymotrypsin promoter, alpha-2-HS promoter α-glycoprotein, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, complement protein promoters (e.g., Clq, Clr, C2, C3, C4, C5, C6, C8, C9, complement factor I, and factor H), complement activator C3, and promoter [ alpha]1-acid glycoprotein. Additional tissue-specific promoters can be found in the tissue-specific promoter database, TiProD (Nucleic Acids Research, J4: D104-D107 (2006).

[0135] In some embodiments, for example, when liver targeting is mediated by a capsid protein, a nucleic acid encoding an enzyme having DNase activity may be operably linked to a promoter allowing for efficient systemic expression (e.g., CMV promoter, chicken beta actin promoter (CBA), EF1a promoter).

[0136] The term "portal vein cancer" is used herein to refer to a cancer (eg, carcinoma, sarcoma, or lymphoma) that has originated in or metastasized to an organ or tissue that drains into the portal vein. Non-limiting examples of such organs and tissues are shown in Figure 1 and include, for example, the liver, colon, small and large intestines, stomach, spleen, pancreas, and gallbladder.

[0137] The term "neurodegeneration" as used herein refers to a distinct clinical pathological condition with progressive loss of neuronal structure and/or function, including neuronal death. Neurodegeneration can be primary or secondary. Non-limiting examples of diseases involving primary neurodegeneration include, for example, Alzheimer's disease (AD), mild cognitive impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion diseases, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism-17 (FTDP-17), dementia with Lewy bodies, vascular dementia, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), agyrophilic granule disease (AGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steele-Richardson-Olszewski), corticodentatonigraal degeneration, Hallervorden-Spatz disease, progressive familial myoclonic epilepsy , strionigral degeneration, torsion dystonia (eg, torsion spasm; deforming muscle dystonia), spasmodic torticollis and other dyskinesias, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration (eg, Friedreich's ataxia and related disorders), Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia , peroneal amyotrophy (Charcot-Marie-Tooth), interstitial hypertrophic polyneuropathy (Dejerine-Sotta), chronic progressive neuropathy, retinitis pigmentosa (retinitis pigmentosa), and hereditary optic nerve atrophy (Leber's disease). Secondary neurodegeneration is caused primarily by necrosis. Non-limiting examples of conditions that can lead to secondary neurodegeneration include neuronal destruction by neoplasm, edema, hemorrhage, stroke, trauma, immune attack, hypoxia, poisoning, metabolic disorders, and infections.

[0138] The phrase "pharmaceutically acceptable" as used in connection with compositions of the invention refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically cause adverse reactions when administered to a subject (e.g., a mammal, such as a human). As used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the USP or other generally accepted pharmacopeia for use in mammals, and more specifically, in humans.

[0139] The terms "cytostatic and/or cytotoxic chemotherapy" and "chemotherapy" are used interchangeably herein to refer to therapy involving the administration of a cytostatic and/or cytotoxic agent.

[0140] The terms "anticancer agent" and "anticancer chemotherapeutic agent" are used herein to refer to any chemical compound that is used to treat cancer. Anticancer chemotherapeutic agents are well known in the art (see, for example, Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)). The description provides specific examples of chemotherapeutic agents.

[0141] The term "side effect of chemotherapy" as used herein refers to an unwanted and unintended, though not necessarily unexpected, outcome of chemotherapy.

[0142] As used herein, the terms "radiotherapy", "radiation therapy", and "RT" are used interchangeably to refer to the medical use of ionizing radiation as part of cancer treatment to damage the DNA of malignant cells, either directly or by creating charged particles within diseased cells that damage DNA. Commonly used types of radiation therapy encompassed by the present invention include, for example, external beam radiation therapy (EBRT or XRT), brachytherapy/sealed source radiation therapy, and systemic radioisotope/open source radiation therapy.

[0143] As used herein, the terms "side effect of radiation therapy" or "side effect of radiation therapy" refer to an unwanted and unintended, although not necessarily unexpected, result of radiation therapy. Developed side effects depend on the site of the body being treated, the dose administered per day, the total dose administered, the general health of the patient, and other treatments being administered at the same time, and may include, for example, skin irritation or injury, fatigue, nausea, vomiting , fibrosis, intestinal damage, memory loss, infertility, or secondary cancer.

[0144] The term "catabolic state" as used herein refers to a condition characterized by rapid weight loss and loss of skeletal fat and muscle mass, which may occur during chemotherapy or chemoradiotherapy. Associated clinical events include, for example, immunosuppression, muscle weakness, susceptibility to pulmonary embolism, thrombophlebitis, and altered stress response.

[0145] As used herein, the terms "viral vector" and "viral construct" refer to a recombinant viral construct that includes one or more heterologous nucleotide sequences (eg, a nucleotide sequence encoding an enzyme having DNase activity). In some embodiments, the viral vector is a replication defective vector. In some embodiments, structural and non-structural viral coding sequences are not present in the viral vector and are provided during production of the viral vector by transfer with a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell line. Depending on the virus, the viral vector may be packaged in a capsid (eg AAV vector) and/or a lipid envelope (eg lentiviral vector).

[0146] The term "polyadenylation signal", as used herein, refers to a nucleic acid sequence that mediates the attachment of a polyadenine region to the 3' end of an mRNA. Non-limiting examples of polyadenylation signals that can be used in expression constructs of the present invention include, for example, SV40 early polyadenylation signal, SV40 late polyadenylation signal, HSV thymidine kinase polyadenylation signal, protamine gene polyadenylation signal, adenovirus 5 EIb polyadenylation signal, bovine growth hormone polyadenylation signal , the polyadenylation signal of the human variant of growth hormone, and the like.

[0147] As used herein and in the appended claims, singular and definite articles include the plural unless the context clearly dictates otherwise.

[0148] In accordance with the present invention, traditional methods of pharmacology and molecular biology can be used within the skill in the art. Such methods are fully explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. (1985)); Transcription and Translation (BD Hames & SJ Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994), among others.

Targeting the DNase enzyme to the liver

[0149] The present inventors surprisingly found that targeting the expression of an enzyme having DNase activity to the liver can cause increased clearance of disease-associated cfDNA throughout the bloodstream, and especially in the portosinusoidal circulation. As shown in the Examples section below, cfDNA can accumulate in the liver, especially in the periendothelial space and space of Disse. Degradation of this cfDNA pool through localized expression of an enzyme with DNase activity can reduce the overall level of cfDNA in the bloodstream.

[0150] As discussed throughout the application, an enzyme having DNase activity can be efficiently expressed in liver cells using viral vectors (e.g., adeno-associated viral (AAV) vectors, retroviral vectors (e.g., lentiviral vectors), or adenovirus vectors), liposomes, nanoparticles carrying the DNase transgene, or naked DNA. The adult liver receives almost a quarter of the cardiac output, filters about 1 liter of blood per minute, and holds 10-15% of the blood volume at any given moment. Such an active blood flow contributes to the rapid accumulation of high levels of vector particles in the liver. The vector particles can pass through the fenestrated endothelium along the sinusoids of the liver directly to the hepatocytes. Such hepatocytes can be transfected with vector particles and continue to produce DNase. The resulting DNase is secreted into the periendothelial space and space of Disse, where it can effectively degrade the concentrated pool of cfDNA.

[0151] The present inventors have found that transfection of liver cells to express an enzyme having DNase activity provides an unexpectedly greater reduction in cfDNA, along with a significant and unexpected improvement in diseases and conditions associated with high levels of cfDNA in the bloodstream.

[0152] In some embodiments, expression of the DNase enzyme in the liver is achieved using nucleic acid expression cassettes that are predominantly expressed in the liver. In some embodiments, such expression cassettes may contain one or more of the following: (a) a hepatic locus control element; (b) a liver promoter located at the 3' end of the liver locus control element; (c) a coding sequence located at the 3' end of a hepatic promoter, said coding sequence encoding a polypeptide, such as deoxyribonuclease; (d) a polyadenylation signal located at 3' of the coding sequence; and (e) an intron located at the 3' end of the liver promoter and the 5' end of the polyadenylation signal. Elements (a), (b), (c), (d) and (e) may be operably linked to express the polypeptide encoded by the coding sequence. In some embodiments, the expression cassettes of the present invention direct expression of a therapeutic amount of a polypeptide in liver cells over a period of at least 100 days (e.g., at least 200 days, at least 300 days, at least 400 days, or at least at least 500 days). In some embodiments, the polypeptide is a DNase. The DNase can be any DNase described herein, such as DNase I.

[0153] The polyadenylation signal can be operably linked to a nucleic acid encoding a DNase. Non-limiting examples of polyadenylation signals include, for example, SV40 early polyadenylation signal, SV40 late polyadenylation signal, HSV thymidine kinase polyadenylation signal, protamine gene polyadenylation signal, adenovirus 5 EIb polyadenylation signal, bovine growth hormone polyadenylation signal, human variant growth hormone polyadenylation signal, and the like. .

[0154] In another aspect, the present invention provides vectors that contain a nucleic acid expression cassette that is predominantly expressed in the mammalian liver. Such an expression cassette may contain, for example: (a) a hepatic locus control element; (b) a liver promoter located at the 3' end of the liver locus control element; (c) a coding sequence located at the 3' end of a hepatic promoter, said coding sequence encoding a polypeptide, such as deoxyribonuclease; (d) a polyadenylation signal located at 3' of the coding sequence; and (e) an intron located at the 3' end of the liver promoter and the 5' end of the polyadenylation signal. Elements (a), (b), (c), (d) and (e) are operably linked to express the polypeptide encoded by the coding sequence. Some vectors according to the present invention are episomal vectors, and some vectors according to the present invention are integrating vectors, such as integrating viral vectors. In some embodiments, the polypeptide is a deoxyribonuclease (DNase). The DNase can be any DNase described herein, such as DNase I.

[0155] In another aspect, the present invention relates to methods for treating a disease or condition associated with elevated levels of cfDNA. The disease or condition may be any disease or condition described herein or in any of US Pat. Nos. 7,612,032; 8388951; 8431123; 8535663; 8710012; 8796004; 8871200; 8916151; 9072733; 9248166; 9770492; US Patent Application Publication No. US20170056482, US20170100463, US20150110769 and International Patent Application Publication No. WO2016/190780, all of which are hereby incorporated by reference in their entirety. In one embodiment, the method includes the steps of: (1) introducing into the subject's liver a vector containing a nucleic acid expression cassette, said expression cassette comprising: (a) a liver-specific promoter; (b) a coding sequence located at the 3' end of a liver-specific promoter, said coding sequence encoding an enzyme having DNase activity (eg, any DNA described herein); (c) a polyadenylation signal located at the 3' end of the coding sequence; and optionally (d) an intron located at the 3' end of the liver promoter and the 5' end of the polyadenylation signal. Elements (a), (b), (c) and (d) are operably linked to express the polypeptide encoded by the coding sequence; and (2) expressing a therapeutic amount of said polypeptide in the liver. In some embodiments of the methods of this aspect of the present invention, a therapeutic amount of the polypeptide is expressed for at least 100 days (eg, at least 200 days, at least 300 days, at least 400 days, and at least 500 days).

[0156] The nucleic acid expression vectors and cassettes of this aspect of the present invention are predominantly expressed in the liver, eg, at least 50% of the expression of the encoded polypeptide occurs in the liver. Advantageously, at least 90% of the expression occurs in the liver. Some of the vectors and expression cassettes according to this aspect of the present invention are exclusively expressed in the liver.

[0157] Hepatic locus control elements can be included in the expression cassettes of the present invention, which are capable of enhancing the expression of nucleic acid molecules in liver cells and provide copy number dependent, position independent expression in the expression cassette. Some areas of liver locus control useful in the expression cassettes of the present invention include, for example, a template attachment site and/or a liver-specific enhancer element.

[0158] In addition to the promoter, the cassette and/or expression vector may contain one or more additional initiation, termination, and/or transcriptional enhancer sequences; RNA processing signals (such as splicing and polyadenylation (poly A) signals); sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (eg, a Kozak consensus sequence such as 5'-GCCGCCACC-3' [SEQ ID NO: 33]); sequences that increase protein stability; and sequences that enhance the secretion of the encoded polypeptide. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Examples of suitable enhancers include, for example, alpha-fetoprotein enhancer, minimal TTR promoter/enhancer, LSP (TH-binding globulin promoter/alpha-1-microglobulin/bicunin enhancer), Serpin1 enhancer, among others. In one embodiment, the expression cassette included one or more expression enhancers. In one embodiment, the expression cassette included two or more expression enhancers. These enhancers may be the same or may be different from each other. For example, an enhancer may include an Alpha mic/bik enhancer. This enhancer may be present in two copies located next to each other. Alternatively, duplicate copies of the enhancer may be separated by one or more sequences.

[0159] In yet another embodiment, the expression cassette further comprises an intron (eg, Promega intron, truncated chimeric intron (T-chimeric intron), introns described in International Patent Application Publication No. WO 2011/126808).

[0160] Optionally, one or more sequences may be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence that can be engineered upstream of the polyA sequence and downstream of the coding sequence (see, for example, MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605-619).

[0161] The enhancer can be an approximately 155 bp ApoE enhancer element derived from apolipoprotein E or ApoE. ApoE is an apolipoprotein that mediates the binding, internalization and catabolism of lipoprotein particles and is a ligand for the low density lipoprotein (ApoB/E) receptor and for the ApoE receptor in liver tissues. The genetic enhancer associated with the ApoE gene is a eukaryotic control element that can increase nucleic acid transcription, especially in the liver.

[0162] An enhancer may include an element from a liver control region (eg, HCR-1 or HCR-2) associated with the ApoE gene. For example, the sequence from HCR-1 is found in SEQ ID NO: 17.

[0163] In yet another embodiment, the cassette and/or expression vector further comprises a post-transcriptional regulatory element (PRE). An example of a post-transcriptional regulatory element is the groundhog hepatitis virus post-transcriptional regulatory element (WPRE). For example, the sequence from WPRE is shown in SEQ ID NO: 16. WPRE may be effective in increasing protein (eg, DNase) expression. WPRE can be modified such that it does not form the active form of the truncated X protein that is encoded in WPRE. One such modified WPRE is shown in at least SEQ ID NO: 16 and in the sequence: ApoEHCR enhancer: hAAT>hDNaseI promoter (hyperactive) correct leader-WPRE Xinact (bases 1576-2212 of SEQ ID NO: 30; see also Figures 10A and 10B).

[0164] In some embodiments, the cassettes of the present invention direct expression of a therapeutic amount of a polypeptide encoded by a coding sequence over an extended period, typically over 200 days, and in some cases over 500 days. Expression of a polypeptide encoded by a coding sequence can be measured by any methods known in the art, such as, for example, antibody-based assays such as Western blot or ELISA assay. Again, as a non-limiting example, expression of a polypeptide encoded by a coding sequence in an expression cassette can be measured by a bioassay that determines the enzymatic or biological activity of the polypeptide.

[0165] Upon entering liver cells (eg, hepatocytes), the vectors may remain episomal (i.e., not integrate into the host cell genome) or may integrate into the host cell genome. Non-limiting examples of episomal vectors include adenoviral vectors, and examples of a vector that integrates into the genome of a host cell include retroviral vectors.

[0166] In some embodiments of the present invention, expression vectors containing a nucleotide sequence encoding an enzyme having DNase activity are delivered to cells in the form of liposomes in which the DNA is associated with one or more lipids, such as DOTMA (1,2-diolcyloxypropyl- 3-trimethylammonium bromide) and DOPE (dioleoylphosphatidylethanolamine). In some embodiments, cationic liposomes containing DOTMA are effective in targeting the expression vectors of the present invention to the liver. In some embodiments, lactoferrin and/or poly-L-lysine and/or polyethylenemine and/or chitosan are conjugated to an expression vector, the conjugate effectively targeting the liver upon direct administration to a subject.

[0167] In some embodiments, the liposomes are smaller than the fenestrations of the endothelial layer lining the hepatic sinusoids in the liver.

[0168] In some embodiments, the expression vector is packaged in a nanovector or encapsulated in a nanovector.

[0169] In some embodiments, the expression vector is packaged in phosphoramidite nanoparticles.

[0170] To increase the availability of nucleic acids of the present invention encoding enzymes having DNase activity, various devices have been developed for target cells, including, for example, catheters or implantable materials containing DNA (GD Chapman et al., Circulation Res. 71 :27-33 (1992)) and needleless jet injection devices that deliver a column of fluid directly into the target tissue under high pressure (PA Furth et al., Anal Biochem. 20:365-368 (1992); (HL Vahlsing et al ., J. Immunol. Meth. 175:11-22 (1994) (FD Ledley et al., Cell Biochem. 18A:226 (1994)).

[0171] Another approach for targeted liver delivery of nucleic acids of the present invention encoding enzymes having DNase activity is to use molecular conjugates that consist of protein or synthetic ligands to which a nucleic acid binding agent is attached to specifically target nucleic acids. acids into cells (RJ Cristiano et al., Proc. Natl. Acad. Sci. USA 90:11548-52 (1993); BA Bunnell et al., Somat. Cell Mol. Genet. 18:559-69 (1992); M. Cotten et al., Proc Natl Acad Sci USA 89:6094-98 (1992)). This gene delivery system has been shown to be capable of targeting many cell types through the use of various ligands (RJ Cristiano et al., Proc. Natl. Acad. Sci. USA 90:11548-52 (1993)). For example, lipofection and malarial circumsporozoite protein have been used for liver-specific gene delivery (Z. Ding et al., J. Biol. Chem. 270:3667-76 (1995)).

[0172] The preferred routes of administration of the expression vectors of the present invention are intrahepatic, intravenous and intraarterial administration. However, other routes of administration that target the liver can also be used, such as, for example, enteral (eg, oral), intramuscular, intraperitoneal, and the like.

[0173] The expression vectors of the present invention can be administered directly to the liver by any of the methods recognized in the art, such as direct liver injection, portal vein injection, or tail vein injection.

[0174] The expression vectors of the present invention can be delivered by (1) creating an access site in a mammalian artery, (2) inserting a guidewire into the artery, (3) inserting a sheath through the guidewire into the artery, (4) advancing the catheter through the sheath into the liver, (5) introduction of an expression vector, for example, rAAV virions, through a catheter into the liver, (6) removal of the catheter and sheath from the artery, and (7) restoration of the access site. The catheter can be introduced into the target organ through an appropriate vein or artery. For example, you can access the liver through the portal vein or hepatic artery. You can access the appropriate vein or artery using methods known in the art, such as the Seldinger method. See, for example, Conahan et al., (1977) JAMA 237:446-447, incorporated herein by reference. Other ways of accessing veins and arteries are also known. See, for example, US patent No. 5944695, included in this application by reference.

[0175] In some other embodiments, one or more blood vessels that carry blood to or from the liver are blocked. The closure device may be, for example, a balloon attached to the tip of the catheter. All arteries and veins entering or exiting the liver can be occluded, so the expression vector is delivered by bloodless liver perfusion, whereby the liver is selectively perfused after normal blood flow to the liver is shut off. This can be done by clamping the hepatic artery, portal vein, suprahepatic vena cava, right adrenal vein, and subhepatic vena cava to achieve complete shutdown of the hepatic vessels. The portal vein is then cannulated with an appropriate caliber catheter while an incision is made in the anterior wall of the subhepatic vena cava and a suction cannula is inserted into the vena cava to collect outflow. The liver is then perfused with the pump, preferably in a single pass. The flow rate can vary from 0.1 ml/min per gram of liver to 10 ml/min per gram of liver. A rate of 1 ml/min per gram of liver is preferred and may be increased to ensure satisfactory perfusion of the organ. After perfusion, the portal and vena cava incisions are sutured, and liver revascularization is performed. Cardoso, et al., Human Gene Therapy 4:411-418 at 412 (1993).

[0176] In other embodiments, expression vectors are delivered to the liver by injection into the hepatic artery.

[0177] In the liver organ, parenchymal cells include hepatocytes, Kupffer cells, and epithelial cells lining the bile ducts and bile ducts. The main constituents of the liver parenchyma are multifaceted hepatocytes (also known as liver cells), which are present at least on one side in the hepatic sinusoid and on the other sides in the bile duct. Liver cells that are not parenchymal cells include cells within blood vessels such as endothelial cells or fibroblast cells.

[0178] In the liver, the hepatic vein is the efferent blood vessel because it normally carries blood from the liver to the inferior vena cava. Also in the liver, the portal vein and hepatic arteries are the afferent blood vessels of the liver, as they normally carry blood to the liver.

[0179] The blood vessels of the liver include the portal vein system, which transports blood from the gastrointestinal tract and other internal organs (eg, spleen, pancreas, and gallbladder) to the liver. Another blood vessel in the liver is the hepatic vein. The hepatic vein can also be reached through the inferior vena cava or another blood vessel that eventually connects to the liver. A needle or catheter can be used to introduce the polynucleotide into the vascular system. The injection can be carried out under direct observation after incision and visualization of the blood vessels of the tissues. Alternatively, the catheter can be inserted at a remote site and positioned so that it is in the vasculature that connects to the target tissue. In some embodiments, a deep injection into the vena cava is performed. In another embodiment, the injection can be performed using a needle that passes through intact skin and enters a vessel that supplies or drains blood from the target tissue.

[0180] In some embodiments, delivery is carried out as follows. The liver and portal vein are visualized through the ventral midline incision. The vector/liposome/naked DNA in 1 ml of various solutions containing heparin to prevent clotting is injected into the portal vein with a needle for about 30 seconds.

[0181] The efficiency of liver delivery, expression and secretion of an enzyme having DNase activity can be determined, for example, by analyzing the level of the enzyme in the blood (for example, blood obtained from the retroorbital venous sinus), for example, using immunological assays (for example, assays with antibodies recognizing the enzyme, such as, for example, radioimmunoassay (RIA) (HGH-TGES 100T kit from Nichols Institute, San Juan Capistrano, CA, USA)). In experimental animal models, the efficiency of liver delivery, expression and secretion of an enzyme having DNase activity can be determined, for example, by sacrificing the animals by displacing the cervical vertebrae, and dividing the liver (average weight is 1.5 g) into six sections consisting of two parts of the middle lobe, two parts of the left lateral lobe, the right lateral lobe and the caudal lobe with a small part of the right lateral lobe. Each of the six sections can be placed separately in homogenizing buffer. Homogenates can be centrifuged and the supernatant analyzed for the presence of a foreign gene product (ie, an enzyme having DNase activity). Injections into the liver can be directed into the inferior vena cava, which has been clamped in two places: proximally and distally from the entrance of the hepatic vein into the inferior vena cava. In particular, an inferior vena cava clamp located downstream of blood flow may be placed between the diaphragm and the entry point of the hepatic vein. An inferior vena cava clamp, located upstream of the blood flow, can be placed just in front of the renal vein entry point. Because the veins of other organs, such as the renal veins, enter the inferior vena cava at this site, not all of the injected fluid reaches the liver. In some animals given retrograde injections into the inferior vena cava, the hepatic artery, mesenteric artery, and portal vein may become occluded (obstructed).

Parvovirus vectors, including adeno-associated viral vectors

[0182] In some embodiments, the vector used in the present invention is a parvovirus vector, such as an adeno-associated viral (AAV) vector. The term "parvovirus" as used herein embraces the Parvoviridae family, including the autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Autonomous parvoviruses include, but are not limited to, mouse minute virus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, musky duck parvovirus, B19 virus, and any other autonomous parvoviruses, currently known or discovered later. Other autonomous parvoviruses are known to those skilled in the art. See, for example, Bernard N. Fields et al., Virology, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0183] In some embodiments, the parvovirus vector is a single stranded parvovirus vector, such as an AAV vector. AAV (genus Dependovirus) commonly infects humans (eg, serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (eg, serotypes 1 and 4) or other warm-blooded animals (eg, AAVs from cattle, dogs, horses and sheep). For more information on parvoviruses and other members of the Parvoviridae, see, for example, Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed. 1996).

[0184] The AAV vectors disclosed herein can be derived from any AAV serotype, including combinations of serotypes (eg, "pseudo-typed" AAV), or from different genomes (eg, single-stranded or self-complementary). "Serotype" is traditionally defined based on the absence of cross-reactivity between antibodies to one virus compared to another virus. Such differences in cross-reactivity usually arise from differences in the sequences of capsid proteins/antigenic determinants (for example, due to differences in the sequences of VP1, VP2 and/or VP3). Non-limiting examples of AAV serotypes that can be used to develop AAV expression vectors of the present invention include, for example, AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 , AAV10, AAV11, AAV12, AAVrh10 (as described, for example, in US Patent No. 9790472, International Patent Application Publication Nos. , 2015, 23(12):1877-1887), AAVhu37 (as described, for example, in International Patent Application Publication No. WO2017180857), AAVrh64R1 (as described, for example, in International Patent Application Publication No. WO2017180857), Anc80 (on based on the predicted progenitor of AAV1, AAV2, AAV8 and AAV9 serotypes, see Zinn et al., Cell Rep., 2015, 12(67): 1056-1068), avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV and any other AAV currently known or later discovered. See, for example, Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

[0185] A number of putative new AAV serotypes and clades have recently been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375- 383). The genomic sequences of various AAV serotypes and autonomous parvoviruses, as well as the sequences of terminal repeats, Rep proteins, and capsid subunits, are known in the art, for example, Srivistava et al., (1983) J. Virology 45:555; Chiorini et al., (1998) J. Virology 71:6823; Chiorini et a1, (1999) J. Virology 73:1309; Bantel-Schaal et al., (1999) J. Virology 73:939; Xiao et al., (1999) J. Virology 73:3994; Muramatsu et al., (1996) Virology 221: 208; Shade et al., (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. sci. USA 99:11854; Moris et al., (2004) Virology 33-: 375-383; registration number in GenBank U89790; registration number in GenBank J01901; registration number in GenBank AF043303; registration number in GenBank AF085716; registration number in GenBank NC 006152; registration number in GenBank Y18065; registration number in GenBank NC 006260; registration number in GenBank NC_006261; International Patent Publications No. WO 00/28061, WO 99/61601, WO 98/11244; and US Pat. No. US 6,156,303, the disclosure of which is incorporated herein by reference to study the nucleic acid and amino acid sequences of parvovirus and AAV.

[0186] The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear single stranded DNA molecule that is less than 5000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique coding nucleotide sequences for non-structural replication proteins (Rep) and structural proteins (VP). The VP proteins (VP1, VP2 and VP3) form the capsid. The terminal 145 nucleotides are self-complementary and arranged in such a way that an energetically stable intramolecular duplex forming a T-shaped hairpin can be formed. These hairpin structures function as the starting point for viral DNA replication by acting as primers for the cellular DNA polymerase complex. Upon infection with wtAAV in mammalian cells, the Rep genes (ie, Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have a function in viral genome replication.

[0187] In some embodiments, recombinant AAV vectors (rAAV) contain one or more nucleotide sequences of interest flanked by at least one parvovirus or AAV inverted terminal repeat sequence (ITR). Such rAAV vectors can be replicated and packaged into viral particles when produced in a packaging cell that expresses AAV rep and cap gene products (ie AAV Rep and Cap proteins). The terms "AAV Cap protein" or "AAV capsid protein" as used herein refers to a polypeptide having at least one functional activity of a native AAV Cap protein (eg, VP1, VP2, VP3). Examples of the functional activities of Cap proteins (e.g., VP1, VP2, VP3) include the ability to induce capsid formation, promote accumulation of single-stranded DNA, facilitate packaging of AAV DNA into capsids (i.e., encapsidation), bind to cell receptors, and facilitate cell entry of the virion. hosts.

[0188] In some embodiments, the AAV vectors may include the desired proteins or protein variants. As used herein, the term "variant" refers to an amino acid sequence that is altered by one or more amino acids. A variant may have "conservative" changes, with the substituted amino acid having similar structural or chemical properties, such as replacing leucine with isoleucine. Less commonly, a variant may have "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor variations may also include deletions or insertions of amino acids, or both.

[0189] AAV vectors are packaged in AAV viral capsids. The sequence of the AAV viral capsid protein defines numerous features of a particular AAV vector. For example, the capsid protein influences the structure and assembly of the capsid, interactions with AAV non-structural proteins such as Rep and AAP proteins, interactions with host body fluids and extracellular matrix, clearance of virus from the blood, vascular permeability, antigenicity, reactivity to neutralizing antibodies, tropism for tissue/organ/cell type, cell attachment and internalization efficiency, intracellular transport routes, virion opening rate, and others. The sequence of the capsid protein (eg VP3) can be changed to improve delivery to the liver.

[0190] The AAV constructs may further comprise a sequence encoding one or more capsid proteins (VP1 and/or VP2 and/or VP3 capsid proteins, preferably only the VP3 capsid protein) that package the aforementioned polynucleotide sequence. The sequences encoding the capsid protein(s) for use in the context of the present invention may be taken from any of the 42 known serotypes such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80, or newly developed AAV-like particles, obtained, for example, using capsid shuffling techniques and/or using AAV capsid libraries. For some non-limiting examples of capsid protein sequences, see, for example, US Pat. Nos. 9,790,472; 9677089; 7282199; International Patent Publication Nos. WO 2015/054653, WO2017/180857 (AAV8, AAV9, AAVrh10, AAVhu37, AAVrh64R1), WO2017/180861 (AAVrh10), WO2017/180854 (AAV8 mutants), and Wang et al., Mol. Ther., 2015, 23(12):1877-1887 (AAV8, AAVrh10, AAV3B and AAVLK03). When the sequences encoding the capsid proteins are derived from a different AAV serotype, such as the ITR, the AAV construct is known as a "hybrid" parvovirus genome (i.e., in which the AAV capsid and AAV terminal repeat(s) are derived from different AAVs), as described in International Patent Application Publication No. WO 00/28004 and Chao et al. (2000) Molecular Therapy 2:619.

[0191] In some embodiments, the capsid protein(s) mediate efficient targeting of the AAV vector to the liver. In some embodiments, the capsid protein(s) mediates the preferential targeting of the AAV vector to the liver. Some capsid proteins (eg VP3 from Anc80, AAV8 and AAV3B) are naturally liver specific. The present invention also encompasses the use of AAV capsid mutants that enhance liver targeting and/or liver specificity. Non-limiting examples of such point mutations in the AAV8 capsid sequence include, for example, S279A, S671A, K137R and T252A, as well as the AAV8 capsid mutations described in International Patent Application Publication No. WO2017/180854 (for example, AAV3G1, AAVT20 or AAVTR1, VP3 mutations in amino acids 263-267 [e.g. 263NGTSG267->SGTH or 263NGTSG267->SDTH] and/or amino acids 457-459 [e.g. 457TAN459->SRP] and/or amino acids 455-459 [e.g. 455GGTAN459->DGSGL] and /or amino acids 583-597).

[0192] The disclosed AAV vectors include a nucleic acid encoding an enzyme having DNase activity, such as, for example, DNase I. In various embodiments, the nucleic acid may also include one or more regulatory sequences for expression and secretion of the encoded enzyme, such as eg, promoter, enhancer, polyadenylation signal, internal ribosome entry site (IRES), protein transduction domain (PTD) coding sequence, secretory signal sequence, and the like. Thus, in some embodiments, the nucleic acid may contain a promoter region operably linked to a coding sequence to cause or improve the expression of a protein of interest in transfected cells. Such a promoter may be universal, cell or tissue specific, strong, weak, regulated, chimeric, etc., for example, to ensure efficient and stable protein production in the liver. The promoter may be homologous to the encoded protein or heterologous, although in general the promoters used in the disclosed methods are functional in human cells. Examples of regulated promoters include, without limitation, promoters containing a Tet on/off element, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters. Other promoters that can be used include tissue-specific promoters, such as those for the liver. Non-limiting examples of liver-specific promoters include, for example, albumin (Alb) promoter, human antitrypsin alpha-1 promoter (hAAT), thyroxine-binding globulin (TBG), hepatic E-control region apolipoprotein promoter, apolipoprotein A-II (APOA2) promoter , serpin peptidase inhibitor, clade A member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter; miRNA 122 (miR-122) promoter, IGF-II liver-specific P1 promoter, mouse transthyretin (MTTR) promoter, and alpha-fetoprotein (AFP) promoter. Non-limiting examples of universal promoters include, for example, viral promoters such as the CMV promoter, RSV promoter, SV40 promoter, etc., and cellular promoters such as the phosphoglycerate kinase (PGK) promoter, the EF1a promoter, the CMVE/CAG promoter system, and the β promoter. -actin.

[0193] The AAV constructs of the present invention may also contain undetectable terminal repeats. The term "undetectable terminal repeat" as used herein refers to terminal repeats that are not recognized and defined (i.e., "broken") by Rep AAV proteins such that the definition of the terminal repeat is significantly reduced (e.g., at least about 50 %, 60%, 70%, 80%, 90%, 95%, 98% or more compared to the determined terminal repeat) or excluded. Such undetectable terminal repeats may be naturally occurring terminal repeat sequences (including their altered forms) and, for example, may be derived from parvovirus, including AAV, or may be derived from another virus, or, as a further alternative, may be partially or completely synthetic. The undetectable terminal repeat may be a non-AAV viral sequence that is not recognized by the AAV Rep proteins, or it may be an AAV terminal repeat that has been modified (eg, by insertion, substitution, and/or deletion) such that it is larger not recognized by AAV Rep proteins. In addition, an undetectable terminal repeat may be any terminal repeat that is not detectable under the conditions used to obtain the viral vector. In addition, the AAV terminal repeat can be modified such that AAV Rep detection by proteins is significantly reduced or eliminated. An undetectable terminal repeat can be any inverted repeat sequence that forms a hairpin structure and cannot be cut by AAV Rep proteins.

[0194] Inverted terminal repeats (ITRs) are typically present in at least two copies in an AAV vector, typically flanking an expression cassette containing the nucleotide sequence(s) encoding an enzyme having DNase activity. ITRs are typically located at the 5' and 3' ends of the nucleotide sequence(s) encoding an enzyme having DNase activity, but are not necessarily adjacent to it. ITRs can be the same or different from each other. The term "terminal repeat" includes any viral terminal repeat and/or partially or fully synthetic sequences that form hairpin structures and function as an inverted terminal repeat, such as the "double-D sequence" as described in US Pat. No. 5,478,745, Samulski et al. . "AAV terminal repeat" may be derived from any AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Anc80, or any other AAV known in the art. present or discovered later. The AAV terminal repeat need not have a wild-type sequence (e.g., the wild-type sequence may be altered by insertion, deletion, processing, or missense mutations) as long as the terminal repeat mediates desired functions, such as replication, cleavage, viral packaging, integration, and/ or rescue of proviruses, etc. The vector construct may include one or more (eg, two) AAV terminal repeats, which may be the same or different. In addition, one or more AAV terminal repeats may be of the same AAV serotype as the AAV capsid, or may be different. In specific embodiments, the vector construct included an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and/or Anc80 terminal repeat.

[0195] Parvovirus ITR nucleotide sequences are typically palindromic sequences containing mostly complementary, symmetrically arranged sequences, also referred to as "A", "B", and "C" regions. The ITR functions as the origin of replication, a site that plays a "cis" role in replication, i.e. a recognition site for trans-acting replication proteins, such as, for example, Rep78 (or Rep68), which recognize the palindrome and specific sequences that are internal to the palindrome. One exception in terms of the symmetry of the ITR sequence is the "D" region of the ITR. It is unique (having no complement within one ITR). The formation of single-stranded DNA breaks occurs at the junction between regions A and D. In this region, the synthesis of new DNA is initiated. The D region is usually on one side of the palindrome and provides direction to the nucleic acid replication step. Parvovirus replicating in a mammalian cell typically has two ITR sequences. However, it is possible to design the ITR so that the binding sites are on both strands of the A regions and the D regions are arranged symmetrically, one on each side of the palindrome. On a double-stranded circular DNA template (eg, a plasmid), nucleic acid replication by Rep78 or Rep68 occurs in both directions, and one ITR is sufficient for parvovirus replication of the circular vector. Thus, a single ITR nucleotide sequence can be used in the context of the present invention. However, two or another even number of standard ITRs may be used.

[0196] Functional ITR sequences are essential for replication, rescue, or packaging of AAV virions. The ITR sequences may be wild type sequences or may be at least 80%, 85%, 90%, 95% or 100% identical to the wild type sequences. ITR sequences can be changed, for example, by insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to direct the packaging of the genome into the capsid envelope and then to transduce expression in a host or target cell.

[0197] The AAV vector may consist of single-stranded or double-stranded (self-complementary) DNA. A single stranded nucleic acid molecule is either a sense or an antisense strand because both polarities are equally capable of expressing genes. The AAV vector may further comprise a marker or reporter gene, such as, for example, encoding an antibiotic resistance gene, a fluorescent protein (e.g., GFP), or a gene encoding a chemically, enzymatically, or otherwise detectable and/or selectable product (e.g., lacZ, aph etc.) known in the art.

[0198] AAV expression vectors may contain a nucleic acid that may include a secretory signal sequence that allows the secretion of an encoded enzyme that has DNase activity from the transduced cell. Non-limiting examples of such secretory signal sequences include, for example, DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, β-glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence.

[0199] In some embodiments, an AAV8 or Anc80-based vector is used as the expression vector. The AAV8 and Anc80 vectors are particularly suitable for targeting and expression in the liver. For example, both the AAV8 and Anc80 vectors can transfect liver cells with greater efficiency than the AAV2 vector. Both AAV8 and Anc80 also induce lower amounts of neutralizing antibodies than some other AAV vectors.

[0200] An AAV8 vector or an Anc80 vector containing a nucleotide encoding an enzyme having DNase activity can be administered intrahepatic (for example, by direct injection into an organ) or systemically, for example, by intravenous injection of an AAV8 or Anc80 vector effective for transfecting liver cells and mediating efficient production of an encoded enzyme having DNase activity and its secretion in the periendothelial space and space of Disse. In some embodiments, an Anc80 capsid protein (e.g., an Anc80 VP1 capsid protein containing the sequence of SEQ ID NO: 3 or SEQ ID NO: 9, or an Anc80L65 VP1 capsid protein containing the sequence of SEQ ID NO: 34, or an Anc80L65 VP1 variant capsid protein , containing the sequence of SEQ ID NO: 35) is encoded by the nucleotide sequence in the expression vector. In some embodiments, an AAV8 capsid protein (eg, AAV8 VP1 or AAV8 VP3) is encoded by a nucleotide sequence in an expression vector. Peripheral administration of the AAV vectors of the present invention may include systemic injections such as, for example, intramuscular, intravenous, intraperitoneal, and intra-arterial injections.

[0201] Desired doses of the DNase enzyme encoding the AAV vectors of the present invention can be easily adapted by one of skill in the art, eg, depending on disease state, subject, treatment regimen, etc. In some embodiments, 10 ⁵ to 10 ¹⁴ recombinant viral particles are administered per dose, eg, 10 ⁶ to 10 ¹¹ , 10 ⁷ to 10 ¹¹ , or 10 ⁸ to 10 ¹⁴ . In other embodiments, exemplary doses to achieve therapeutic effects may include titers of at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , or 10 ¹¹ or more recombinant viral particles.

[0202] The exogenous targeting sequence(s) may replace or replace part or all of the major capsid subunit (eg, VP3). As a further alternative, more than one exogenous targeting sequence can be introduced into the virion capsid, eg two, three, four, five or more sequences. In alternative embodiments, insertions and substitutions can be made in minor subunits of the capsid (eg, VP1 and VP2). For AAV capsids, insertions or substitutions in VP2 or VP3 can be made.

[0203] The tropism of a native virion can be reduced or eliminated by insertion or substitution of an amino acid sequence. Alternatively, insertion or replacement of an exogenous amino acid sequence can target the virion to specific cell type(s). The exogenous targeting sequence may be any amino acid sequence encoding a protein or peptide that alters the tropism of the virion. In specific embodiments, the target peptide or protein may be natural or, alternatively, fully or partially synthetic. Examples of peptides and proteins include ligands and other peptides that bind to cell surface receptors present on liver cells, including ligands capable of binding the apolyprotein E Sr-B1 receptor, galactose- and lactose-specific lectins, low-density lipoprotein receptor ligands, asialoglycoprotein (galactose terminal) and the like.

[0204] Alternatively, the exogenous targeting sequence may be an antibody or an antigen-recognizing fragment thereof. As used herein, the term "antibody" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodies may be monoclonal or polyclonal and may be of any origin, including (for example) mouse, rat, rabbit, horse or human, or may be chimeric antibodies. The term "antibody" also encompasses bispecific or "bridge" antibodies known to those skilled in the art. Antibody fragments within the scope of the present invention include, for example, Fab, F(ab')2 and Fc fragments, and corresponding fragments derived from antibodies other than IgG. Such fragments can be obtained by known methods. The exogenous amino acid sequence inserted into the capsid of the virion may be one that facilitates purification or detection of the virion. For example, the exogenous amino acid sequence may include a polyhistidine sequence that can be used to purify the virion on a nickel column, as is known to those skilled in the art, or an antigenic peptide or protein that can be used to purify the virion by standard immunopurification methods. Alternatively, the amino acid sequence may encode a receptor ligand or any other peptide or protein that can be used to purify the modified virion by affinity purification or any other methods known in the art (e.g., purification methods based on different size, density, charge or isoelectric point, ion exchange chromatography or peptide chromatography).

[0205] Alternatively, the exogenous targeting sequence may be an antibody or an antigen-recognizing fragment thereof. As used herein, the term "antibody" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodies may be monoclonal or polyclonal and may be of any origin, including (for example) mouse, rat, rabbit, horse or human, or may be chimeric antibodies. The term "antibody" also encompasses bispecific or "bridge" antibodies known to those skilled in the art. Antibody fragments within the scope of the present invention include, for example, Fab, F(ab')2 and Fc fragments, and corresponding fragments derived from antibodies other than IgG. Such fragments can be obtained by known methods.

[0206] The exogenous amino acid sequence inserted into the capsid of the virion may be one that facilitates purification or detection of the virion. According to this aspect of the invention, it is not necessary that the exogenous amino acid sequence also alter the modified parvovirus virion. For example, the exogenous amino acid sequence may include a polyhistidine sequence that can be used to purify the virion on a nickel column, as is known to those skilled in the art, or an antigenic peptide or protein that can be used to purify the virion by standard immunopurification methods. Alternatively, the amino acid sequence may encode a receptor ligand or any other peptide or protein that can be used to purify the modified virion by affinity purification or any other methods known in the art (e.g., purification methods based on different size, density, charge or isoelectric point, ion exchange chromatography or peptide chromatography).

Adenovirus vectors

[0207] The adenovirus genome is a 36 kb linear double-stranded DNA containing many heavily spliced transcripts. At both ends of the genome are inverted terminal repeats (ITRs). Genes are divided into early (E1-E4) and late (L1-L5) transcripts.

[0208] In some embodiments, recombinant adenoviral vectors have two deleted genes: E1 and E3 (dE1/E3). The E1 deletion renders the viral vector replication defective. E1 can be provided by adenovirus packaging cell lines (eg T 293 or 911). E3 is involved in host immunity evasion and is not essential for virus reproduction. Deletion of E1 and E3 results in a transgene packing capacity of >8 kb. In some embodiments, the constructs comprise a left and a right arm to facilitate homologous recombination of the transgene into an adenoviral plasmid.

[0209] Any of the accepted types of human adenovirus can be used, such as, for example, an Ad5-based adenovirus vector. Ad5-based vectors use the Coxsackie adenovirus receptor (CAR) to enter cells. In some embodiments, human adenovirus type 5 (dE1/E3) is used as a vector. Most adenoviral vectors are designed such that the transgene replaces the Ad E1a, E1b and/or E3 genes; subsequently, the replication-defective vector propagates in human 293 cells, which provide the function of the deleted gene upon transfer. Adenovirus vectors can transduce various tissue types in vivo, including non-dividing, differentiated cells such as liver cells. Ordinary vectors Ad have a large capacity. An example of the use of the Ad vector in a clinical trial includes polynucleotide therapy for antitumor immunization by intramuscular injection (Sterman et al., Hum. Gene Ther . 7:1083-9 (1998)). Additional examples of the use of adenoviral vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther . 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther . 2:205-18 (1995); Alvarez et al., Hum. Gene Ther . 5:597-613 (1997); Topf et al., Gene Ther . 5:507-513 (1998); Sterman et al., Hum. Gene Ther . 7:1083-1089 (1998).

Hepatitis B vectors and other hepatotropic vectors

[0210] In some embodiments, liver-specific delivery and expression of an enzyme having DNase activity can be achieved by using viral vectors that naturally target hepatocytes. Vectors derived from hepatotropic viruses, such as hepatitis B viruses (HBV) or other viruses from the hepadnaviridae family, can be administered via the bloodstream and targeted to hepatocytes using the same receptors as wild-type viruses. Hepatitis B virus (HBV) is the prototype of the hepadnaviridae, a family of small enveloped DNA viruses with strong host and tissue specificity (Ganem, 1996). Hepadnaviruses have been found in mammals such as humans (HBV), marmots (WHV) and ground squirrels (GSHV), as well as birds such as Peking ducks (DHBV) and gray herons (HHBV).

[0211] In one embodiment of the present invention, HBV vectors retain all of the cis-acting elements required for viral genome replication. However, the present invention does not limit the position of the two new cis-acting elements (ie, α-element and β-element) in the specified position on the map. In one embodiment, it is contemplated that to accommodate a larger insert without exceeding the package size limit, the position of new cis-acting elements can be changed without compromising the vector function.

[0212] In one embodiment of the method, a sequence encoding an enzyme having DNase activity is inserted between the α-element and DR2 of the HBV vector. In another embodiment of the method, it is assumed that the sequence encoding the enzyme with DNase activity is inserted between the 5'-epsilon and the α-element of the HBV vector.

Delivery of naked DNA

[0213] In some embodiments, an expression vector containing a sequence encoding an enzyme having DNase activity is administered to a subject as naked DNA. Such naked DNA can be delivered, for example, to a hepatic blood vessel at distal or proximal locations.

Wrapped viral vectors

[0214] In various embodiments of the present invention, an enveloped viral particle can be used to deliver to the liver a nucleic acid encoding an enzyme having DNase activity.

[0215] In some embodiments, the viral particle described herein is derived from a virus of the Retroviridae family. In one particular embodiment, the viral particle described herein is a retroviral particle. In another specific embodiment, the viral particle described herein is a lentiviral particle. Compared to other gene transfer systems, lentiviral and retroviral vectors offer a wide range of advantages, including their ability to transduce various cell types, stably integrate the transferred genetic material into the genome of the target host cell, and express the transduced gene at significant levels. Vectors derived from gamma-retroviruses, such as murine leukemia virus (MLV), have been used in clinical gene therapy studies (Ross et al., Hum. Gen Ther. 7:1781-1790, 1996).

[0216] In one specific embodiment, at least one viral element associated with the nucleotide of interest is a retroviral element. In another specific embodiment, at least one viral element associated with the nucleotide of interest is a lentiviral element. In one particular embodiment, at least one viral element associated with the nucleotide of interest is a Psi (ψ) packing signal.

[0217] In one particular embodiment, the lentiviral particle does not contain gp120 surface coat protein and/or gp41 transmembrane coat protein. In another specific embodiment, the lentiviral particle comprises a mutant gp120 surface coat protein and/or a mutant gp41 transmembrane coat protein.

[0218] In some embodiments, the viral particles described herein are replication deficient and contain only an incomplete genome of the virus from which they are derived. For example, in some embodiments, lentiviral and retroviral particles do not contain the genetic information of the gag, env, or pol genes (which may be involved in viral particle assembly), which is a known minimum requirement for successful lentivirus or retrovirus replication. In these cases, the minimum set of viral proteins needed to assemble the vector particle is provided when transferred using a packaging cell line. In one particular embodiment, the env, tat, vive, vpu, and nef genes are absent from the HIV-1 derived lentiviral particles and are provided upon transfer or rendered inactive by the use of a frameshift mutation(s).

[0219] In some embodiments, the RNA molecule included in the lentiviral or retroviral particles (and encoding an enzyme having DNase activity) included the psi and LTR packaging signal. In order to achieve expression of a nucleotide sequence encoding an enzyme having DNase activity in liver cells, such a sequence is usually placed under the control of non-limiting examples of ubiquitous promoters, including, for example, viral promoters such as the CMV promoter, RSV promoter, SV40 promoter, etc. , and cellular promoters such as the phosphoglycerate kinase (PGK) promoter, the EF1a promoter, the CMVE/CAG promoter system, and the β-actin promoter.

[0220] In some embodiments of lentiviral and retroviral particles, an RNA molecule, along with the proteins encoded by gag and pol, are provided upon transfer by a packaging cell line, then assembled into vector particles that then infect cells, reverse-transcribe the RNA molecule, which includes the nucleotide sequence , encoding an enzyme with DNase activity under the control of a promoter, and either integrate the specified genetic information into the genome of target cells, or remain episomal (if one or more components required for integration are impaired). If the genetic information for the proteins encoded by gag and pol is not present on the transduced RNA molecule, the vector particles are replication deficient, i.e. the transduced cell will thus not generate a new generation of these vector particles, which ensures the safety of clinical use.

[0221] In some embodiments of any of the above methods, one or more viral elements encode human immunodeficiency virus (HIV) component(s), bovine immunodeficiency virus (BIV) component(s), feline immunodeficiency virus (FIV) component(s). ), simian immunodeficiency virus (SIV) component(s), equine infectious anemia virus (EIAV) component(s), mouse stem cell virus (MSCV) component(s), mouse leukemia virus (MLV) component(s), component(s) s) avian leukemia virus (ALV), component(s) of feline leukemia virus (FLV), component(s) of bovine leukemia virus (BLV), component(s) of human T-lymphotropic virus (HTLV), component(s) feline sarcoma virus, component(s) of avian reticuloendotheliosis virus, component(s) of goat arthritis virus (CAEV), and/or component(s) of visna maedi virus (VMV).

[0222] In combination with the viral particles described herein, methods for detecting and/or isolating cells are described herein. In some embodiments, such a method uses viral particles containing a selectable marker (e.g., neomycin resistance) and/or a reporter (e.g., GFP or eGFP) as a nucleotide sequence of interest to target cells using, for example, exposure to a compound for selection or sorting of fluorescence-activated cells (FACS).

[0223] In some embodiments, the retroviral vectors described herein are derived from mouse leukemia virus (MLV). Retroviral vectors encoding MLV are widely available to those skilled in the art, such as PINCO (Grignani et al., 1998) or the pBabe vector series (Morgenstern and Land, 1990).

[0224] In some embodiments, the lentiviral particles described herein are derived from a lentivirus, such as human immunodeficiency virus (HIV). Suitable vectors encoding HIV and other suitable viruses can be easily identified and/or obtained by a person skilled in the art.

[0225] In one specific embodiment, the transfer vector comprises a cytomegalovirus (CMV) enhancer/promoter sequence; R and U5 sequences from HIV 5'LTR; flap HIV-1 signal; internal enhancer; internal promoter; the nucleotide sequence of interest; an element that reacts to the woodchuck hepatitis virus; an amber suppressor tRNA sequence; a U3 element with a deletion of its enhancer sequence; chicken beta-globin insulator; and the R and U5 3′ HIV LTR sequences.

[0226] The present invention also contemplates enveloped viral vectors containing a heterologous targeting sequence or molecule inserted or substituted in the native envelope. A heterologous targeting sequence or molecule may provide an altered tropism to the liver or increase the efficiency of delivery of the vector to the liver.

Methods and modes of introduction of expression vectors encoding the DNase enzyme

[0227] In the methods of the present invention, expression vectors containing an encoding nucleic acid and an enzyme having DNase activity can be administered to a patient at one time or over a series of treatments; one or more times a day.

[0228] The effective amount of the expression vector to be administered will vary from patient to patient. Accordingly, effective amounts are best determined by the physician administering the compositions, and appropriate dosages can be readily determined by one of ordinary skill in the art. Through analysis of blood (serum, plasma) or tissue levels of the enzyme encoded by the vector that has DNase activity and comparison with baseline prior to administration, it can be determined whether the amount administered is too low, in the correct range, or too high. Suitable regimens for initial and subsequent administration also vary, but typical are initial administration followed, if necessary, by subsequent administrations. Subsequent administrations can be administered at various intervals, from daily to weekly, monthly, yearly, and up to once every few years.

[0229] Doses of an expression vector encoding an enzyme having DNase activity depend on the type of condition being treated, the severity and course of these side effects, the patient's medical history, the opinion of the attending physician, and the response to previous treatment, such as, for example, chemotherapy or radiation therapy and therapy with DNase proteins. In some embodiments, an effective amount of the expression vector is an amount that results in a DNase protein level in the hepatic portal vein of 0.5 to 100 mg/L or 1,000 to 200,000 Kunitz units (KU)/L, 0.5 up to 50 mg/l or from 1000 to 100000 Kunitz units (KU)/l, from 1.5 to 50 mg/l or from 3000 to 100000 KU/l, from 10 to 50 mg/l or from 20000 to 100000 KU/ l.

[0230] Administration of an expression vector, e.g., an AAV or viral vector encoding a DNase enzyme and/or a DNase enzyme according to the methods of the present invention may be by any suitable route, including, for example, intravenous, intra-arterial, and intrahepatic administration.

Cancer treatment

[0231] In one aspect, a method of treating or eliminating cancer in a subject suffering from cancer is provided, which comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme that has DNase activity (e.g., DNase I, such as, for example, recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (eg DNase X, DNase gamma or DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence containing a secretory signal sequence). In some embodiments, the DNase enzyme contains the sequence set forth in SEQ ID NO: 2 (mutant precursor sequence containing a secretory signal sequence as well as mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without secretory signal sequence). In some embodiments, DNase enzyme refers to the sequence shown in SEQ ID NO: 5 (mature mutant sequence lacking a secretory signal sequence and containing mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter or a liver-specific enhancer element.

[0232] The amount and method of administration of the expression vectors of the present invention should be effective in treating cancer, for example, by one or more of the following: reduction in tumor size, reduction in tumor growth rate, reduction in rate of metastasis, enhancement of the therapeutic effects of chemotherapy and/or radiation therapy , prolonging life, reducing the rate of weight loss associated with cancer or treatment, or increasing the rate of weight gain.

[0233] In some embodiments, administration of an expression vector encoding an enzyme having DNase activity can be combined with administration of the same enzyme as the protein (e.g., intravenously, intra-arterially, intrahepatic, intramuscularly, intraperitoneally, enterally, etc.). The enzyme can be, for example, any of the DNase enzymes (for example, DNase I (for example, recombinant human DNase I or bovine pancreatic DNase I), DNase I analogues (for example, DNase X, DNase gamma or DNAS1L2), DNase II (for example, DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).

[0234] Methods of treatment using vectors according to the present invention can be used in subjects suffering from a wide range of types of cancer. The present invention is particularly suitable for the treatment of "portal vein cancer", which includes any carcinoma, sarcoma, melanoma or lymphoma arising in or metastasizing in organs, tissues and structures that drain into the portal vein (see figure 1). Non-limiting examples of portal vein cancer include, for example, peritoneal carcinomatosis, lymphomas (e.g., B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphomas, Hodgkin's lymphoma), gastric cancer, colon cancer, intestinal cancer, colorectal cancer, pancreatic cancer, liver cancer, spleen cancer, bile duct cancer, gallbladder cancer, sarcomas in tissue draining the portal vein (eg, angiosarcoma, endotheliosarcoma, hemangiosarcoma, leiomyosarcoma, other sarcomas, and epithelial carcinoma in such tissues), and metastatic liver disease of any origin.

[0235] Non-limiting examples of other cancers that can be treated with the methods of the present invention include, for example, breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g., non-small cell lung cancer (NSCLC)), cancer kidney, thyroid cancer, leukemia (eg, chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia), head and neck cancer, esophageal cancer, rectal cancer, ovarian cancer, uterine endometrial cancer, vaginal cancer, cervical cancer , bladder cancer, neuroblastoma, sarcoma, osteosarcoma, malignant melanoma, squamous cell carcinoma, bone cancer (including both primary bone cancer (eg, osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, adamantinoma, and giant cell tumor) and secondary (metastatic) bone cancer), soft tissue sarcoma, basal cell carcinoma, angiosarcoma, hemangiosa rcoma, myxosarcoma, liposarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, adenocarcinoma, papillary strenocarcinemia cystadenocarcinoma, bronchogenic carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, medullary carcinoma, sarcomutymoma etc.

[0236] For combined cancer treatment, an expression vector, for example, an AAV vector, or a lentiviral vector, or an adenoviral vector encoding an enzyme having DNase activity, can be administered before, concomitantly, or after a chemotherapeutic agent (or radiation therapy).

[0237] In some embodiments, the methods of the present invention are used to prevent cancer in a patient, eg, a patient at high risk of predisposition to cancer.

[0238] In some embodiments, the methods of the present invention are used to prevent cancer following a positive blood-based DNA screening test (eg, liquid biopsy) to detect predisposition to cancer.

[0239] In some embodiments, the methods of the present invention are used to prevent cancer after evaluating a tumor susceptibility gene. Examples of tumor susceptibility genes include, but are not limited to, ABL1 (ABL), ABL2(ABLL, ARG), AKAP13 (HT31, LBC. BRX), ARAF1, ARHGEF5 (TIM), ATF1, AXL, BCL2, BRAF (BRAF1) genes. , RAFB1), BRCA1, BRCA2(FANCD1), BRIP1, CBL (CBL2), CSF1R (CSF-1, FMS, MCSF), DAPK1 (DAPK), DEK (D6S231E), DUSP6(MKP3,PYST1), EGF, EGFR ( ERBB, ERBB1), ERBB3 (HER3), ERG, ETS1, ETS2, EWSR1 (EWS, ES, PNE), FES (FPS), FGF4 (HSTF1,KFGF), FGFR1, FGFR10P (FOP), FLCN, FOS (c- fos), FRAP1, FUS (TLS), HRAS, GLI1, GLI2, GPC3, HER2 (ERBB2, TKR1, NEU), HGF (SF), IRF4 (LSIRF, MUM1), JUNB, KIT(SCFR), KRAS2 (RASK2) , LCK, LCO, MAP3K8(TPL2, COT, EST), MCF2 (DBL), MDM2, MET(HGFR, RCCP2), MLH type, MMD genes, MOS (MSV), MRAS (RRAS3), MSH type, MYB genes ( AMV), MYC, MYCL1 (LMYC), MYCN, NCOA4 (ELE1, ARA70, PTC3), NF1 type, NMYC, NRAS, NTRK1 (TRK, TRKA), NUP214 (CAN, D9S46E), OVC, TP53 (P53), PALB2 , PAX3 (HUP2), STAT1, PDGFB (SIS), PIM genes, PML (MYL), PMS (PMSL) genes, PPM1D (WIP1), PTEN (MMAC1), PVT1, RAF1 (CRAF), RB1 ( RB), RET, RRAS2 (TC21), ROS1 (ROS, MCF3), SMAD type, SMARCB1(SNF5, INI1), SMURF1, SRC (AVS), STAT1, STAT3, STAT5, TDGF1 (CRGF), TGFBR2, THRA (ERBA , EAR7, etc.), TFG (TRKT3), TIF1 (TRIM24, TIF1A), TNC (TN, HXB), TRK, TUSC3, USP6 (TRE2), WNT1 (INT1), WT1 and VHL.

[0240] A susceptibility gene can be assessed using one or more of the following methods: next generation sequencing, whole genome sequencing, exome sequencing, ELISA-based method, and PCR amplification. Other methods can also be used to evaluate a susceptibility gene.

[0241] In another aspect, the present invention provides a method for treating or eliminating cancer in a subject in need thereof, said method comprising administering to the subject an expression vector containing a nucleic acid encoding an enzyme having deoxyribonuclease (DNase) activity, said vector expression ensures the synthesis of the DNase enzyme in the liver of the specified subject. In some embodiments, cancer is accompanied by an increase in the total level or relative content of extracellular DNA (ecDNA) of microbial origin in the blood of the specified subject. In several embodiments, the method includes determining the level of microbial-derived cfDNA, or the ratio of microbial-derived cfDNA to the total level of cfDNA in the subject's blood. Non-limiting examples of methods for detecting cfDNA of microbial origin include, for example, PCR, RT-PCR, next generation sequencing (NGS) and whole genome sequencing (WGS). In some embodiments, cfDNA of microbial origin can be detected, for example, by PCR or RT-PCR for 16S ribosomal RNA, 16S ribosomal DNA, or an intergenic spacer region. In some embodiments, determining the total level or relative content of microbial-derived cfDNA comprises comparing the level of microbial-derived cfDNA or the ratio of microbial-derived cfDNA to the total blood level of cfDNA in the subject with a control level or ratio (e.g., a predetermined standard or a corresponding level or ratio defined for age-matched non-cancer subjects).

[0242] In one embodiment of any of the methods of the present invention, the subject is a human.

Elimination of toxicity of anti-cancer chemotherapy or radiation therapy

[0243] In one aspect, the present invention provides a method for preventing or eliminating toxicity or a condition associated with cytostatic and/or cytotoxic chemotherapy in a subject with cancer and receiving or conditionally receiving said therapy, the method comprising administering to the subject a therapeutically effective amount of an expression vector coding for an enzyme that has DNase activity (for example, DNase I, (for example, recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (for example, DNase X, DNase gamma or DNAS1L2), DNase II (for example , DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence containing a secretory signal sequence). In some embodiments, the DNase enzyme contains the sequence set forth in SEQ ID NO: 2 (mutant precursor sequence containing a secretory signal sequence as well as mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without secretory signal sequence). In some embodiments, DNase enzyme refers to the sequence shown in SEQ ID NO: 5 (mature mutant sequence lacking a secretory signal sequence and containing mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter and/or a liver-specific enhancer element.

[0244] The amount and method of administration of the expression vector is effective in eliminating at least one side effect of said chemotherapy.

[0245] A condition associated with cytostatic and/or cytotoxic chemotherapy may include, for example, (i) a catabolic state leading to weight loss, (ii) bone marrow toxicity and/or catabolic changes in blood biochemistry, (iii) cardiotoxicity (eg, myocardial necrosis), (iv) gastrointestinal toxicity, (v) immune suppression, (vi) neutropenia, and (vii) weight loss.

[0246] In some embodiments, the cancer is associated with elevated levels of cfDNA in the blood or cerebrospinal fluid (CSF) or intestines of a patient, and the level is above a control level (e.g., the level of cfDNA in the blood, CSF, or intestines of a healthy individual of the same age or average level cfDNA in the blood, cerebrospinal fluid, or intestines of several healthy individuals of the same age).

[0247] In some embodiments, the effects of chemotherapy are associated with increased levels of cfDNA in the blood or cerebrospinal fluid, ascitic fluid, or intestines of a patient that is above a control level (e.g., levels of cfDNA in the blood or cerebrospinal fluid or intestines of an age-matched individual with a similar cancer profile). not receiving chemotherapy, or the average level of cfDNA in the blood, cerebrospinal fluid, or intestines of several age-matched individuals who have cancer but are not receiving chemotherapy).

[0248] In another aspect, the present invention provides a method for increasing the efficacy of cytostatic and/or cytotoxic chemotherapy in a subject having cancer and receiving or conditionally receiving said therapy, the method comprising administering to the subject a therapeutically effective amount of an expression vector (e.g., Anc80 vector or AAV8 AAV or a lentiviral vector or an adenoviral vector) encoding an enzyme that has DNase activity (for example, DNase I, (for example, recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (for example, DNase X, DNase gamma or DNAS1L2), DNase II (DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase). The amount and method of administration of the expression vector is effective to prevent or alleviate at least one side effect of said chemotherapy and to prevent or alleviate toxicity associated with said chemotherapy. In some embodiments, any of the DNase enzymes (e.g., DNase I, (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma, or DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase) is also administered, for example intravenously.

[0249] The expression vectors of the present invention can be used to alleviate toxicity and/or increase the efficacy of a wide variety of different chemotherapeutic agents. Non-limiting examples of such agents include antimetabolites such as pyrimidine analogs (eg, 5-fluorouracil [5-FU], floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists, and related inhibitors (eg, mercaptopurine, thioguanine, pentostatin, and 2 -chlordeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (eg vinblastine, vincristine and vinorelbine), microtubule disruptors such as taxanes (eg paclitaxel, doxetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins (eg, etoposide, teniposide), DNA disruptors (eg, actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, nedaplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, aclarubicin, pyrarubicin , hexamethylmelaminoxaliplatin, ifosfamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, nimustine, ranimustine, estramustine, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics (eg dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mitramycin), pleomycin, peplomycin, mitomycins (eg mitomycin C), actinomycins (eg actinomycin D), zinostatin stimalamer); enzymes (eg, L-asparaginase); neocarcinostatin; antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (eg mechlorethamine, cyclophosphamide and analogs, imidazole carboxamide, melphalan, chlorambucil, nitrogen mustard-N-oxide hydrochloride, ifosfamide), ethyleneimines and methylmelamines (eg hexamethylmelamine, thiotepa, carboquone, triethylene thiophosphoramide), alkylsulfonates (eg busulfan, isoprosulfan tosylate), nitrosoureas (eg carmustine (BCNU) and its analogues, streptozocin), trazene-dacarbazinine (DTIC); epoxide type compounds (eg mitobronitol); antiproliferative/antimitotic antimetabolites such as folic acid analogs (eg methotrexate); platinum coordination complexes (eg, cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (eg, estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (eg, letrozole, anastrozole); antisecretory agents (eg, breveldin); immunosuppressive agents (eg, cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (eg, TNP-470, genistein, bevacizumab) and growth factor inhibitors (eg, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (eg, trastuzumab); cell cycle inhibitors and differentiation inducers (eg tretinoin); mTOR inhibitors, topoisomerase inhibitors (eg, doxorubicin (adriamycin), amsacrine, campotothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, irinotecan); kinase inhibitors of growth factor signal transduction; inducers of mitochondrial dysfunction; agents that destroy chromatin; sobuzoxan; tretinoin; pentostatin; flutamide; sodium porfimer; fadrozole; procarbazine; aceglatone; compounds for radioimmunotherapy (RIT) (eg, ibritumomab tiuxetan, iodine ( ¹³¹ I) tositumomab); and compounds for targeted radionuclide therapy (TRT) (eg, samarium-153-EDTMP, strontium-89-chloride).

[0250] Non-limiting examples of side effects of cytostatic and/or cytotoxic chemotherapy that can be prevented or ameliorated by administration of an expression vector according to the methods of the present invention include, for example, bone marrow toxicity, neutropenia, myelopathy (e.g., leukopenia, granulocytopenia, lymphopenia, thrombocytopenia, erythropenia); hematopathy (eg, plasma fibrinogenopenia); catabolic changes in blood biochemistry; disorders of the gastrointestinal tract (eg, nausea, vomiting, anorexia, weight loss, feeling of heaviness in the stomach, diarrhea, constipation, stomatitis, esophagitis); pulmonary insufficiency (eg, chronic pneumonia, pulmonary fibrosis, ARDS, ALS, pulmonary embolism); dermatopathy (eg, keratinization, pachymenia, chromatosis, epilation, rash, change of nails, alopecia due to cancer); nervous system disorders (eg, paresthesia, depression, deep areflexia, neuroparalysis, hearing impairment, allolalia, disorientation, neurological manifestations, cerebellar ataxia, drowsiness, coma, dizziness, frequent urination, frequent urge to defecate); endocrine disorders (eg, pituitary disorder, adrenal disorder, hyperglycemia, hypoglycemia); genital disorders (eg, hyposexuality, oligospermia, gynecomastia, menstrual disorders); cardiovascular disorders (eg, myocardial necrosis, cardiomyopathy, arrhythmia, low blood pressure, tachycardia, heart failure); hepatopathy, pancreatic disorder, nephropathy, bladder disorder, hyperuricemia, immunosuppression, and infections.

[0251] The expression vectors of the present invention can also be used to alleviate the toxicity and increase the efficacy of various types of radiation therapy, including, for example, external beam radiation therapy (EBRT or XRT), brachytherapy/sealed source radiation therapy, and systemic radioisotope therapy/radiotherapy with open source. Non-limiting examples of side effects of radiation therapy that can be prevented or alleviated by the introduction of an expression vector in accordance with the methods of the present invention include, for example, skin irritation or damage, fatigue, nausea, vomiting, fibrosis, intestinal damage, memory loss, infertility, and secondary crayfish.

[0252] The expression vectors of the present invention can be used to improve the effectiveness of cancer immunotherapy, including, for example, antibodies (naked monoclonal antibodies, conjugated monoclonal antibodies, chemically labeled antibodies, bispecific monoclonal antibodies), cancer vaccines, immune checkpoint inhibitors (drugs targeting PD-1 or PD-L1, CTLA-4), non-specific immunotherapeutic drugs and adjuvants, CAR-T therapies.

Treatment of neurodegeneration

[0253] In one aspect, the present invention provides a method for treating or alleviating neurodegeneration. The term "neurodegeneration" as used herein refers to a distinct clinical pathological condition with progressive loss of neuronal structure and/or function, including neuronal death. Neurodegeneration can be primary or secondary.

[0254] In some embodiments, neurodegeneration is associated with elevated levels of cfDNA (e.g., microbial cfDNA or human cfDNA) in the blood or cerebrospinal fluid (CSF) or intestines of the patient, the level being above a control level (e.g., blood cfDNA, CSF, or the intestines of a healthy individual of the same age or the average level of cfDNA in the blood or cerebrospinal fluid or the intestines of several healthy individuals of the same age). In one embodiment, the neurodegeneration is associated with a neurodegenerative disorder. Non-limiting examples covering neurodegenerative diseases include, for example, Alzheimer's disease (e.g., late Alzheimer's disease), mild cognitive impairment (MCI), Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), prion-induced diseases, progressive supranuclear palsy (PSP), progressive supranuclear palsy (Steele-Richardson-Olszewski), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), agyrophilic granule disease (AGD), Pick's disease (PiD), frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism-17 (FTDP-17), multiple sclerosis, CADASIL syndrome, ankylosing spondylitis, dental rubro-pallidoluis atrophy, Kennedy's disease, Friedreich's ataxia, cerebellar ataxia, spinocerebellar ataxia, dementia with Lewy bodies, vascular dementia, Danish-type familial dementia, British-type familial dementia, spinal muscle atrophy, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, corticodentatonic degeneration, Hallervorden-Spatz disease, progressive familial myoclonic epilepsy, strionigral degeneration, torsion dystonia (eg, torsion spasm; deforming muscular dystonia), spastic torticollis and other dyskinesias, familial tremor, Gilles de la Tourette syndrome, degeneration of the cerebellar cortex, spinocerebellar degeneration (eg, Friedreich's ataxia and related disorders), Shy-Drager syndrome, primary lateral sclerosis, hereditary spastic paraplegia, peroneal amyotrophy (Charcot-Marie-Tooth), interstitial hypertrophic polyneuropathy (Dejerine-Sotta), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa) and hereditary atrophy of the optic nerve (Leber's disease), secondary neurodegeneration caused by necrosis (for example, destruction of neurons by neoplasm , edema, hemorrhage, stroke, trauma, immune attack, hypoxia, poisoning, metabolic disorders, and infections), and various taupathies (eg, primary age-related tauopathy (PART)/senile dementia with a predominance of neurofibrillary glomeruli, with NFTs similar to AD, but without plaques, CTE, more zn Lytico-Body, ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis). In some embodiments, neurodegeneration is associated with dysfunction of the nervous system, such as, for example, schizophrenia or bipolar disorder, depressive disorder, autism, autism spectrum disorders, chronic fatigue syndrome, obsessive-compulsive disorder, generalized anxiety disorder (GAD), major depressive disorder ( MDD) or social anxiety disorder (SAD). In some embodiments, the neurodegenerative disorder is caused by, secondary to, or associated with amyloidosis. In some embodiments, a neurodegenerative disorder is a protein misfolding disease. In some embodiments, the neurodegenerative disorder is caused by, secondary to, or associated with diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, amyloidosis (e.g., amyloidosis with hereditary cerebral hemorrhage, primary systemic amyloidosis, secondary systemic amyloidosis, serum amyloidosis, senile systemic amyloidosis, hemodialysis-associated amyloidosis, Finnish-type hereditary systemic amyloidosis, atrial amyloidosis, lysozyme systemic amyloidosis, insulin-related amyloidosis, fibrinogen α-chain amyloidosis), asthma, or prion disease.

[0255] In some embodiments, neurodegeneration is caused by, secondary to, or associated with the formation of misfolded protein due to the presence of DNA (eg, human, microbial, extracellular, or intracellular). In some embodiments, neurodegeneration is caused by the formation of a misfolded protein due to the presence of DNA (eg, human, microbial, extracellular, or intracellular).

[0256] In some embodiments, neurodegeneration is caused by, secondary to, or associated with the formation of misfolded protein, including, but not limited to, β-amyloid, tau protein, α-synuclein, SOD1, TDP-43, IAPP, ADan, ABri, fusion protein (FUS) in sarcoma, Notch3, glial fibrillary acidic protein, seipin, transthyretin, serpins, apolipoproteins, amyloid β-peptide, lactoferrin, galectin-7, corneodesmosin.

[0257] In some embodiments, the methods of the present invention can be used to prevent the development of a neurodegenerative disease or other protein misfolding disease after evaluating the presence or activity of a predisposition gene associated with the disease. Examples of disease-associated susceptibility genes include, but are not limited to, ADAR1, MDA5 (IFIH1), RNAseH subunits, SamHD1, TREX, TBK1, Optineurin, P62 (sequestosome 1), progranulin, TDP43, FUS, VCP, CHMP2B, profilin -1, amyloid-β, Tau, α-synuclein, PINK, Parkin, LRRK2, DJ-1, GBA, ATPA13A2, EXOSCIII, TSEN2, TBC1D23, risk factor alleles, PLCG2, TREM2, APOE, TOMM40, IL-33, glucocerebrosidase , ataxin2, C9orf72, SOD1 and FUS. A predisposition gene can be assessed using one or more of the following methods: next generation sequencing, whole genome sequencing, exome sequencing, ELISA-based method, and PCR amplification. Other methods can also be used to evaluate a susceptibility gene.

[0258] In one embodiment of the above methods, prevention or treatment of the formation of misfolded protein aggregates in biological fluids and tissues of mammals is achieved through the use of the expression vectors described herein, including vectors containing the sequence of SEQ ID NO: 30 (enhancer ApoEHCR-hAAT promoter -hDNaseI (hyperactive) correct leader-WPRE Xinact) or SEQ ID NO: 31 (ApoEHCR enhancer-hAAT-hDNaseI wild-type promoter-WPRE Xinact5653). In the sequence of SEQ ID NO: 30, bases 1-320 correspond to the APOE HCR enhancer, bases 321-717 correspond to the human alpha-1 antitrypsin promoter, bases 718-726 correspond to the Kozak sequence, bases 727-1575 correspond to a hyperactive human DNase I variant with a natural fully correct leader sequence, and bases 1576-2212 correspond to the inactivated WPRE X protein. In SEQ ID NO: 31, bases 1-320 correspond to the APOE HCR enhancer, bases 321-717 correspond to the human alpha-1 antitrypsin promoter, bases 718-726 correspond to the sequence Kozak, bases 727-1575 correspond to wild-type human DNase I with a natural fully correct leader sequence, and bases 1576-2212 correspond to the inactivated WPRE X protein.

[0259] As described in International Patent Application Publication No. WO 2016/190780, cfDNA from the gut, blood, and cerebrospinal fluid of patients suffering from neurodegeneration induces neuronal cell death and apoptosis, and treatment with a recombinant DNase protein that degrades such cfDNA improves nerve function. systems in these patients. As shown in the Examples section below, the use of the vectors of the present invention for liver-specific DNase expression provides an additional improvement in treatment over DNase protein administration.

[0260] Evaluation of the effectiveness of the treatment of neurodegeneration and neuroinflammation can be carried out using any methods known in the art, for example, in accordance with generally accepted clinical diagnostic criteria for cognitive decline, such as MMSE, PANSS, physical function and / or functional tasks ( see, for example, Holmes et al., (1999) The British Journal of Psychiatry, 174(1), 45-50; Os et al., (2006) Acta Psychiatrica Scandinavica, 113(2), 91-95; O 'Shea et al., (2002) Physical therapy, 82(9), 888-897; Rochester et al., Arch. Phys. Med. Rehabil. (2004) 85(10), 1578-1585).

[0261] In some embodiments of the present invention, neurodegeneration is caused or aggravated by increased intestinal permeability and translocation of intestinal bacteria and DAMPs of microbial origin, including microbial cfDNA. Such cfDNA can cause neuroinflammation and a range of neurodegenerative diseases. cfDNA derived from the gut microbiota undergoes a "first pass" through the portosinusoidal circulation in the liver. The NLRP3 inflammasome is considered a key factor in the development of neuroinflammation. For example, the NLRP3 inflammasome can undergo significant activation in the brain, such as in the cerebral cortex. Aberrant activation of NLRP3 inflammasome signaling has been demonstrated to contribute to the pathology of a wide range of neurological diseases (Song, 2017).

[0262] In some embodiments, the present invention provides a method of treating neurodegeneration in a subject suffering from neurodegeneration, which comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme that has DNase activity (e.g., DNase I, (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (eg DNase X, DNase gamma or DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase). The amount and method of administration of the expression vector (eg, AAV vector, or lentiviral vector, or adenoviral vector, or liposome, or naked DNA) should be effective to treat one or more of the symptoms or effects of such neurodegeneration.

[0263] Without being limited by theory, after delivering a nucleic acid encoding an enzyme having DNase activity to the liver using the vectors of the present invention, the enzyme is produced by liver cells and secreted into the periendothelial space and space of Disse, where the enzyme effectively degrades the concentrated pool of cfDNA, present in them.

[0264] In some embodiments, the use of the expression vectors of the present invention is combined with administration (e.g., intravenous, intra-arterial, intrahepatic, intramuscular, intraperitoneal, or enteral administration) of a DNase protein (e.g., DNase I, (e.g., recombinant human DNase I or DNase I bovine pancreas), DNase I analogs (eg DNase X, DNase gamma or DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase). Without being limited by theory, systemic administration of DNase protein in combination with the above described DNase production in liver cells using the expression vectors of the present invention rapidly degrades cfDNA both in the circulation and in any concentrated pool of cfDNA in the liver.

[0265] In some embodiments, expression vectors encoding an enzyme having DNase activity can be administered in combination with other treatments suitable for the treatment of neurodegenerative diseases or other encompassed nervous system dysfunctions (e.g., bipolar disorder, migraine, schizophrenia, epilepsy). Non-limiting examples of such adjunctive treatments include, for example, histone acetyltransferase activators, cyclin-dependent protein kinase 5 inhibitors, neurotrophin mimetics, semaphorin-4D blockers, microsomal prostaglandin E-synthase-1 inhibitors, levodopa, and N-methyl-d-aspartate (NMDA) receptor inhibitors (e.g. , memantine).

Treatment of delayed-type hypersensitivity reactions

[0266] In one aspect, a method is provided for treating a delayed-type hypersensitivity reaction (e.g., graft-versus-host disease (GVHD)) of a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme that has DNase activity ( DNase I such as, for example, recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (eg DNase X, DNase gamma or DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence containing a secretory signal sequence). In some embodiments, the DNase enzyme contains the sequence set forth in SEQ ID NO: 2 (mutant precursor sequence containing a secretory signal sequence as well as mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without secretory signal sequence). In some embodiments, DNase enzyme refers to the sequence shown in SEQ ID NO: 5 (mature mutant sequence lacking a secretory signal sequence and containing mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter or a liver-specific enhancer element.

[0267] While not wishing to be bound by theory, neutrophil activation can release granular proteins along with double-stranded DNA (dsDNA) to form extracellular fibers known as NETs. Complement is activated by NET. Complement activation can lead to the symptoms seen in delayed-type hypersensitivity reactions.

[0268] In some embodiments, a delayed-type hypersensitivity reaction is associated with an elevated level of cfDNA in the blood or CSF or intestines of the patient, the level being above a control level (e.g., the level of cfDNA in the blood or cerebrospinal fluid or intestines of a healthy individual of the same age or average level cfDNA in the blood or CSF or intestines of several healthy individuals of the same age). The introduction of an expression vector encoding an enzyme with DNase activity can inhibit the formation of NET and lead to less complement activation.

[0269] Without being limited by theory, systemic administration of the DNase enzyme, in combination with the above-described production of DNase in liver cells using expression vectors, more rapidly degrades cfDNA both in the bloodstream and in any concentrated pool of cfDNA in the liver.

[0270] The expression vector encoding the DNase enzyme can also be administered in combination with one or more other treatments suitable for the treatment of delayed type hypersensitivity reaction, such as, for example, cyclosporine, tacrolimus (FK506), methylprednisolone, prednisone, ATG, sirolimus , mycophenolate mofetil, anti-interleukin-2 (IL-2) receptor, anti-CD5-specific immunotoxin, T-cell immunotoxin containing ricin A-chain (XomaZyme), addition of ex vivo cultured mesenchymal cells obtained from unrelated donors, to conventional steroid therapy, and any combination thereof.

Treatment of atherosclerosis

[0271] In one aspect, provided is a method for treating atherosclerosis in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme that has DNase activity (e.g., DNase I, such as, for example, recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (eg DNase X, DNase gamma or DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence containing a secretory signal sequence). In some embodiments, the DNase enzyme contains the sequence set forth in SEQ ID NO: 2 (mutant precursor sequence containing a secretory signal sequence as well as mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without secretory signal sequence). In some embodiments, DNase enzyme refers to the sequence shown in SEQ ID NO: 5 (mature mutant sequence lacking a secretory signal sequence and containing mutations that attenuate actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter or a liver-specific enhancer element.

[0272] In some embodiments, atherosclerosis is associated with an elevated level of cfDNA in the blood or CSF or intestines of a patient, the level being above a control level (e.g., the level of cfDNA in the blood or cerebrospinal fluid or intestines of a healthy individual of the same age, or the average level of cfDNA in the blood or cerebrospinal fluid or intestines of several healthy individuals of the same age).

[0273] An expression vector, such as Anc80 or another AAV or lentiviral vector encoding a DNase enzyme, can optionally be administered with an additional DNase enzyme. In some embodiments of any of the DNase enzymes (e.g., DNase I, (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma, or DNAS1L2), DNase II (eg DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin or acetylcholinesterase) is also administered, for example intravenously. Without being limited by theory, systemic administration of the DNase enzyme, in combination with the above-described DNase production in liver cells, rapidly degrades cfDNA both in the circulation and in any concentrated pool of cfDNA in the liver.

[0274] The expression vector encoding the DNase enzyme can also be administered in combination with other treatments suitable for the treatment of atherosclerosis.

Pharmaceutical compositions, formulations and dosage forms

[0275] The AAV vectors described herein can be administered in any suitable form, for example, in the form of a liquid solution or suspension, in the form of a solid form suitable for dissolution or suspension in a liquid prior to injection. The vectors may be formulated with any suitable and pharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc. For example, for injection, a suitable carrier or diluent may be isotonic saline, a buffer, sterile pyrogen-free water, or, for example, sterile pyrogen-free phosphate buffered saline. Expression vectors can be formulated into pharmaceutical compositions and dosage forms. Such pharmaceutical compositions and dosage forms may be prepared in any conventional manner using one or more physiologically acceptable carriers and/or excipients. The vector particles can be prepared for administration, for example by injection. Pharmaceutically acceptable carriers are defined in part by the particular composition administered, as well as by the particular route used to administer the composition. Accordingly, there is a wide range of suitable formulations of pharmaceutical compositions available, as described below (see, for example, Remington's Pharmaceutical Sciences , 17th ed., 1989).

[0276] Formulations also include suspensions in liquid or emulsified liquids. Active ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the composition may contain minor amounts of adjuvants such as wetting or emulsifying agents, pH buffering agents, stabilizing agents, or other agents that enhance the effectiveness of the pharmaceutical composition.

[0277] The compositions used in the methods of the present invention may conveniently be presented in unit dosage form and may be prepared by methods known in the art. The amount of active ingredients that can be combined with the carrier material to form a single dosage form will vary depending on the patient being treated and the particular route of administration. The amount of active ingredients that can be combined with the carrier material to form a single dosage form is usually that amount of the compound that produces a therapeutic effect.

[0278] Pharmaceutical compositions suitable for parenteral administration may additionally contain one or more additional active ingredients (for example, a DNase protein or other active compound) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders from which sterile injectable solutions or dispersions may be prepared immediately before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes to render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions according to the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

[0279] These compositions may also contain preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be necessary to include isotonic agents such as sugars, sodium chloride, and the like in the compositions.

EXAMPLES

[0280] The present invention is also described and demonstrated using the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or any exemplary term. Likewise, the present invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the present invention may become apparent to those skilled in the art upon reading this disclosure, and such changes may be made without departing from the scope and spirit of the present invention. Thus, the present invention is to be limited only by the terms of the appended claims together with the full scope of the equivalents to which are listed in this claims.

EXAMPLE 1 Limited ability of DNase enzyme to cleave circulating extracellular DNA (cfDNA) in patients with tumor

[0281] Nine patients with late-stage metastatic disease admitted to the Department of Thoracic Surgery at the Kosciuszko Hospital (St. Petersburg, Russia) underwent a three-week course of intravenous treatment with bovine pancreatic DNase I protein with a specific activity of 2500 KU/mg (Samson , Russia). The treatment was carried out according to the following schedule: in the first week, 300 mg was administered daily through six 30-minute intravenous infusions; at week 2, 450 mg was administered daily via six 30-minute intravenous infusions; and in week III, 600 mg was administered daily by six 30-minute intravenous infusions.

[0282] Electrophoretic profiling of circulating cfDNA was performed for each patient before the first DNase I protein infusion, at the end of week II, and immediately after the last DNase I protein infusion. DNA was isolated from blood plasma by the classical phenol-chloroform method. Blood cfDNA electrophoresis was performed in 1% agarose gel. DNA was visualized with ethidium bromide. Treatment efficacy was assessed one week after the last DNase I protein infusion using helical computed tomography based on RECIST criteria (see, for example, RECIST 1.1, published January 2009, http://www.irrecist.com). Summary patient data and individual blood electrophoretic data are presented in Figures 2A-2IJ. In general, according to the results of RECIST, there was a slight decrease in the diameter of solid tumors (figures 2A-2H). Most patients still had cfDNA in their circulation after the third week.

[0283] Despite continuous intravenous infusions of high doses of DNase I enzyme with dose escalation, only one patient with recurrent rectal carcinoma (PVIII) had complete clearance of circulating cfDNA in plasma, measured by gel electrophoresis. Patient PVIII had stable disease with no signs of progression according to the RECIST criteria. Three other patients who were qualified as patients with stable disease according to the RECIST criteria (PIX, PVII, PI) had little or no increase in circulating plasma cfDNA as measured by gel electrophoresis.

[0284] Five patients who were qualified as patients with progressive disease according to the RECIST criteria (PII, PIII, PIV, PV, and PVI) had a significant increase in circulating plasma cfDNA as measured by gel electrophoresis. In addition, in 8 of 9 patients (88%), three-week continuous intravenous infusions of high doses of DNase enzyme with increasing doses did not provide therapeutically effective clearance of circulating cfDNA in plasma.

EXAMPLE 2. Accumulation of extracellular DNA in the liver of animals with a tumor

[0285] Approximately 12×10 ⁶ human colon carcinoma LS174T cells were inoculated subcutaneously into the left flank of each of twelve female nu/nu mice (IBC RA breed) at 8 weeks of age. Mice received a single intravenous injection of 99 mTc-labeled anti-DNA antibodies according to the following schedule: three mice received an injection at D5, three mice received an injection at D10, three mice received an injection at D15, and three mice received an injection at D25. Mice were sacrificed two hours after injection and tissues and blood were collected for gamma radiometry.

[0286] DNA-binding antibodies AC-30-10 (Genetex) were labeled with 99mTc as follows: 0.024 mg/ml antibody solution in 0.3 M citrate buffer (pH 4.2) was mixed with 1 mCi of 99mTc-pertechnetate in 0, 25 ml of saline, and mixed with a solution of 10 mg / ml of SnCl2 dihydrate in 0.1 N. HCl. After 5 min incubation at room temperature, 1.9 ml of saline and 825 μl of IgM solution were added. Doses for single injections were prepared as follows: 16.6 μCi (36852000CPM, approximately 0.047 μg 99mTc-IgM) of 99mTc-IgM solution was mixed with 3.3 μg of unlabeled IgM and diluted with saline to a volume of 100 μl. The gamma signal was quantified in each tissue using an automatic gamma counter. Measurements were made in units of CMP. 99mTc IgM concentrations were quantified based on gamma radiometry, tissue weight, specific radioactivity of administered IgM preparations, and 99mTc half-life.

[0287] The distribution of radioactivity in tissues is presented in Table 1 below:

Table 1 Accumulation of anti-DNA antibodies in tumor-bearing mice (% injected dose/g tissue)

D5 D10 D15 D25 Brain 0.020±0.0 0.056±0.04 0.078±0.05 0.507±0.36 Spleen 0.06±0.02 0.154±0.07 0.451±0.18 0.501±0.18 kidneys 0.21±0.02 0.412±0.26 2.44±3.05 0.593±0.15 Liver 1.07±0.23 2.382±0.97 3.542±1.62 6.41±3.76 Tumor 0.08±0.02 0.257±0.22 1.443±0.79 3.374±4.8 Heart 0.13±0.16 0.323±0.27 0.407±0.31 0.334±0.06 Blood 0.22±0.02 0.384±0.06 0.719±0.64 1.123±0.3

[0288] Figure 3 shows a gamma immunoscintigraphic image of a mouse with a tumor obtained on day 15, one hour after injection of 99mTc labeled anti-DNA antibodies. The liver area (b) in the middle right of the image gave a stronger radioactive signal than the tumor area (a) in the lower left of the image. Thus, the greatest accumulation of circulating cfDNA was observed in the liver.

EXAMPLE 3. Entry of tumor cfDNA from the liver into the blood

[0289] Six 6-week-old female nude mice (IBCh RAS breed) were injected subcutaneously with 2x10 ⁶ MCF-7 cells in the left flank. On the 20th day after the injection, the tumor was removed surgically. On day 25, all mice were anesthetized and 500 μl blood samples were taken from each mouse from the portal vein, hepatic artery, and hepatic vein. Plasma was separated from blood cells by centrifugation at 2000 g for 10 minutes; the pure plasma phase was transferred from above into a new tube and centrifuged at 14,000 g. The supernatant was transferred to a fresh DNA extraction tube. cfDNA was extracted from plasma samples using the QIAamp DNA Blood Mini Kit according to the manufacturer's instructions. Human beta globin sequence in extracellular DNA was quantified in each sample by RT-PCR (iCycler iQ5b Bio-Rad) with human beta globin primer CAACTTCATCCACGTTCACC (SEQ ID NO: 10). The Ct values of the human beta globin sequence in blood plasma taken from various vessels are presented in Table 2 below:

table 2

Mouse No. Portal vein hepatic artery hepatic vein one 29.49±0.161 31.86±0.817 23.59±0.109 2 29.2±0.379 28.62±0.278 23.85±0.218 3 29.94±0.874 30.09±0.347 24.22±0.096 four →0 →0 →0 5 30.26±0.21 29.42±0.341 27.89±0.112 6 30.44±0.151 30.26±0.176 25.78±0.155

[0290] Despite the fact that the tumor was removed surgically, only one mouse plasma did not contain circulating cfDNA of human origin. MCF-7 tumors do not metastasize when grown subcutaneously. The absence of metastatic nodules in the liver or lungs was further confirmed by macroscopic and microscopic examination. Circulating cfDNA derived from a human tumor xenograft was found in the blood of mice, with the largest amounts found in the hepatic vein. These results confirm that the liver sinusoids are the depot source of circulating cfDNA.

EXAMPLE 4 Effects of transgenic delivery of DNase I and intravenous delivery of recombinant DNase I protein in a model of pancreatic cancer

[0291] The AAV vector for liver-specific transgene expression of DNase I (4658_pAAV-AFP-Alb-huDNaseI_mut) was custom-made by Sirion Biotech (Martinsried, Germany). The vector map is shown in Figure 5A. The AAV vector is based on AAV serotype 8, which is effective in the liver. The vector contains the sequence set forth in SEQ ID NO: 2 which encodes a mutant human DNase I enzyme comprising several mutations that make the enzyme less sensitive to actin inhibition and more catalytically active. The DNase I coding sequence also contains a secretory signal sequence. The DNase I coding sequence is operably linked at the 5' end to the mAFP/Alb enhancer/promoter system (liver-specific mouse alpha-fetoprotein enhancer and liver-specific mouse albumin minimal promoter), and at the 3' end to the hGH polyA signal. The expression cassette is further flanked by AA8 L-ITR and R-ITR.

[0292] The huDNaseI_mut expression cassette was cloned into the AAV transfer vector and produced in 293T cells by co-transfection of the transfer vector with AAV8 packaging plasmids. Virus particles were purified and concentrated to 1×10 ¹³ GC/ml and stored frozen in PBS.

[0293] A control vector was also generated, identical to 4658_pAAV-AFP-Alb-huDNaseI_mut, but with a non-liver-specific CMVE/CAG promoter/enhancer system instead of a liver-specific mAFP/Alb-enhancer/promoter system.

[0294] Human recombinant DNase I protein (rhDNase I) was manufactured by Catalent (Madison, USA) and stored frozen at 10 mg/ml stock solution in 1 mM CaCl ₂ and 150 mM NaCl.

[0295] In the experiment, 56 female BALB/c nude mice (IBCh RAS) aged 6-8 weeks and weighing 18-22 g were used. Mice were kept in individual ventilated cages at controlled temperature and humidity, four animals in each cage. Pancreatic tumor cells MIA PaCa-2 (1×10 ⁷ ) in 0.2 ml PBS supplemented with BD Matrigel (1:1) were subcutaneously injected into the right flank of each mouse to develop the tumor. Treatment was started on day 17 after tumor inoculation, when the average tumor size reached approximately 170 mm ³ . Each group consisted of eight mice with tumors. The test vectors and compositions were administered to mice according to a predetermined schedule as shown in Table 3 below:

Table 3 Study Design

Group N ^a Treatment Dose, mg/kg Path Schedule one eight Vehicle control 0.8% NaCl - IP BIW*3 weeks 2 eight DNaseI_mut
mAFP/Alb AAV8
+ Gemcitabine 1.00x10 ¹¹ GC/kg
60mg/kg BB
IP D15 single dose
BIW*3 weeks 3 eight DNaseI_mut CMVE/CAG AAV8
+ Gemcitabine 1.00x10 ¹¹ GC/kg
60mg/kg BB
IP D15 single dose
BIW*3 weeks four eight rhDNaseI protein
+ Gemcitabine 5mg/kg
60mg/kg IP
IP TDx2 weeks
BIWx2 weeks 5 eight rhDNaseI protein
+ Gemcitabine 15mg/kg
60mg/kg IP
IP TDx2 weeks
BIWx2 weeks 6 eight rhDNaseI protein
+ Gemcitabine 50mg/kg
60mg/kg IP
IP TDx2 weeks
BIWx2 weeks 7 eight Gemcitabine 60mg/kg IP BIWx2 weeks

[0296] The size of the tumor was measured twice a week in two directions using a caliper, and the volume was expressed in mm ³ according to the formula: V = 0.5 a x b ² where a and b are the long and short diameters of the tumor, respectively. During routine monitoring, animals were checked daily for any effects of tumor growth and treatment on normal behavior such as mobility, food and water intake by visual inspection only, weight loss or gain (measured twice a week), eyes/hair tangles, and any other anomalous effects. On day 35, the study was terminated, mice were sacrificed humanely with CO ₂ and blood samples were taken from the central and hepatic veins. Liver tissue samples were also taken and immersed in formalin for fixation.

[0297] Circulating cfDNA was measured in plasma using the method described in H. Goldstein, “A rapid direct fluorescent assay for cell-free DNA quantification in biological fluids”, Annals of Clinical Biochemistry, Vol 46, Issue 6, pp. 488-494. ^SYBR® Gold Nucleic Acid Gel Stain (Invitrogen) was first diluted 1:1000 with dimethyl sulfoxide and then 1:8 in PBS. 10 μl of plasma samples were applied to 96-well plates. 40 µl of diluted SYBR® Gold (final dilution 1:10,000) was added to each well and fluorescence was measured with a 96-well fluorometer at an emission wavelength of 535 nm and an excitation wavelength of 485 nm.

[0298] Serum DNase I activity was measured using ORG590 (Orgentec) according to the manufacturer's protocol. Detection was performed using a microplate photometer (Multiscan FC) at 450 nm with a correction wavelength of 620 nm. cfDNA in liver tissue was quantified by immunohistochemistry (IHC) on paraffin-embedded liver sections using AC-30-10 mouse IgG-DNA-binding antibodies (Sigma) as primary antibodies and Novolink Novocastra polymer detection systems (Leica Biosystems).

[0299] The mean tumor volume over time in female BALB/c nude mice bearing MIA PaCa-2 xenografts treated with the test vectors and formulation (according to Table 3) is shown in Table 4 below:

Table 4 Mean tumor volume over time

Day Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 17 172±14 171±15 171±15 172±15 170±14 170±14 170±13 21 214±18 147±17 166±14 207±17 164±16 202±16 164±14 24 242±18 117±16 161±17 235±18 193±25 220±14 197±17 28 275±18 86±19 152±20 274±21 202±37 239±28 246±10 31 299±19 58±11 119±17 310±31 189±41 212±23 220±8 35 335±32 44±12 107±19 368±40 167±36 197±27 264±14

[0300] The number of animals with weight loss at day 35 in each group of female BALB/c nude mice having MIA PaCa-2 xenografts treated with test vectors and composition (according to Table 3) is shown in Table 5 below:

Table 5. Number of animals with weight loss on day 35

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 BWL>10% 3 of 8 0 of 8 4 out of 8 8 out of 8 6 out of 8 3 of 8 8 out of 8 BWL>20% 0 of 8 0 of 8 0 of 8 3 of 8 1 of 8 0 of 8 4 out of 8

[0301] The results of the quantitative determination of circulating cfDNA in the blood and the enzymatic activity of DNase I in the central and hepatic vein are shown in table 6 below:

Table 6

Extracellular DNA in the blood
ng/ml DNase activity in the blood
mKuU/ml DNase activity in the liver
vein
mKuU/ml Group 1 173±5 1.75 1.55 Group 2 27±3 6.72 8.15 Group 3 105±8 5.78 5.35 Group 6 133±13 4.95 4.75 Group 7 241±11 1.15 1.05

[0302] IHC micrographs of liver tissue of different groups are shown in Figure 4.

[0303] The data from Tables 4-6 above and Figure 4 clearly show that liver-specific transgenic expression of the DNase I enzyme in combination with chemotherapy not only provides significantly better results in terms of antitumor efficacy, but is also able to completely reduce chemotherapy-induced loss body weight. The recombinant DNase I enzyme administered twice daily at three increasing doses, as well as DNase I transgenic expression, which does not target the liver, demonstrated significantly less antitumor efficacy and reduction in chemotherapy-induced toxicity than liver-specific DNase I transgenic expression. treated with the mAFP/Alb AAV8 DNase vector had the least circulating cfDNA, the least cfDNA remaining in the liver, and the highest DNase I enzymatic activity in the hepatic vein.

EXAMPLE 5 Effect of DNase I Transgenic Delivery on Orthotopic and Non-Orthotopic Growth of Pancreatic Cancer

[0304] Twelve 8-week-old SCID mice (IBCh RAS) were used in the experiment, divided into 4 experimental groups. Six mice received 4×10 ⁶ MIA PaCa-2 pancreatic cancer cells expressing luciferase, subcutaneously in the right thigh, in 100 μl of Matrigel (non-orthotopic model). Six mice were implanted in the caudal pancreas with 4×10 ⁶ MIA PaCa-2 pancreatic cancer cells expressing luciferase in 100 μl of Matrigel (orthotopic model). The following day, 3 mice with an orthotopic MIA PaCa-2 implant received an intravenous injection of mAFP/Alb AAV8 of vector 4658_pAAV-AFP-Alb-huDNaseI_mut at a dose of 1.00×10 ¹¹ GC/kg, and 3 mice with an orthotopic MIA PaCa-2 implant received placebo injection (PBS). Three mice with a non-orthotopic MIA PaCa-2 implant received an intravenous injection of DNaseI mAFP/Alb AAV8 vector at a dose of 1.00×10 ¹¹ GC/kg, and three mice with a non-orthotopic MIA PaCa-2 implant received a placebo (PBS) injection. Four weeks later, at the end of the experiment, tumors were assessed by bioluminescent imaging using the AMI-1000 imaging system. The bioluminescence data are summarized in Table 7 below and representative images are presented in Figure 6.

Table 7

Bioluminescence (ph/s/ROI) x10 ⁸ Orthotopic MIA PaCa-2 Nonorthotopic MIA PaCa-2 huDNaseI_mut mAFP/Alb AAV8 placebo huDNaseI_mut mAFP/Alb AAV8 placebo Mouse 1 0.0 1.13 1.71 5.8 Mouse 2 0.0 1.41 2.13 6.1 Mouse 3 0.0 1.74 1.05 5.65

[0305] Thus, transgenic delivery of the DNase I enzyme to the liver completely blocks the development of tumor growth in an orthotopic manner in the area drained by the portal vein, that is, in the pancreas. The development of the same pancreatic cancer xenograft in a non-orthotopic manner was also significantly suppressed by DNase I mAFP/Alb AAV8 injection, but not to the extent that tumor growth was completely excluded.

EXAMPLE 6 Effect of DNase I transgene delivery to the liver and lungs on breast carcinoma growth in a breast cancer model

[0306] Vector ADV-207186 was purchased from Vector Biolabs (Malvern, PA). The vector map is shown in Figure 5C. This vector is derived from human adenovirus type 5 (dE1/E3) (which has a natural tropism for the liver) and contains the wild-type human DNase I coding sequence, which includes a secretory signal sequence (SEQ ID NO: 1). The DNase I coding sequence is operably linked at the 5' end to the non-specific CMV hepatic promoter and at the 3' end to the hGH polyA signal. The expression cassette is further flanked by ATT-L1 and ATT-L2.

[0307] For the study, female mice of the MMTV-PyMT breed of the Institute of Bioorganic Chemistry of the Russian Academy of Sciences were used. When each mouse developed at least three palpable tumors of at least 3 mm × 5 mm, which usually occurred at 8 weeks of age, six mice were intravenously injected with 0.2 × 10 ¹¹ PFU per ADV-207186 mouse and six mice were injected with 0. 2×10 ¹¹ PFU per mouse ADV-207186 intranasally under anesthesia. Mice were sacrificed at 14 weeks of age. Six mice were used as controls. The size of the tumor was measured in two directions with a caliper and the volume was expressed in mm ³ according to the formula: V = 0.5 a x b ² where a and b are the long and short diameters of the tumor, respectively. TGI was calculated for each group according to the formula: TGI (%) = [1-(Ti-T0)/(Vi-V0)] ×100; Ti is the mean tumor volume in the treatment group on a given day, T0 is the mean tumor volume in the treatment group on the day of treatment, Vi is the mean tumor volume of the carrier control group on the same day with Ti, and V0 is the mean tumor volume in the carrier group at day of treatment. At the end of the study, blood was taken from the central vein. Circulating cfDNA and serum DNase I activity was measured as described in Example 4. Data are presented in Table 8 below.

Table 8

Treatment Tumor volume (mm ³ ) TGI (%) Extracellular DNA, ngml/ Serum DNase activity
mKuU/ml Control 355±32 -- 137±13 2.15 DNase I Ad Type 5 (dE1/E3) AV IN 209±28 78 110±7 4.95 DNase I Ad Type 5 (dE1/E3) AV IV 54±22 164 34±11 5.2

[0308] The data show that liver targeting of an adenoviral vector carrying a DNase I transgene provides superior suppression of breast tumor growth and blood clearance of circulating cfDNA compared to delivering a DNase I transgene to the bronchoalveolar system, despite approximately the same level of enzymatic activity DNase in the blood.

EXAMPLE 7. Effect of transgene expression of actin-resistant hyperactive DNase I in the liver and muscles on the level of microbial cfDNA and the level of neuroinflammation

[0309] Increased intestinal permeability and translocation of the gastrointestinal microbiota and damage-associated molecular structures (DAMPs) of microbial origin, including microbial cfDNA, cause neuroinflammation and a spectrum of neurodegenerative diseases. cfDNA derived from the microbiota of the gastrointestinal tract undergoes a "first pass" through the portosinusoidal circulation. Additionally, the effect of delivery of the hyperactive actin-resistant DNase I transgene to muscle and liver, the effect of injections of wild-type DNase I protein on the level of microbial cfDNA in the brain and liver, and the level of neuroinflammation in the cerebrospinal fluid and brain after bacteriophage-induced hypersensitivity syndrome were studied. intestinal permeability.

[0310] Healthy adult male Wistar rats (n = 24; 12 weeks of age, 240-280 g) were housed in individual cages in room P3 on a 12-hour light/dark cycle at 22 to 25°C and 60 ± 5% atmospheric humidity. All animals had free access to food and water in accordance with the Guidelines for the Care and Use of Laboratory Animals.

[0311] A DNA molecule encoding a hyperactive actin-resistant DNase I mutant (SEQ ID NO: 5) with a DNase I secretory signal sequence replaced with an IL2 secretory signal sequence was synthesized (GeneCust) and cloned into the pLV2 lentiviral vector (Clontech) under control EF1a promoter (non-liver specific). The resulting construct is pLV2-IL2ss-DNaseI mut and is shown in Figure 9. The 293T lentiviral packaging cell line (Clontech) was cultured in DMEM (Gibco). HEK 293T cells were transfected with the pLV2-based pLV2-IL2ss-DNAseI mut lentiviral vector using Lipofectamine 2000 (Life Technology). 48 hours after transfection, 5 ml of the supernatant was collected. Lentiviruses were collected by filtration through a 0.45 μm polyethersulfone membrane filter (Millipore), centrifugation for 30 minutes at 20,000 g. The resulting pellet was resuspended in 1 ml PBS.

[0312] In 24 rats, increased intestinal permeability was induced by applying a mixture of bacteriophages against the Enterobacteriaceae, Staphylococcaceae, and Streptococcaceae families to cause microbiota diseases. The mixture included a mixture of Salmonella bacteriophages from Microgen (Moscow, Russia) containing bacteriophages against S. paratyphi, S. typhimurium, S. heidelberg, S. newport, S. choleraesuis, S. oranienburg, S. infans, S. dublin, S. enteritidis, S. anatum and S. Newlands and a mixture of Pyobacteriophage phages from Microgen containing phages against seven bacterial species, Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli . Phages (1.5 ml, 1×10 ⁶ pfu/ml of each phage mixture) were added to drinking water according to the manufacturer's instructions and administered orally for 10 days. On day 7 of phage treatment, the rats were randomized into 4 groups as follows: six rats received an intrahepatic injection of 1.0 x 10 ¹² LVP in a volume of 200 μl; six rats received an injection of 1.0 × 10 ¹² LVP in a volume of 200 μl into the thigh muscle; six rats received twice daily intravenous injections of recombinant DNase I protein at a dose of 50 mg/kg from days 7 to 12. The remaining six rats were used as untreated control animals with increased intestinal permeability. An additional six healthy rats were used as healthy controls. On day 12, all rats were sacrificed and blood samples were collected to measure DNase activity in the blood. Brain autopsies were collected to analyze NLRP3 inflammasome expression and quantify the 16S universal bacterial RNA gene.

[0313] The NLRP3 inflammasome is considered a key factor in the development of neuroinflammation. Aberrant activation of NLRP3 inflammasome signaling has been demonstrated to contribute to the pathology of a wide range of neurological diseases (Song, 2017). NLRP3 expression was assessed by collecting 50 mg cortical tissue samples. RNA was isolated using the SV total RNA isolation system (Promega). Superscrit III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen) was used for reverse transcriptase PCR using 5'-GTTCTGAGCTCCAACCATTCT-3' (SEQ ID NO: 11) as NLRP3 forward primer and 5'-CACTGTGGGTCCTTCATCTTT- 3' (SEQ ID NO: 12) as NLRP3 reverse primer. 16S universal primers for bacterial RNA genes (forward: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3' (SEQ ID NO: 13); reverse: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 14)) were used to quantify the presence of microbial origin liver and brain. Gene expression analysis was performed using the Ct comparative method.

[0314] Serum DNase I activity was measured using ORG590 (Orgentec) according to the manufacturer's protocol. Detection was performed using a microplate photometer (Multiscan FC) at 450 nm with a correction wavelength of 620 nm. The results are presented in Table 9 below:

Table 9

Group DNase activity in blood, mKuU/ml Mean ΔCt values for universal bacterial 16S from RT-PCR, liver Mean ΔCt values for universal bacterial 16S from RT-PCR, brain Mean ΔCt values for brain NLRP3 from RT-PCR results healthy control 4.35 28.315 28.723 18.992 Control with increased intestinal permeability 2.33 24.617 26.315 17.352 DNase I protein (mass) 7.15 25.752 27.417 17.341 Delivery to the liver pLV2-IL2ss-DNaseI mut 9.73 27.717 28.212 18.882 Delivery to muscle pLV2-IL2ss-DNaseI mut 8.77 25.112 27.573 17.931

[0315] The data show that the induction of increased intestinal permeability resulted in the suppression of endogenous blood DNase enzyme activity, a significant increase in the presence of microbial cfDNA in the liver and cerebral cortex, and a significant increase in the activity of the NLRP3 inflammasome component in the cortical tissue of the brain. Delivery of the hyperactive actin-resistant lentiviral DNase I transgene to the liver, compared with delivery of the same transgene into muscle tissue or intravenous injections of the DNase I protein, almost completely prevented the increase in microbial cfDNA in the liver and brain and the activation of brain inflammasomes, despite maintaining a comparable enzymatic DNase activity in the central circulation.

EXAMPLE 8. Effect of transgenic delivery of hyperactive actin-resistant DNase I and intravenous delivery of recombinant DNase I protein on GVHD

[0316] Different models of MHC class I and II, from C57BL/6 (H-2b) to BALB/c (H-2d), were used to establish GVHD (graft versus host disease). All recipients were females of the same age, 2-6 months old at the time of bone marrow transplantation (BMT). Single cell suspensions of bone marrow cells and splenocytes were prepared in saline for injection. To create BMT chimeras, BALB/c recipients received 1200 rad TBI (source of 137Cs) divided into 2 doses. The mice then received cells from C57BL/6 mice: 5×10 ⁶ bone marrow cells and 10×10 ⁶ splenocytes.

[0317] Experiments were designed to compare the following two treatments: wild-type mouse DNase I protein (50 mg/kg) daily and wild-type mouse DNase I mAFP/Alb AAV8 liver-specific vector as a single intravenous injection. Beginning on the same day as BMT, recipients were treated with DNase I by the intrapenitoneal route twice daily or a single intravenous delivery of mAFP/Alb AAV8 at 1.00 x 10 ¹¹ GC/kg. For histopathology, mice were sacrificed 16 days after transplantation. Tissues were placed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. For quantification of donor-derived T cells, spleen (SP), peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP) were harvested from recipient mice 16 days post-transplantation. Cells isolated from these tissues were stained with antibodies to CD3, CD4, CD8 and H-2Db. Donor-derived T cells (H-2Db+) and CD4+/H-2Db+ and CD8+/H-2Db+ subsets were quantified by FACS.

[0318] Survival data are presented in Table 10 below:

Table 10

Treatment D0 D8 D16 D24 D32 dead/alive DNase I 10/10 9/10 8/10 6/10 5/10 DNase I mAFP/Alb AAV8 10/10 10/10 10/10 10/10 10/10

[0319] Data on liver histopathology in recipient mice treated with DNase I and recipient mice treated with mAFP/Alb AAV8 DNase I are shown in Figure 7. Left panel shows lymphoplasmacytic infiltration of the portal tracts, segmental loss of bile duct epithelial cells, areas of focal necrosis of hepatocytes and a significant amount of nucleophilic material on the surface of vessels in animals that received only DNase I protein (and not DNase mAFP/Alb AAV8). The right panel shows that the livers of animals treated with mAFP/Alb AAV8 DNase I have a much lower inflammatory cell density, no necrosis, and much less nucleophilic material at the vessel junction.

[0320] Data on donor T cells in recipient mice treated with DNase I and recipient mice treated with mAFP/Alb AAV8 DNase I are presented in Figure 8. In particular, Figure 8 shows a significant reduction in the total number received from donors T cells derived from CD4+ and CD8+ donors in peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) in recipients treated with mAFP/Alb AAV8 DNase I compared with recipients treated with DNase I protein.

[0321 The results show that liver-specific transgenic expression of the DNase I enzyme results in a doubling of animal survival compared to DNase I protein treatment. This improvement in survival was accompanied by a significant reduction in the total number of donor-derived T cells derived from CD4+ and CD8+ T cell donors. in PLN, MLN, and PP in recipients treated with DNase I mAFP/Alb AAV8 compared to recipients treated with DNase I protein. surface from nucleophilic material.

EXAMPLE 9 AAV vector cloning: ApoEHCR enhancer-hAAT-hDNaseI promoter (hyperactive) correct leader-WPRE Xinact (VR-18013AD)

[0322] The expression vector without the AAV promoter pAAV-MCS-Promoterless was obtained from Cell Bio labs (catalog no. VPK-411; figure 11). The integrity of the ITR was verified by performing SmaI digestion followed by gel purification to evaluate for the presence of the following expected bands: 2681 bp, 666 bp. and 112 b.p. Expected streak at 11 p.p. will not be visible. If the expected bands were present, the plasmid was amplified by growth in SURE cells (Agilent Technologies) or similar bacteria. The 5' EcoRI site and the 3' HindIII site (ApoEHCR enhancer-hAAT-hDNaseI promoter (hyperactive) correct leader-WPRE Xinact) were added to SEQ ID NO: 30. A polynucleotide of SEQ ID NO: 30 (with EcoRI 5' sites and HindIII 3' sites) was synthesized and then digested with EcoRI-HF and HindIII-HF (available from NEB). Band 2.2 kb. extracted from the gel. The promoterless pAAV-MCS vector was digested with EcoRI-HF and HindIII-HF with a final 3.5 kb band gel extraction. The cleaved vector and the polynucleotide of SEQ ID NO: 30 (insert) were ligated for one hour at room temperature in a 3:1 molar ratio of insert to vector. The ligated construct was transformed into SURE electrocompetent cells and plated on LB-agar plates (Amp). Colonies were selected and minipreparations were prepared for screening. SmaI digestion was first carried out to assess the integrity of the ITR to determine if the following expected bands were present: 2681 bp, 1650 bp. and 1311 b.p. Then EcoRI-HF and HindIII-HF were digested to assess the presence of the insert. If the insert were present, the following bands would be visible: 3435 b.p. and 2218 b.p. The VR-18013AD vector map is shown in Figure 10B.

[0323] Clones were selected if the expected bands were present in each of the SmaI and EcoRI-HF and HindIII-HF hydrolysates. For selected clones, a stock solution was prepared in glycerol for long-term storage. The plasmid was also amplified and sequenced. Clones containing the correct sequence plasmid were then cultured and purified with endo-free Gigaprep (ALTA Biotech). The purified plasmid is suitable for MEE packaging into AAV capsids.

EXAMPLE 10 AAV vector cloning: ApoEHCR enhancer-promoter hAAT-hDNaseI (wild type)-WPRE Xinact (VR-18014AD)

[0324] The expression vector without the AAV promoter pAAV-MCS-Promoterless was obtained from Cell Bio labs (catalog no. VPK-411; figure 11). The integrity of the ITR was checked by performing SmaI digestion and gel purification to assess whether the plasmid produced the following expected bands: 2681 bp, 666 bp. and 112 b.p. Expected streak at 11 p.p. will not be visible. If the expected bands were present, the plasmid was amplified by growth in SURE cells (Agilent Technologies) or similar bacteria. The 5' EcoRI site and the 3' HindIII site (ApoEHCR enhancer-hAAT-hDNaseI promoter (wt)-WPRE Xinact) were added to SEQ ID NO: 31. A polynucleotide of SEQ ID NO: 31 (with EcoRI 5' sites and HindIII 3' sites) was synthesized and then digested with EcoRI-HF and HindIII-HF (available from NEB). Band 2.2 kb. extracted from the gel. The promoterless pAAV-MCS vector was digested with EcoRI-HF and HindIII-HF with a final 3.5 kb band gel extraction. The cleaved vector and the polynucleotide of SEQ ID NO: 31 (insert) were ligated for one hour at room temperature in a 3:1 molar ratio of insert to vector. The ligated construct was transformed into SURE electrocompetent cells and plated on LB-agar plates (Amp). Colonies were selected and minipreparations were prepared for screening. First, SmaI digestion was performed to assess the integrity of the ITR to determine if the following expected bands were present: 2681 bp, 1650 bp. and 1311 b.p. Then EcoRI-HF and HindIII-HF were digested to assess the presence of the insert. If the insert were present, the following bands would be visible: 3435 b.p. and 2218 b.p. The VR-18014AD vector map is shown in Figure 10C.

[0325] Clones were selected if the expected bands were present in each of the SmaI and EcoRI-HF and HindIII-HF hydrolysates. For selected clones, a stock solution was prepared in glycerol for long-term storage. The plasmid was also amplified and sequenced. Clones containing the correct sequence plasmid were then cultured and purified with endo-free Gigaprep (ALTA Biotech). The purified plasmid is suitable for MEE packaging into AAV capsids.

EXAMPLE 11 Generation of AAV Vectors ApoEHCR enhancer-promoter hAAT-hDNaseI (hyperactive) correct leader-WPRE Xinact (VR-18013AD) and ApoEHCR enhancer-promoter hAAT-hDNaseI (wild type)-WPRE Xinact (VR-18014AD)

[0326] Expression plasmids VR-18013AD and VR-18014AD were manufactured by GenScript USA Inc. (Piscataway, NJ 08854, USA). The corresponding AAV vector hDNAse I (hyperactive) VR-18013AD and the AAV vector hDNAse I (wild type) VR-18014AD were generated using large-scale polyethyleneimine transfections of Anc80L65 AAV cis, AAV trans, and adenovirus helper plasmids in close to confluent monolayers of HEK293 cells with further purification using a scaled iodixanol gradient using traditional protocols. Purified vectors of ApoEHCR enhancer-hAAT promoter-hDNaseI (hyperactive) correct leader-WPRE Xinact (VR-18013AD transgene cassette containing SEQ ID NO: 30; see vector map in Figure 10B) and ApoEHCR enhancer-hAAT promoter-hDNaseI (wild type)-WPRE Xinact (VR-18014AD (map shown in Figure 10C, the vector transgene cassette has the sequence (SEQ ID NO: 31) vectors were re-prepared in PBS with 35 mM NaCl and 0.001% PF68, which contained x10 ¹³ GC/mL (VR-18014AD) and 5.0 x10 ¹² GC/mL (VR-18013AD).

[0327] The purified AAV vector VR-18013AD was analyzed by SDS-PAGE, the results are shown in Figure 12. The three bands at 60, 72 and 90 kDa were observed in a ratio of approximately 1:1:10, corresponding to VP1-3 proteins.

[0328] For transduction, VR-18014AD and VR-18013AD were prepared as sterile, clear, colorless, highly purified solutions of recombinant VR-18014AD and VR-18013AD particles.

EXAMPLE 12 Liver-specific transduction of AAV vectors: ApoEHCR enhancer-hAAT promoter-hDNaseI (hyperactive) correct leader-WPRE Xinact (VR-18013AD) and ApoEHCR enhancer-hAAT promoter-hDNaseI (wild type)-WPRE Xinact (VR-18014AD)

[0329] The transduction efficiency of the following two gene therapy vectors was studied: ApoEHCR enhancer-hAAT promoter-hDNaseI (hyperactive) regular leader-WPRE Xinact and ApoEHCR enhancer-hAAT promoter-hDNaseI (wild-type)-WPRE Xinact. These gene therapy vector candidates were studied in vitro using a cell line derived from human hepatoma HEP G2 (ATCC HB-8065) and endothelial-derived human liver adenocarcinoma SK-HEP1 (ATCC HTB-52).

[0330] Cells were seeded in 48-well plates at 5x10 ⁵ cells/ml and grown in DMEM Adv, 10% FBS. 24 hours after seeding, VR-18013AD or VR-18014AD was added to wells using MOIs of 10 ⁴ GC/well, 10 ⁵ GC/well, and 10 ⁶ GC/well, or a blank control. Three repetitions were performed for each well.

[0331] DNase activity was analyzed using a fluorescent probe (Terekhov et al., PNAS 2017; www.pnas.org/content/pnas/114/10/2550.full.pdf), which is an oligonucleotide hairpin labeled with a fluorescent dye and a quencher . The increase in fluorescence was monitored using a Variscan Flash plate reader (Thermo Scientific). The culture medium was diluted 20 to 200 times with 20 mM Tris-HCl pH 7.5, 1 mM MnCl ₂ and 0.3 mM CaCl ₂ reaction buffer. A fluorescent probe with a concentration of 0.25 μM was used. Complete hydrolysis of the fluorescent probe under reaction conditions resulted in 28 RFU fluorescence under assay conditions. The background fluorescence of the probe was below 0.3 RFU. A calibration curve for estimating DNase concentration was generated using serial dilutions of pure DNase I Pulmozyme "Dornase alfa" (Roche). The DNase concentration in the samples was assessed using linear regression. The data is shown in figures 13A and 13B.

[0332] The data in Figures 13A and 13B show that the vectors ApoEHCR enhancer-hAAT promoter-hDNaseI (hyperactive) correct leader-WPRE Xinact (VR-18013AD) and ApoEHCR enhancer-hAAT promoter-hDNaseI (wild type)-WPRE Xinact (VR -18014AD) efficiently transduce only cells of hepatocyte origin and induce high expression and secretion of DNase I enzyme or wild-type DNase I enzyme with biologically increased expression from transduced G2 cells. An estimate of DNase concentration was obtained using serial dilutions of pure DNase I Pulmozyme "Dornase alfa" (Roche). Transduction appears to be safe for the cells as there was no evidence of cell death within 72 hours of observation even at the highest doses of VR-18013AD and VR-18014AD of 10 ⁶ GC/cell and there were no morphological differences between any of the transduced cells compared to control cells.

EXAMPLE 13 ApoEHCR enhancer-hAAT promoter-hDNaseI (hyperactive) correct leader-WPRE Xinact (VR-18013AD) for the treatment of Alzheimer's disease

[0333] The therapeutic effect of a single intravenous injection of VR-18013AD (ApoEHCR enhancer-hAAT promoter-hDNaseI (hyperactive) correct leader-WPRE Xinact) was studied in 3xTg-AD mice expressing three mutant human transgenes: presenilin (PS)1 (M146V), βAPP (Swedish mutation) and tau (P301L). These transgenic mice develop both amyloid and tau abnormalities.

[0334] The effect of VR-18013AD on learning and memory deficits was assessed using the following behavioral tests: 1) Y-maze alternation test, which measures spatial working memory and depends on the integrity of the limbic and non-limbic pathways; and 2) the situational fear state test (CFC), which is highly dependent on hippocampal and amygdala function, but also on the cerebral cortex.

[0335] The aim of the study was to quantify the effect of a single intravenous injection of VR-18013AD on amyloid deposition and tau phosphorylation in 3xTg-AD using immunohistochemistry. The effect of VR-18013AD on tau hyperphosphorylation was studied in 3xTg-AD mice using the AT8 mAb specific for pSer202/Thr205 epitopes, which are among the earliest phosphorylated tau epitopes in AD patients. Total tau protein was also determined in treated and untreated 3xTg-AD mice. Overactive microglia can cause extremely detrimental neurotoxic effects; therefore, attenuation of the microglial response has been proposed as a potential therapeutic approach in AD.

[0336] The experimental design is shown in Table 11 below:

Table 11

Research methods D0 D30 D60-D180 D180 Injection VR-18013AD or PB X *Y-maze alternation test X *CFC test X *Quantitative determination of Abeta X *Total and phosphorylated tau protein X *Microglial activation X

[0337] On day 0, the following groups of mice were treated: (a) 13 six-month-old wild type (WT) mice were treated with phosphate buffered saline (PBS); (b) 15 six-month-old 3xTg-AD mice were injected with phosphate-buffered saline (PBS) alone; and (c) 10 six month old 3xTg-AD mice were injected with VR-18013AD at 1.0×10 ¹¹ GC/mouse. The following data has been received. In Figure 14, the Y-maze behavioral test shows a slight improvement in VR-18013AD treated 3xTg-AD mice compared to 3xTg-AD control mice. In Figure 15, the results of the situational fear state test show a slight improvement in VR-18013AD treated 3xTg-AD mice compared to 3xTg-AD control mice. The data in Figure 16 show that microglial activation was significantly reduced in VR-18013AD treated 3xTg-AD mice compared to 3xTg-AD control mice, especially in the cerebral cortex.

[0338] The data in FIG. 17 show that there is a significant reduction in microglial activation in the cortical region of 3xTg-AD mice treated with VR-18013AD compared to control 3xTg-AD mice. The data in Figure 18 show that VR-18013AD treatment significantly reduced amyloid deposition in VR-18013AD treated 3xTg-AD mice compared to 3xTg-AD control mice. The data in Figure 19 show that VR-18013AD treatment significantly reduced the deposition of hyperphosphorylated tau protein in 3xTg-AD mice treated with VR-18013AD compared to 3xTg-AD control mice. The photographs in figure 20 show a significant decrease in p-tau in the hippocampus after treatment with VR-18013AD. Thus, a single intravenous injection of VR-18013AD shows significant therapeutic activity in a mouse model of Alzheimer's disease.

EXAMPLE 14 VR-18014AD for the treatment of peritoneal carcinomatosis

[0339] Twelve BALB/c mice (20-22 g) of the IBCh RAS breed were kept at a controlled temperature of 20-22°C. On day 1, mice were intraperitoneally injected with 200 μl of cell suspension containing 5×10 ⁵ CT26 NNMU induced undifferentiated colon carcinoma cells. On day 6 after tumor transplantation, mice were randomly assigned to either the control group (six mice, single intravenous injection of PBS) or treatment group (six mice, single intravenous injection of VR-18014AD at 1.0 x 10 ¹¹ GC/mouse). Morphometric evaluation and measurement of the number of tumor nodes in each group was performed on day 16 after inoculation of tumor cells after euthanasia of mice. Mice in the control group had 14-22 tumor nodes per mouse, with some of the nodes forming agglomerates. In contrast, mice injected with VR-18014AD had only 2-5 smaller nodes with no evidence of agglomeration. Representative morphometric images of control and VR-18014AD treated mice are shown in Figure 21.

EXAMPLE 15 CfDNA isolated from leaky gut mice induces tau aggregation and liver-specific DNase gene transfer inhibits the ability of cfDNA to induce tau aggregation

[0340] Aggregated tau protein is associated with more than 20 neurological disorders, including Alzheimer's disease. In 18 rats, intestinal permeability was induced as indicated in Example 7. The test vectors and compositions were administered to mice according to a predetermined schedule as shown in Table 12 below:

Table 12

Treatment group Number of animals Induction of increased intestinal permeability Treatment Euthanasia and blood sampling Single intravenous injection VR-18013AD 10 ¹² Gc/kg 3 D0 D5 D9 Single intravenous injection VR-18013AD 10 ¹⁰ Gc/kg 3 D0 D5 D9 Single intravenous injection VR-18014AD 10 ¹² Gc/kg 3 D0 D5 D9 Single intravenous injection VR-18014AD 10 ¹⁰ Gc/kg 3 D0 D5 D9 Single intravenous injection ADV-207186 0.2x10 ¹² PFU/kg 3 D0 D5 D9 Liver pLV2-IL2ss-DNaseI mut 1.0 × 10 ¹³ LVP/kg 3 D0 D5 D9 Control (single PBS injection) 3 D0 D5 D9 Single intravenous injection of a dose of DNase 50 mg/kg 3 D0 D8 D9

[0341] Extraction of cfDNA was performed as follows. Peripheral blood samples (5 ml) were collected by cardiopuncture into cell-free DNA BCT tubes. Plasma was separated from the cell fraction by first centrifugation at 800 g at 4°C for 10 min. Plasma was further centrifuged at 13,000 g at 4° C. for 10 minutes to pellet and remove any remaining cells, and then stored in 1 ml aliquots at -80° C. until DNA extraction.

[0342] QIAamp circulating nucleic acid (Qiagen) was used for cfDNA extraction according to the manufacturer's protocol.

[0343] The cfDNA was assessed for tau protein aggregation. Full length human tau protein (4R2N) was used. For analysis, a solution containing 1 mg/ml tau monomer in the presence of 2.5 μM heparin in 100 mM HEPES, pH 7.4, 100 mM NaCl was incubated in the presence of 25 μl of extracted cfDNA at 20°C with cycling (1 min of shaking). at 500 rpm, then 29 min without shaking). The total sample volume was 200 µl; the solution also contained 5 μM thioflavin T. Aggregation over time was monitored by ThT fluorescence (435 nm excitation, 485 nm emission).

[0344] The results are shown in Table 13 below, indicating the mean and SEM for three repetitions:

Table 13

Time (h) Aggregation, ThT fluorescence Single IV injection VR-18013AD 10 ¹⁰ Gc/kg Single IV injection VR-18013AD 10 ¹² Gc/kg Single IV injection VR-18014AD 10 ¹⁰ Gc/kg VR-18014AD 10 ¹²
Single IV injection GC/kg Single IV injection ADV-207186 0.2x10 ¹² PFU/kg Liver pLV2-IL2ss-DNaseI mut 1.0 × 10 ¹³ LVP/kg Control (single PBS injection) Single IV dose of DNase 50 mg/kg 24 0 0 0 0 0 0 0 0 48 0 0 0 0 16+4 0 12+2 8+2 72 0 0 0 0 18+2 0 16+5 13+4 96 0 0 0 0 48+7 0 34+6 39+9 120 0 0 0 0 52+12 0 45+11 44+7 144 0 0 0 0 60+9 0 67+8 53+5 168 0 0 0 0 96+18 0 88+12 74+6 192 4+3 0 0 0 121+25 0 109+10 92+12 216 8+4 10+2 15+5 7+2 143+38 13+5 158+24 116+18 240 22+4 12+5 34+9 15+3 237+42 27+5 249+38 162+29 264 42+14 27+5 49+9 30+4 275+43 39+7 286+42 191+44 288 56+4 39+8 65+11 36+3 364+58 46+7 351+41 249+36 312 75+6 54+8 89+13 44+10 384+49 52+11 336+34 285+31 336 98+8 67+10 109+15 76+8 360+62 95+138 342+53 302+44

[0345] The data in Table 13 clearly demonstrate that liver-specific transgenic expression of the DNase I enzyme significantly suppresses the ability of cfDNA isolated from the blood of animals with increased intestinal permeability to promote tau protein aggregation upon injection of the recombinant DNase I enzyme, as well as the use of non-specific for the liver, transgenic expression of the DNase I enzyme is much less effective.

Links

Song, L. et al., “NLRP3 Inflammasome in Neurological Diseases, from Functions to Therapies” Front. cell. Neurosci., 2017, Vol. 11, no. 63.

Fleischhacker M. et al., “Circulating nucleic acids (CNAs) and cancer: a survey” Biochim Biophys Acta, 2007, 1775(1): 181-232.

Demers, M. at al., “Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis” PNAS, 2012, 109(32):13076-13081.

Garcia-Olmo D.C. and García-Olmo, D. “Biological role of cell-free nucleic acids in cancer: the theory of genometastasis” Crit Rev Oncolog., 2013, 18:153-161.

Sawyers 2008 C.L., “The cancer biomarker problem” Nature ,2008, 452(7187):548-552.

Butt A.N. et al. Overview of circulating nucleic acids in plasma/serum. Ann. N.Y. Acad. Sci., 2008, 1137:236-242.

Schwarzenbach H. et al., “Detection and monitoring of cell-free DNA in blood of patients with colorectal cancer” Ann. N.Y. Acad. Sci., 2008, 1137:190-196.

Zenaro E, et al., Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin. Nat Rev Nephrol., 2015, Author manuscript; available in PMC 2017 Jul 14.

Fushi, W. et al., “Extracellular DNA in Pancreatic Cancer Promotes Cell Invasion and Metastasis” Cancer Res., 2013, 73:4256-4266.

Tohme, S. et al., “Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress” Cancer Res., 2016 Mar 15, 76(6): 1367-1380.

Patutina, O. et al., Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I. Biochimie., 2011 Apr, 93(4): 689-96.

Dan Li, DNase I Treatment Reduces GVHD in Mice. Biology of Blood and Marrow Transplantation, Volume 21, Issue 2, Page S339.

* * *

[0346] The present invention is not limited in scope to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. It is intended that such modifications fall within the scope of the appended claims.

[0347] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated herein by reference in their entirety as if they were physically present in the present description.

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SEQUENCE LIST

<110> Genkin, Dmitry Dmitrievich

Tets, Georgy Viktorovich

Tets, Viktor Veniaminovich

<120> TREATMENT OF DISEASES THROUGH THE EXPRESSION OF AN ENZYME,

HAVING DEOXYRIBONUCLEASE (DNase) ACTIVITY, IN THE LIVER

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115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Pro Ser Gly Val Gly Ser Asn Thr Met Xaa Ala Gly Gly Gly Ala Pro

195 200 205

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

210 215 220

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

225 230 235 240

Ser Thr Arg Thr Ala Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln

245 250 255

Ile Ser Ser Gln Ser Gly Xaa Ser Thr Asn Asp Asn Thr Tyr Phe Gly

260 265 270

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

275 280 285

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

290 295 300

Arg Pro Lys Xaa Leu Asn Phe Lys Leu Phe Asn Ile Gln Val Lys Glu

305 310 315 320

Val Thr Thr Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Leu Arg Thr Gly Asn Asn Phe Xaa Phe Ser Tyr Thr Phe Glu Asp Val

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Ser Met Ala Asn Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr

465 470 475 480

Arg Gln Gln Arg Val Ser Lys Thr Xaa Asn Gln Asn Asn Asn Asn Ser Asn

485 490 495

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

500 505 510

Leu Val Asn Pro Gly Pro Ala Met Ala Thr His Lys Asp Asp Glu Asp

515 520 525

Lys Phe Phe Pro Met Ser Gly Val Leu Ile Phe Gly Lys Gln Gly Ala

530 535 540

Gly Asn Ser Asn Val Asp Leu Asp Asn Val Met Ile Thr Xaa Glu Glu

545 550 555 560

Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Xaa Tyr Gly Thr Val

565 570 575

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

580 585 590

Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp Gln Xaa Arg Asp Val

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser

675 680 685

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

690 695 700

Thr Asn Val Asp Phe Ala Val Asp Thr Asn Gly Val Tyr Ser Glu Pro

705 710 715 720

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

725 730

<210> 4

<211> 260

<212> PRT

<213> Homo sapiens

<400> 4

Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met

1 5 10 15

Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr

20 25 30

Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val

35 40 45

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

50 55 60

Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr

65 70 75 80

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

85 90 95

Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg Glu

100 105 110

Pro Ala Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe

115 120 125

Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile

130 135 140

Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu

145 150 155 160

Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val

165 170 175

Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe

180 185 190

Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His

195 200 205

Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala

210 215 220

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

225 230 235 240

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

245 250 255

Val Met Leu Lys

260

<210> 5

<211> 260

<212> PRT

<213> Homo sapiens

<400> 5

Leu Lys Ile Ala Ala Phe Asn Ile Arg Thr Phe Gly Arg Thr Lys Met

1 5 10 15

Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr

20 25 30

Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val

35 40 45

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

50 55 60

Tyr Val Val Ser Glu Pro Leu Gly Arg Lys Ser Tyr Lys Glu Arg Tyr

65 70 75 80

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

85 90 95

Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg Glu

100 105 110

Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe

115 120 125

Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile

130 135 140

Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu

145 150 155 160

Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val

165 170 175

Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe

180 185 190

Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His

195 200 205

Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala

210 215 220

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

225 230 235 240

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

245 250 255

Val Met Leu Lys

260

<210> 6

<211> 22

<212> PRT

<213> Homo sapiens

<400> 6

Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu

1 5 10 15

Leu Gln Gly Ala Val Ser

twenty

<210> 7

<211> 20

<212> PRT

<213> Homo sapiens

<400> 7

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

twenty

<210> 8

<211> 214

<212> DNA

<213> Homo sapiens

<400> 8

actagttcca gatggtaaat atacacaagg gatttagtca aacaattttt tggcaagaat 60

attatgaatt ttgtaatcgg ttggcagcca atgaaataca aagatgagtc tagttaataa 120

tctacaatta ttggttaaag aagtatatta gtgctaattt ccctccgttt gtcctagctt 180

ttctcttctg tcaaccccac acgcctttgg cacc 214

<210> 9

<211> 726

<212> PRT

<213> Artificial sequence

<220>

<223> Anc80 VP3 capsid protein

<220>

<221> other_characteristic

<222> (165)..(165)

<223> Xaa is Lys or Arg

<220>

<221> other_characteristic

<222> (201)..(201)

<223> Xaa represents Ala or Ser

<220>

<221> other_characteristic

<222> (261)..(261)

<223> Xaa represents Ala or Gly

<220>

<221> other_characteristic

<222> (306)..(306)

<223> Xaa is Arg or Lys

<220>

<221> other_characteristic

<222> (406)..(406)

<223> Xaa is Glu or Gln

<220>

<221> other_characteristic

<222> (454)..(454)

<223> Xaa is Thr or Glu

<220>

<221> other_characteristic

<222> (486)..(486)

<223> Xaa represents Ala or Thr

<220>

<221> other_characteristic

<222> (554)..(554)

<223> Xaa is Ser or Asn

<220>

<221> other_characteristic

<222> (568)..(568)

<223> Xaa is Gln or Glu

<220>

<221> other_characteristic

<222> (579)..(579)

<223> Xaa represents Ser or Ala

<220>

<221> other_characteristic

<222> (600)..(600)

<223> Xaa is Asn or Asp

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Gly Val Gly Ser Asn Thr Met Xaa Ala Gly Gly Gly Ala Pro Ala

195 200 205

Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asn Trp

210 215 220

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

225 230 235 240

Arg Thr Ala Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser

245 250 255

Ser Gln Ser Gly Xaa Ser Thr Asn Asp Asn Thr Tyr Phe Gly Tyr Ser

260 265 270

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

275 280 285

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

290 295 300

Lys Xaa Leu Asn Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr

305 310 315 320

Thr Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val

325 330 335

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

340 345 350

Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile

355 360 365

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

370 375 380

Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg

385 390 395 400

Thr Gly Asn Asn Phe Xaa Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe

405 410 415

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

420 425 430

Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Thr Thr Ser Gly

435 440 445

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

450 455 460

Ala Asn Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln

465 470 475 480

Arg Val Ser Lys Thr Xaa Asn Gln Asn Asn Asn Ser Asn Phe Ala Trp

485 490 495

Thr Gly Ala Thr Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn

500 505 510

Pro Gly Pro Ala Met Ala Thr His Lys Asp Asp Glu Asp Lys Phe Phe

515 520 525

Pro Met Ser Gly Val Leu Ile Phe Gly Lys Gln Gly Ala Gly Asn Ser

530 535 540

Asn Val Asp Leu Asp Asn Val Ile Thr Xaa Glu Glu Glu Glu Ile Lys Thr

545 550 555 560

Thr Asn Pro Val Ala Thr Glu Xaa Tyr Gly Thr Val Ala Thr Asn Leu

565 570 575

Gln Ser Xaa Asn Thr Ala Pro Ala Thr Gly Thr Val Asn Ser Gln Gly

580 585 590

Ala Leu Pro Gly Val Trp Gln Xaa Arg Asp Val Tyr Leu Gln Gly Pro

595 600 605

Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro

610 615 620

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

625 630 635 640

Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Thr Thr Phe Ser Pro Ala

645 650 655

Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val

660 665 670

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

675 680 685

Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Thr Asn Val Asp Phe Ala

690 695 700

Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg

705 710 715 720

Tyr Leu Thr Arg Asn Leu

725

<210> 10

<211> 20

<212> DNA

<213> Homo sapiens

<400> 10

caacttcatc cacgttcacc 20

<210> 11

<211> 21

<212> DNA

<213> Homo sapiens

<400> 11

gttctgagct ccaaccattc t 21

<210> 12

<211> 21

<212> DNA

<213> Homo sapiens

<400> 12

cactgtgggt ccttcatctt t 21

<210> 13

<211> 50

<212> DNA

<213> Homo sapiens

<220>

<221> other_characteristic

<222> (42)..(42)

<223> n is a, c, g, or t

<400> 13

tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50

<210> 14

<211> 55

<212> DNA

<213> Homo sapiens

<400> 14

gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55

<210> 15

<211> 397

<212> DNA

<213> Homo sapiens

<400> 15

gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga gggccagcta 60

agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg gacacaggac 120

gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg aagctgtaca 180

ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact 240

tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct 300

cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct 360

cagcttcagg caccaccact gacctgggac agtgaat 397

<210> 16

<211> 637

<212> DNA

<213> Homo sapiens

<400> 16

agtggcggcc gctcgagcta gcggccgctc tagaagataa tcaacctctg gattacaaaa 60

tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 120

ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 180

tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 240

gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 300

gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 360

ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 420

tgttgtcggg gaaatcatcg tccttttcctt ggctgctcgc ctgtgttgcc acctggattc 480

tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 540

gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc 600

ggatctccct ttgggccgcc tccccgcatc ggactag 637

<210> 17

<211> 320

<212> DNA

<213> Homo sapiens

<400> 17

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg 320

<210> 18

<211> 840

<212> DNA

<213> Homo sapiens

<400> 18

atgaagctgc tgggggcgct gctggcactg gcggccctac tgcaggggggc cgtgtccctg 60

aagatcgcag ccttcaacat caggacattt gggaggacca agatgtccaa tgccaccctc 120

gtcagctaca ttgtgcagat cctgagccgc tatgacatcg ccctggtcca ggaggtcaga 180

gacagccacc tgactgccgt ggggaagctg ctggacaacc tcaatcagga tgcaccagac 240

acctatcact acgtggtcag tgagccactg ggacggaaga gctataagga gcgctacctg 300

ttcgtgtaca ggcctgacca ggtgtctgcg gtggacagct actactacga tgatggctgc 360

gagccctgcg ggaacgacac cttcaaccga gagccattca ttgtcaggtt cttctcccgg 420

ttcacagagg tcagggagtt tgccattgtt cccctgcatg cggccccgggg ggacgcagta 480

gccgagatcg acgctctcta tgacgtctac ctggatgtcc aagagaaatg gggcttggag 540

gacgtcatgt tgatgggcga cttcaatgcg ggctgcagct atgtgagacc ctcccagtgg 600

tcatccatcc gcctgtggac aagccccacc ttccagtggc tgatccccga cagcgctgac 660

accacagcta cacccacgca ctgtgcctat gacaggatcg tggttgcagg gatgctgctc 720

cgaggcgccg ttgttcccga ctcggctctt ccctttaact tccaggctgc ctatggcctg 780

agtgaccaac tggcccaagc catcagtgac cactatccag tggaggtgat gctgaagtga 840

<210> 19

<211> 849

<212> DNA

<213> Homo sapiens

<400> 19

atgaggggca tgaagctgct gggggcgctg ctggcactgg cggccctact gcaggggggcc 60

gtgtccctga agatcgcagc cttcaacatc aggacatttg ggaggaccaa gatgtccaat 120

gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 180

gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 240

gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggaagag ctataaggag 300

cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 360

gatggctgcg agccctgcgg gaacgacacc ttcaaccgag agccattcat tgtcaggttc 420

ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 480

gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 540

ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 600

tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 660

agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 720

atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 780

tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 840

ctgaagtga 849

<210> 20

<211> 66

<212> DNA

<213> Homo sapiens

<400> 20

atgaggggca tgaagctgct gggggcgctg ctggcactgg cggccctact gcaggggggcc 60

gtgtcc 66

<210> 21

<211> 783

<212> DNA

<213> Homo sapiens

<400> 21

ctgaagatcg cagccttcaa catcaggaca tttgggagga ccaagatgtc caatgccacc 60

ctcgtcagct acattgtgca gatcctgagc cgctatgaca tcgccctggt ccaggaggtc 120

agagacagcc acctgactgc cgtggggaag ctgctggaca acctcaatca ggatgcacca 180

gacacctatc actacgtggt cagtgagcca ctgggacgga aggctataa ggagcgctac 240

ctgttcgtgt acaggcctga ccaggtgtct gcggtggaca gctactacta cgatgatggc 300

tgcgagccct gcgggaacga caccttcaac cgagagccat tcattgtcag gttcttctcc 360

cggttcacag aggtcaggga gtttgccatt gttcccctgc atgcggcccc gggggacgca 420

gtagccgaga tcgacgctct ctatgacgtc tacctggatg tccaagagaa atggggcttg 480

gaggacgtca tgttgatggg cgacttcaat gcgggctgca gctatgtgag accctcccag 540

tggtcatcca tccgcctgtg gacaagcccc accttccagt ggctgatccc cgacagcgct 600

gacaccacag ctacacccac gcactgtgcc tatgacagga tcgtggttgc agggatgctg 660

ctccgaggcg ccgttgttcc cgactcggct cttcccttta acttccaggc tgcctatggc 720

ctgagtgacc aactggccca agccatcagt gaccactatc cagtggaggt gatgctgaag 780

tga 783

<210> 22

<211> 846

<212> DNA

<213> Homo sapiens

<400> 22

atgaggggca tgaagctgct gggggcgctg ctggcactgg cggccctact gcaggggggcc 60

gtgtccctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 120

gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 180

gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 240

gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggaacag ctataaggag 300

cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 360

gatggctgcg agccctgcgg gaacgacacc ttcaaccgag agccagccat tgtcaggttc 420

ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 480

gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 540

ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 600

tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 660

agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 720

atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 780

tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 840

ctgaag 846

<210> 23

<211> 780

<212> DNA

<213> Homo sapiens

<400> 23

ctgaagatcg cagccttcaa catccagaca tttggggaga ccaagatgtc caatgccacc 60

ctcgtcagct acattgtgca gatcctgagc cgctatgaca tcgccctggt ccaggaggtc 120

agagacagcc acctgactgc cgtggggaag ctgctggaca acctcaatca ggatgcacca 180

gacacctatc actacgtggt cagtgagcca ctgggacgga acagctataa ggagcgctac 240

ctgttcgtgt acaggcctga ccaggtgtct gcggtggaca gctactacta cgatgatggc 300

tgcgagccct gcgggaacga caccttcaac cgagagccag ccattgtcag gttcttctcc 360

cggttcacag aggtcaggga gtttgccatt gttcccctgc atgcggcccc gggggacgca 420

gtagccgaga tcgacgctct ctatgacgtc tacctggatg tccaagagaa atggggcttg 480

gaggacgtca tgttgatggg cgacttcaat gcgggctgca gctatgtgag accctcccag 540

tggtcatcca tccgcctgtg gacaagcccc accttccagt ggctgatccc cgacagcgct 600

gacaccacag ctacacccac gcactgtgcc tatgacagga tcgtggttgc agggatgctg 660

ctccgaggcg ccgttgttcc cgactcggct cttcccttta acttccaggc tgcctatggc 720

ctgagtgacc aactggccca agccatcagt gaccactatc cagtggaggt gatgctgaag 780

<210> 24

<211> 284

<212> PRT

<213> Mus musculus

<400> 24

Met Arg Tyr Thr Gly Leu Met Gly Thr Leu Leu Thr Leu Val Asn Leu

1 5 10 15

Leu Gln Leu Ala Gly Thr Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr

20 25 30

Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Ser Val Tyr Phe Val

35 40 45

Lys Ile Leu Ser Arg Tyr Asp Ile Ala Val Ile Gln Glu Val Arg Asp

50 55 60

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

65 70 75 80

Lys Pro Asp Thr Tyr Arg Tyr Val Val Ser Glu Pro Leu Gly Arg Lys

85 90 95

Ser Tyr Lys Glu Gln Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser

100 105 110

Ile Leu Asp Ser Tyr Gln Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn

115 120 125

Asp Thr Phe Ser Arg Glu Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr

130 135 140

Thr Glu Val Gln Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Thr

145 150 155 160

Glu Ala Val Ser Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val

165 170 175

Trp Gln Lys Trp Gly Leu Glu Asp Ile Met Phe Met Gly Asp Phe Asn

180 185 190

Ala Gly Cys Ser Tyr Val Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu

195 200 205

Arg Thr Ser Pro Ile Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr

210 215 220

Thr Val Thr Ser Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly

225 230 235 240

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

245 250 255

Phe Gln Ala Glu Tyr Gly Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser

260 265 270

Asp His Tyr Pro Val Glu Val Thr Leu Arg Lys Ile

275 280

<210> 25

<211> 22

<212> PRT

<213> Mus musculus

<400> 25

Met Arg Tyr Thr Gly Leu Met Gly Thr Leu Leu Thr Leu Val Asn Leu

1 5 10 15

Leu Gln Leu Ala Gly Thr

twenty

<210> 26

<211> 262

<212> PRT

<213> Mus musculus

<400> 26

Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr Phe Gly Glu Thr Lys Met

1 5 10 15

Ser Asn Ala Thr Leu Ser Val Tyr Phe Val Lys Ile Leu Ser Arg Tyr

20 25 30

Asp Ile Ala Val Ile Gln Glu Val Arg Asp Ser His Leu Val Ala Val

35 40 45

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

50 55 60

Tyr Val Val Ser Glu Pro Leu Gly Arg Lys Ser Tyr Lys Glu Gln Tyr

65 70 75 80

Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ile Leu Asp Ser Tyr Gln

85 90 95

Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Ser Arg Glu

100 105 110

Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr Thr Glu Val Gln Glu Phe

115 120 125

Ala Ile Val Pro Leu His Ala Ala Pro Thr Glu Ala Val Ser Glu Ile

130 135 140

Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Trp Gln Lys Trp Gly Leu

145 150 155 160

Glu Asp Ile Met Phe Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val

165 170 175

Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu Arg Thr Ser Pro Ile Phe

180 185 190

Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Val Thr Ser Thr His

195 200 205

Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Ala Leu Leu Gln Ala Ala

210 215 220

Val Val Pro Asn Ser Ala Val Pro Phe Asp Phe Gln Ala Glu Tyr Gly

225 230 235 240

Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser Asp His Tyr Pro Val Glu

245 250 255

Val Thr Leu Arg Lys Ile

260

<210> 27

<211> 66

<212> DNA

<213> Mus musculus

<400> 27

atgcggtaca cagggctaat gggaacactg ctcaccttgg tcaacctgct gcagctggct 60

gggact 66

<210> 28

<211> 789

<212> DNA

<213> Mus musculus

<400> 28

ctgagaattg cagccttcaa cattcggact tttggggaga ctaagatgtc caatgctacc 60

ctctctgtat actttgtgaa aatcctgagt cgctatgaca tcgctgttat ccaagaggtc 120

agagactccc acctggttgc tgttgggaag ctcctggatg aactcaatcg ggacaaacct 180

gacacctacc gctatgtagt cagtgagccg ctgggccgca aaagctacaa ggaacagtac 240

ctttttgtgt acaggcctga ccaggtgtct attctggaca gctatcaata tgatgatggc 300

tgtgaaccct gtggaaatga caccttcagc agagagccag ccattgttaa gttcttttcc 360

ccatacactg aggtccaaga atttgcgatc gtgcccttgc atgcagcccc aacagaagct 420

gtgagtgaga tcgacgccct ctacgatgtt tacctagatg tctggcaaaa gtggggcctg 480

gaggacatca tgttcatggg agacttcaat gctggctgca gctacgtcac ttcctcccag 540

tggtcctcca ttcgccttcg gacaagcccc atcttccagt ggctgatccc tgacagtgcg 600

gacaccacag tcacatcaac acactgtgct tatgacagga ttgtggttgc tggagctctg 660

ctccaggctg ctgttgttcc caactcggct gttccttttg atttccaagc agaatacgga 720

ctttccaacc agctggctga agccatcagt gaccattacc cagtggaggt gacactcaga 780

aaaatctga 789

<210> 29

<211> 855

<212> DNA

<213> Mus musculus

<400> 29

atgcggtaca cagggctaat gggaacactg ctcaccttgg tcaacctgct gcagctggct 60

gggactctga gaattgcagc cttcaacatt cggacttttg gggagactaa gatgtccaat 120

gctaccctct ctgtatactt tgtgaaaatc ctgagtcgct atgacatcgc tgttatccaa 180

gaggtcagag actcccacct ggttgctgtt gggaagctcc tggatgaact caatcgggac 240

aaacctgaca cctaccgcta tgtagtcagt gagccgctgg gccgcaaaag ctacaaggaa 300

360

gatggctgtg aaccctgtgg aaatgacacc ttcagcagag agccagccat tgttaagttc 420

ttttccccat acactgaggt ccaagaattt gcgatcgtgc ccttgcatgc agccccaaca 480

gaagctgtga gtgagatcga cgccctctac gatgtttacc tagatgtctg gcaaaagtgg 540

ggcctggagg acatcatgtt catgggagac ttcaatgctg gctgcagcta cgtcacttcc 600

tcccagtggt cctccattcg ccttcggaca agccccatct tccagtggct gatccctgac 660

agtgcggaca ccacagtcac atcaacacac tgtgcttatg acaggattgt ggttgctgga 720

gctctgctcc aggctgctgt tgttcccaac tcggctgttc cttttgatt ccaagcagaa 780

tacggacttt ccaaccagct ggctgaagcc atcagtgacc attacccagt ggaggtgaca 840

ctcagaaaaa tctga 855

<210> 30

<211> 2212

<212> DNA

<213> Artificial sequence

<220>

<223> Homo sapiens

<400> 30

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg gatcttgcta ccagtggaac agccactaag gattctgcag 360

tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc acgccacccc 420

ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact cctttcggta 480

agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact 540

cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg gtgaccttgg 600

ttaatattca ccagcagcct ccccgttgc ccctctggat ccactgctta aatacggacg 660

aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac agtgaatgcc 720

gccaccatga ggggcatgaa gctgctgggg gcgctgctgg cactggcggc cctactgcag 780

ggggccgtgt ccctgaagat cgcagccttc aacatcagga catttgggag gaccaagatg 840

tccaatgcca ccctcgtcag ctacattgtg cagatcctga gccgctatga catcgccctg 900

gtcggagagg tcagagacag ccacctgact gccgtgggga agctgctgga caacctcaat 960

caggatgcac cagacaccta tcactacgtg gtcagtgagc cactgggacg gaagagctat 1020

aaggagcgct acctgttcgt gtacaggcct gaccaggtgt ctgcggtgga cagctactac 1080

tacgatgatg gctgcgagcc ctgcgggaac gacaccttca accgagagcc attcattgtc 1140

aggttcttct cccggttcac agaggtcagg gagtttgcca ttgttcccct gcatgcggcc 1200

ccggggggacg cagtagccga gatcgacgct ctctatgacg tctacctgga tgtccaagag 1260

aaatggggct tggaggacgt catgttgatg ggcgacttca atgcggggctg cagctatgtg 1320

agaccctccc agtggtcatc catccgcctg tggacaagcc ccaccttcca gtggctgatc 1380

cccgacagcg ctgacaccac agctacaccc acgcactgtg cctatgacag gatcgtggtt 1440

gcagggatgc tgctccgagg cgccgttgtt cccgactcgg ctcttccctt taacttccag 1500

gctgcctatg gcctgagtga ccaactggcc caagccatca gtgaccacta tccagtggag 1560

gtgatgctga agtgaagtgg cggccgctcg agctagcggc cgctctagaa gataatcaac 1620

ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 1680

cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 1740

1800

ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 1860

gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 1920

cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 1980

ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 2040

ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 2100

cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 2160

gccctcagac gagtcggatc tccctttgggg ccgcctcccc gcatcggact ag 2212

<210> 31

<211> 2212

<212> DNA

<213> Artificial sequence

<220>

<223> Homo sapiens

<400> 31

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg gatcttgcta ccagtggaac agccactaag gattctgcag 360

tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc acgccacccc 420

ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact cctttcggta 480

agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact 540

cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg gtgaccttgg 600

ttaatattca ccagcagcct ccccgttgc ccctctggat ccactgctta aatacggacg 660

aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac agtgaatgcc 720

gccaccatga ggggcatgaa gctgctgggg gcgctgctgg cactggcggc cctactgcag 780

ggggccgtgt ccctgaagat cgcagccttc aacatccaga catttgggga gaccaagatg 840

tccaatgcca ccctcgtcag ctacattgtg cagatcctga gccgctatga catcgccctg 900

gtcggagagg tcagagacag ccacctgact gccgtgggga agctgctgga caacctcaat 960

caggatgcac cagacaccta tcactacgtg gtcagtgagc cactgggacg gaacagctat 1020

aaggagcgct acctgttcgt gtacaggcct gaccaggtgt ctgcggtgga cagctactac 1080

tacgatgatg gctgcgagcc ctgcgggaac gacaccttca accgagagcc agccattgtc 1140

aggttcttct cccggttcac agaggtcagg gagtttgcca ttgttcccct gcatgcggcc 1200

ccggggggacg cagtagccga gatcgacgct ctctatgacg tctacctgga tgtccaagag 1260

aaatggggct tggaggacgt catgttgatg ggcgacttca atgcgggctg cagctatgtg 1320

agaccctccc agtggtcatc catccgcctg tggacaagcc ccaccttcca gtggctgatc 1380

cccgacagcg ctgacaccac agctacaccc acgcactgtg cctatgacag gatcgtggtt 1440

gcagggatgc tgctccgagg cgccgttgtt cccgactcgg ctcttccctt taacttccag 1500

gctgcctatg gcctgagtga ccaactggcc caagccatca gtgaccacta tccagtggag 1560

gtgatgctga agtgaagtgg cggccgctcg agctagcggc cgctctagaa gataatcaac 1620

ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 1680

cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 1740

1800

ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 1860

gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 1920

cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 1980

ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 2040

ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 2100

cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 2160

gccctcagac gagtcggatc tccctttgggg ccgcctcccc gcatcggact ag 2212

<210> 32

<211> 849

<212> DNA

<213> Homo sapiens

<400> 32

atgaggggca tgaagctgct gggggcgctg ctggcactgg cggccctact gcaggggggcc 60

gtgtccctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 120

gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 180

gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 240

gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggaacag ctataaggag 300

cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 360

gatggctgcg agccctgcgg gaacgacacc ttcaaccgag agccagccat tgtcaggttc 420

ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 480

gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 540

ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 600

tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 660

agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 720

atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 780

tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 840

ctgaagtga 849

<210> 33

<211> 9

<212> DNA

<213> Homo sapiens

<400> 33

gccgccacc 9

<210> 34

<211> 736

<212> PRT

<213> Artificial sequence

<220>

<223> Anc80L65 VP1 capsid protein

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Lys Gln Gly Ala Gly Asn Ser Asn Val Asp Leu Asp Asn Val Met Ile

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

<210> 35

<211> 730

<212> PRT

<213> Artificial sequence

<220>

<223> Anc80L65 VP1 capsid protein variant

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gln Ile Ser Ser Gln Ser Gly Gly Ser Thr Asn Asp Asn Thr Tyr Phe

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Gly Pro Ser Ser Ala Asn Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys

465 470 475 480

Tyr Arg Gln Gln Arg Val Ser Lys Thr Thr Asn Gln Asn Asn Asn Ser

485 490 495

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

500 505 510

Ser Leu Val Asn Pro Gly Pro Ala Met Ala Thr His Lys Asp Asp Glu

515 520 525

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

530 535 540

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

545 550 555 560

Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Glu Tyr Gly Thr Val

565 570 575

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

580 585 590

Asn Ser Gln Gly Ala Leu Pro Gly Val Trp Gln Asp Arg Asp Val Tyr

595 600 605

Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Gln Val Ser Val Glu Ile Glu Glu Leu Gln Lys Glu Asn Ser Lys Arg

675 680 685

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

690 695 700

Val Asp Phe Ala Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro

705 710 715 720

Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730

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Claims (24)

1. A method of treating a disease or condition in a subject in need thereof, characterized in that the disease or condition is accompanied by accumulation of extracellular DNA (cfDNA) in the portosinusoidal bloodstream of the subject's liver, said method comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated expression vector a virus (rAAV) containing (i) a capsid protein and (ii) a nucleic acid containing a promoter operably linked to a nucleotide sequence encoding an enzyme that has deoxyribonuclease (DNase) activity, where the promoter is a liver-specific promoter. 2. The method according to claim 1, characterized in that the liver-specific promoter mediates a significantly increased expression of the enzyme in the liver compared to other tissues and organs. 3. The method of claim 1, wherein the capsid protein comprises one or more mutations that improve the efficiency and/or specificity of vector delivery to the liver compared to the corresponding wild-type capsid protein. 4. The method according to claim 1, characterized in that the enzyme that has DNase activity is DNase I or its mutant, and said DNase I mutant contains one or more mutations that increase DNase activity. 5. The method according to claim 1, characterized in that the nucleic acid contains the sequence of SEQ ID NO: 30 or SEQ ID NO: 31. 6. The method according to p. 5, characterized in that the DNase I mutant contains mutations Q9R, E13R, N74K and A114F. 7. The method according to claim 1, characterized in that the sequence encoding an enzyme having DNase activity contains a sequence encoding a secretory signal sequence, and said secretory signal sequence mediates efficient secretion of the enzyme into the sinusoidal system when the vector is expressed in the liver. 8. The method according to claim 7, characterized in that the secretory signal sequence contains the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6), or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7), or MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). 9. The method of claim 1, wherein the promoter is an α1-antitrypsin (AAT) promoter or an albumin promoter. 10. The method according to p. 1, characterized in that the capsid protein contains the sequence of SEQ ID NO: 34. 11. The method according to p. 1, characterized in that the AAV belongs to serotype 8 or Anc 80. 12. The method according to p. 1, characterized in that the nucleic acid additionally contains one or more enhancers located upstream or downstream of the promoter. 13. The method of claim 12 wherein the enhancer is an apolipoprotein E (ApoE) enhancer. 14. The method according to p. 12, characterized in that the enhancer contains the sequence of SEQ ID NO: 17. 15. The method according to p. 1, containing (i) a capsid protein containing the sequence of SEQ ID NO: 34, and (ii) a nucleic acid containing the nucleotide sequence of SEQ ID NO: 30. 16. The method of claim. 1, characterized in that the disease is selected from the group consisting of cancer, neurodegenerative disease, atherosclerosis and delayed hypersensitivity reaction. 17. The method according to claim 16, characterized in that the disease is a cancer that occurs and / or metastasizes in organs, tissues and / or structures from which the outflow occurs in the portal vein. 18. The method according to claim 16, characterized in that the cancer is selected from the group consisting of peritoneal carcinomatosis, lymphoma, gastric cancer, colon cancer, intestinal cancer, colorectal cancer, pancreatic cancer, liver cancer, bile duct cancer, bile duct cancer bladder, sarcoma and metastatic liver disease of any origin. 19. The method of claim 16 wherein the delayed hypersensitivity reaction is graft-versus-host disease (GVHD). 20. The method of claim. 1, characterized in that the rAAV vector or vector composition is administered at a dose and regimen that is sufficient to reduce the level of extracellular DNA (cfDNA) in the portosinusoidal bloodstream of the specified subject. 21. A method of treating a disease or condition in a subject in need thereof, wherein the disease or condition is accompanied by an accumulation of extracellular DNA (cfDNA) in the portosinusoidal circulation of the subject's liver, said method comprising administering to the subject an expression vector containing a nucleic acid containing a promoter operably linked with a sequence encoding an enzyme having deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter and/or the vector includes one or more molecules capable of targeting the nucleic acid to liver cells. 22. The method of claim. 21, wherein the disease is selected from the group consisting of cancer, neurodegenerative disease, atherosclerosis, and delayed hypersensitivity reaction. 23. The method according to claim 21, characterized in that the subject is a human. 24. The method of claim 21, wherein said method further comprises selecting a subject with an increased level of extracellular DNA (cfDNA) in the bloodstream compared to the level of cfDNA in the bloodstream of a normal healthy subject.

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