WO2021011365A1 - Treatment of diseases by expression of an enzyme which has a deoxyribonuclease (dnase) activity - Google Patents

Treatment of diseases by expression of an enzyme which has a deoxyribonuclease (dnase) activity Download PDF

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Publication number
WO2021011365A1
WO2021011365A1 PCT/US2020/041574 US2020041574W WO2021011365A1 WO 2021011365 A1 WO2021011365 A1 WO 2021011365A1 US 2020041574 W US2020041574 W US 2020041574W WO 2021011365 A1 WO2021011365 A1 WO 2021011365A1
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Prior art keywords
promoter
vector
sequence
dnase
seq
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PCT/US2020/041574
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French (fr)
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Dmitry Dmitrievich Genkin
Georgy Viktorovich Tets
Viktor Veniaminovich Tets
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Dmitry Dmitrievich Genkin
Georgy Viktorovich Tets
Viktor Veniaminovich Tets
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Priority to CA3146976A priority Critical patent/CA3146976A1/en
Publication of WO2021011365A1 publication Critical patent/WO2021011365A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C12Y301/21001—Deoxyribonuclease I (3.1.21.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the invention relates to the delivery and/or expression of an enzyme which has a deoxyribonuclease (DNase) activity to liver, nervous system and/or intestine resulting in enhanced clearance of cell free DNA (cfDNA) useful for treatment of various diseases and conditions, including cancer and neurodegeneration.
  • DNase deoxyribonuclease
  • cfDNA cell free DNA
  • cfDNA in their plasma and serum as compared to healthy individuals (Fleischhacker, 2007).
  • circulating cfDNA originates both from dying non-tumor cells, tumor cells and neutrophils. Tumors predispose neutrophils to release extracellular DNA traps that contribute to the establishment of a pro-thrombotic state, cachexia and organ failure in cancer patients (Demmers, 2012). Tumor originated cfDNA can contribute to the development of metastasis and chemotherapy resistance (Garcia-Ol mo, 2013)
  • the quantity of circul ating cfDNA increases as the tumor progresses (Sawyers, 2008) reaching the maximal levels in patients with advanced disease and metastatic disease (Butt, 2008). It has been shown that higher quantities of circulating cfDNA significantly correlate with poor patient survival (Schwarzenbach, 2008).
  • Neurodegeneration is a separate clinical pathological condition with progressive loss of structure and/or function of neurons, including death of neurons.
  • Molecular pathways leading to neurodegeneration are highly disease-specific (e.g., accumulation of abnormally folded amyloid-beta and tau proteins in the brain in Alzheimer's disease patients; accumulation of alpha- synuclein in Parkinson's disease; accumulation of mutant Huntingtin in Huntington's disease; accumulation of TDP-43 and FUS protein aggregates in Amyelotropic Lateral Sclerosis (ALS); accumulation of mitochondrial DNA mutations and broken mitochondria division mechanics in aging) and result in neuronal cell death at advanced stages of disease progression.
  • ALS Amyelotropic Lateral Sclerosis
  • kidney damage see, e.g., U.S. Patent No. 9,770,492
  • delayed- type hypersensitivity reactions such as, e.g., graft-versus-host disease [GVHD])
  • GVHD graft-versus-host disease
  • reduction of fertility see, e.g., U.S. Patent No. 8,916,151
  • age-specific sperm motility impairment see, e.g., U.S. Patent No. 8,871,200
  • aging see, e.g., U.S. Pat. Appl. Pub. No. US20150110769. All of these patents and applications are incorporated by reference herein in their entireties.
  • RhDNase I also suppressed the development of metastatic disease by 60-90% when administered daily at 0.02-2.3 mg/kg in lung carcinoma and hepatoma metastatic disease model (Patutina, 2011).
  • Li Li (Li, 2015) reported successful use of DNasel to treat GVHD in mice.
  • the present invention addresses these and other needs by providing methods and compositions for delivery and/or expression of an enzyme which has a deoxyribonuclease (DNase) activity to liver, nervous system and/or intestine resulting in enhanced clearance of cell free DNA (cfDNA) useful for treatment of various diseases and conditions, including cancer and neurodegeneration.
  • DNase deoxyribonuclease
  • the invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/nervous system-specific tandem promoter.
  • rAAV adeno-associated virus
  • the liver-specific/nervous system-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system as compared to other tissues and organs.
  • the liver-specific/nervous system- specific tandem promoter comprises a liver-specific promoter selected from the group consisting of an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyru
  • the liver-specific promoter is an anti-trypsin (AAT) promoter.
  • the AAT promoter is a human a 1 -anti -trypsin (hAAT) promoter.
  • the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 15.
  • the liver-specific promoter comprises the sequence SEQ ID NO: 15.
  • the liver-specific promoter consists of the sequence SEQ ID NO: 15.
  • the liver-specific promoter is an albumin promoter.
  • the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 8.
  • the liver-specifi c/nervous system- specific tandem promoter comprises a nervous system-specific promoter selected from the group consisting of F4/80 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, CMV promoter, MEF2 promoter, FoxP2 promoter, Ibal promoter, TTR promoter, CDl lb promoter, c- fes promoter, NSE promoter, synapsin promoter, CamKII promoter, a-CaMKII promoter, VGLUT1 promoter, and glial fibrillary acidic protein (GFAP) promoter.
  • the nervous system-specific promoter is a synapsin promoter.
  • the synapsisn promoter is a human synapsin promoter.
  • the nervous system- specific promoter is a F4/80 promoter.
  • the nervous system-specific promoter is a CMV promoter.
  • the nervous system-specific promoter is a TMEM119 promoter.
  • the nervous system-specific promoter is a MEF2 promoter.
  • the nervous system-specific promoter is a FoxP2 promoter.
  • the liver-specific/nervous system-specific tandem promoter comprises a nervous-specific promoter comprising a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to any one of the sequences SEQ ID NO: 36 and 38-42.
  • the nervous system-specific promoter comprises any one of the sequences SEQ ID NO: 36 and 38-42.
  • the nervous system-specific promoter consists of any one of the sequences SEQ ID NO: 36 and 38-42.
  • the invention provides a recombinant adeno-associated virus
  • rAAV liver-specific/intestine-specific tandem promoter
  • DNase deoxyribonuclease
  • the liver-specific/intestine-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs.
  • the liver-specific/intestine-specific tandem promoter comprises a liver-specific promoter selected from the group consisting of an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruv
  • the liver-specific promoter is an anti-trypsin (AAT) promoter.
  • the AAT promoter is a human a 1 -anti -trypsin (hAAT) promoter.
  • the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 15.
  • the liver-specific promoter comprises the sequence SEQ ID NO: 15.
  • the liver-specific promoter consists of the sequence SEQ ID NO: 15.
  • the liver-specific promoter is an albumin promoter.
  • the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 8.
  • the liver-specific/intestine-specific tandem promoter comprises an intestine-specific promoter selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti-CMV promoter, MUC2 promoter, villin promoter, and T3 b promoter.
  • the intestine-specific promoter is a villin promoter.
  • the villin promoter promoter is a human villin promoter.
  • the intestine-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37.
  • the liver-specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter.
  • the liver-specific/nervous system-specific tandem promoter is part of the liver-specific/nervous system-specific/intestine-specific tandem promoter, which mediates a substantially increased expression of the enzyme in the liver and/or nervous system and/or intestine as compared to other tissues and organs.
  • the liver- specific/nervous system-specific/intestine-specific tandem promoter comprises an intestine- specific promoter selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti- CMV promoter, MUC2 promoter, villin promoter, and T3 b promoter.
  • the intestine-specific promoter is a villin promoter.
  • the villin promoter promoter is a human villin promoter.
  • the intestine-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37.
  • the liver-specific/nervous system- specific/intestine-specific tandem promoter comprises a hAAT promoter, a human synapsin promoter and a human villin promoter.
  • the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
  • the enzyme which has a DNase activity is DNase I or a mutant or derivative thereof.
  • the DNase l is a human DNase I or a mutant or derivative thereof.
  • the DNase I mutant comprises one or more mutations in an actin binding site.
  • the one or more mutations in the actin-binding site are selected from a mutation at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combinations thereof.
  • one of the mutations in the actin-binding site is a mutation at Ala-114.
  • the DNase I mutant comprises one or more mutations increasing DNase activity.
  • one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof. In one embodiment, one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combinations thereof.
  • the DNase I mutant comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 5. In one embodiment, the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F.
  • the DNase I mutant comprises the sequence of SEQ ID NO: 5. In one embodiment, the DNAse I mutant consists of the sequence of SEQ ID NO: 5. In one embodiment, the DNase I mutant comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 2. In one embodiment, the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 2. In one embodiment, the DNAse I mutant consists of the sequence of SEQ ID NO: 2.
  • the DNase I mutant comprises 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
  • the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 4. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 23. In one embodiment, the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 1. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 22. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 22.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 32. In one embodiment, the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 24. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 29. In one embodiment, the nucleic acid encodes a DNase I comprising the sequence of SEQ ID NO: 26.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 28. In one embodiment, the nucleic acid encodes a DNase I mutant comprising the sequence SEQ ID NO: 5. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 21. In one embodiment, the nucleic acid encodes a DNase I mutant comprising the sequence SEQ ID NO: 2.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 19.
  • the enzyme which has a DNase activity is a fusion protein comprising (i) a DNase enzyme or a fragment thereof linked to (ii) an albumin or an Fc or a fragment thereof.
  • the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme.
  • the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, the albumin secretory signal sequence, the b-glucuronidase secretory signal sequence, the alkaline protease secretory signal sequence, and the fibronectin secretory signal sequence.
  • the secretory signal sequence comprises the sequence MRGMKLLGALLAL AALLQGAV S (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the secretory signal sequence consists of the sequence MRGMKLLGALLAL AALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of MRGMKLLGALLAL AALLQGAVS (SEQ ID NO: 6) or a sequence having at least 85% or at least 90% or at least 95% sequence identity to the sequence of MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 20. In one embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence of SEQ ID NO: 20. In one embodiment, the secretory signal sequence comprises the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one embodiment, the secretory signal sequence consists of the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25).
  • the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence SEQ ID NO: 27.
  • nucleic acid of the invention comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.
  • the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or nervous system as compared to the corresponding wild-type capsid protein. In one embodiment of any of the vectors described above and comprising liver-specific and intestine- specific promoters, the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or intestine as compared to the corresponding wild-type capsid protein.
  • the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or nervous system and/or intestine as compared to the corresponding wild-type capsid protein.
  • the improved efficiency and/or specificity of the delivery of the vector to the liver results in a substantially increased expression of the enzyme in the liver as compared to other tissues and organs.
  • the improved efficiency and/or specificity of the delivery of the vector to the nervous system results in a substantially increased expression of the enzyme in the nervous system as compared to other tissues and organs.
  • the improved efficiency and/or specificity of the delivery of the vector to the intestine results in a substantially increased expression of the enzyme in the intestine as compared to other tissues and organs.
  • the capsid protein comprisesVP3.
  • the one or more mutations in the capsid protein are selected from the group consisting of S279A, S671A, K137R, T252A, and any combinations thereof.
  • the one or more mutations in the capsid protein include mutation K137R.
  • the capsid protein comprises the sequence SEQ ID NO:34.
  • the capsid protein consists of the sequence SEQ ID NO:34
  • the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAVhu37, AAVrh64Rl, Anc 80, AAV-KP1, and AAV-F.
  • the AAV is from serotype 8 or Anc 80.
  • the nucleic acid further comprises two AAV inverted terminal repeats (ITRs), wherein the ITRs flank the nucleotide sequence encoding the enzyme which has a DNase activity.
  • ITRs AAV inverted terminal repeats
  • the nucleic acid further comprises one or more enhancers located upstream or downstream of the promoter.
  • the one or more enhancers are selected from the group consisting of an apolipoprotein E (ApoE) enhancer, an alpha fetoprotein enhancer, a TTR enhancer, an LSP enhancer, an al-microglobulin/bikunin enhancer, an albumin gene enhancer (Ealb), a nPE2 enhancer, a Gal4 enhancer, a foxP2 enhancer, aMefZ enhancer, a CMV enhancer, and any combination thereof.
  • ApoE apolipoprotein E
  • the enhancer is an apolipoprotein E (ApoE) enhancer. In one embodiment, the enhancer is a hepatic control region (HCR) enhancer. In one embodiment, the enhancer comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to SEQ ID NO: 17. In one embodiment, the enhancer comprises the sequence SEQ ID NO: 17. In one embodiment, the enhancer consists of the sequence SEQ ID NO: 17.
  • the nucleic acid further comprises a polyadenylation signal operably linked to the nucleotide sequence encoding the enzyme which has a DNase activity.
  • the nucleic acid further comprises a Kozak sequence.
  • the Kozak sequence comprises the sequence 5 '-GCCGCC ACC-3 ' (SEQ ID NO: 33).
  • the nucleic acid further comprises a post-transcriptional regulatory element.
  • the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE).
  • WPRE woodchuck hepatitis post-transcriptional regulatory element
  • the WPRE does not encode a functional X protein.
  • the post- transcriptional regulatory element comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to SEQ ID NO: 16.
  • the post- transcriptional regulatory element comprises the sequence SEQ ID NO: 16.
  • the post-transcriptional regulatory element consists of the sequence SEQ ID NO: 16.
  • the invention provides pharmaceutical compositions comprising any of the rAAV vectors described above and a pharmaceutically acceptable carrier and/or excipient.
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and the nervous system in a subject in need thereof, comprising administering to the subject any of the above-described rAAV vectors comprising liver-specific and nervous system-specific promoters or pharmaceutical compositions comprising such vectors.
  • DNase deoxyribonuclease
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject any of the above-described rAAV vectors comprising liver-specific and intestine-specific promoters or pharmaceutical compositions comprising such vectors.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of any of the above-described rAAV vectors or pharmaceutical compositions comprising such vectors.
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and nervous system in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific/nervous system-specific tandem promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and nervous system in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver and nervous system cells when administered in vivo.
  • DNase deoxyribonuclease
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver- specific/intestine-specific tandem promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver and intestine cells when administered in vivo.
  • DNase deoxyribonuclease
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver, nervous system and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific/nervous system-specific/intestine-specific tandem promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver, nervous system and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver, nervous system and intestine cells when administered in vivo.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specifi c/nervous system-specific tandem promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver and nervous system cells.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specifi c/intestine-specific tandem promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver and intestinal cells.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specifi c/nervous system-specific/intestine-specific tandem promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver, nervous system and intestinal cells.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and nervous system of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and instestine of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
  • DNase deoxyribonuclease
  • the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver, nervous system and intestine of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
  • DNase deoxyribonuclease
  • Non-limiting examples of the neurodegenerative disease treatable by the methods of the invention include, e.g., Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, CADASIL Syndrome, Friedreich’s ataxia, cerebellar ataxia, spinocerebellar ataxia, dementia (e.g., fronto-temporal dementia, frontotemporal dementia with parkinsonism- 17 (FTDP-17), familial Danish dementia, and familial British dementia), prion- caused diseases, Lewy body diseases, an amyloidosis (e.g., hereditary cerebral hemorrhage with amyloidosis, senile systemic amyloidosis, an amyloidosis with a hereditary cerebral hemorrhage, a primary systemic amyloidosis, a secondary systemic amyloidosis, a serum amyloidosis, a senile systemic amyloidosis, a
  • the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, an amyloidosis, asthma, or prion disease.
  • Non-limiting examples of neurodevelopmental disease treatable by the methods of the invention include, e.g., autism, neural tube defects, attention deficit hyperactivity disorder, Dawn syndrome, cerebral palsy, Rett syndrome, Landau-Kleffner syndrome, Alexander disease, Alpers' disease, alternating hemiplegia, Angelman syndrome, ataxias and cerebellar or spinocerebellar degeneration, ataxia telangiectasia, attention deficit-hyperactivity disorder, autism spectrum disorders, Asperger syndrome, Batten disease, Canavan disease, Tourette syndrome, and impairments in vision and/or hearing.
  • autism e.g., autism, neural tube defects, attention deficit hyperactivity disorder, Dawn syndrome, cerebral palsy, Rett syndrome, Landau-Kleffner syndrome, Alexander disease, Alpers' disease, alternating hemiplegia, Angelman syndrome, ataxias and cerebellar or spinocerebellar degeneration, ataxia telangiectasia, attention deficit-hyperactivity disorder, autism spectrum
  • Non-limiting examples of onclological diseases treatable by the methods of the invention include, e.g., astrocytoma, gliomas, glioblastoma, ependymoma, medulloblastoma, central nervous system (CNS) lymphoma, craniopharyngioma, glioblastoma, meningioma, pituitary carcinomas, pituitary adenomas, neurofibromatosis, embryonal tumors, tumors of the pineal region, tumors of meninges, choroid plexus tumors, neuronal and mixed neuronal-glial tumors, germ cell tumors, anaplastic astrocytoma, central neurocytoma, choroid plexus carcinoma, choroid plexus papilloma, dysembryoplastic neuroepithelial tumor, giant-cell glioblastoma, gliosarcoma, hemangiopericytoma
  • Non-limiting examples of autoimmune diseases treatable by the methods of the invention include, e.g., autoimmune encephalitis, autoimmune-related epilepsy, CNS vasculitis, Hashimoto’s encephalopathy, neurosarcoidosis, Neuro-Behcet’s disease, cerebral lupus, diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, asthma, celiac disease, ankylosing spondylitis, vasculitis, pemphigus vulgaris, inflammatory bowel disease (IBD), Crohn's disease, and ulcerative colitis.
  • autoimmune encephalitis e.g., autoimmune encephalitis, autoimmune-related epilepsy, CNS vasculitis, Hashimoto’s encephalopathy, neurosarcoidosis, Neuro-Behcet’s disease, cerebral lupus, diabetes, rheumatoid arthritis, systemic lupus erythemato
  • Non-limiting examples of psychiatric diseases treatable by the methods of the invention include, e.g., anxiety disorders, psychotic disorders, schizophrenia, bipolar disorder, depression, and post-traumatic stress disorder.
  • Non-limiting examples of infections treatable by the methods of the invention include, e.g., adenoviral infections, hepatitis B, hepatitis G, poxvirus infections, herpesvirus infections, papillomavirus infections, HIV infections, meningitis (bacterial, fungal, or viral), bacterial persistence in brain, fungal persistence in brain, pancreatitis, and peritonitis.
  • adenoviral infections e.g., adenoviral infections, hepatitis B, hepatitis G, poxvirus infections, herpesvirus infections, papillomavirus infections, HIV infections, meningitis (bacterial, fungal, or viral), bacterial persistence in brain, fungal persistence in brain, pancreatitis, and peritonitis.
  • the vector further comprises one or more molecules capable of targeting of the nucleic acid to liver and/or nervous system and/or intestinal cells.
  • the liver-specific/nervous system- specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system as compared to other tissues and organs.
  • the liver-specific/intestine- specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs.
  • the liver-specific/nervous system- specific/intestine-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system and/or intestine as compared to other tissues and organs.
  • the liver-specific promoter is selected from the group consisting of an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykina
  • AAT al -anti -tryps
  • the liver-specific promoter is an anti-trypsin (AAT) promoter.
  • the AAT promoter is a human al -anti -trypsin (hAAT) promoter.
  • the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 15.
  • the liver-specific promoter comprises the sequence SEQ ID NO: 15.
  • the liver-specific promoter consists of the sequence SEQ ID NO: 15.
  • the liver-specific promoter is an albumin promoter.
  • the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 8.
  • the nervous system-specific promoter is selected from the group consisting of F4/80 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, CMV promoter, MEF2 promoter, FoxP2 promoter, Ibal promoter, TTR promoter, CDl lb promoter, c-fes promoter, NSE promoter, synapsin promoter, CamKII promoter, a-CaMKII promoter, VGLUT1 promoter, and glial fibrillary acidic protein (GFAP) promoter.
  • the nervous system-specific promoter is a synapsin promoter.
  • the synapsisn promoter is a human synapsin promoter.
  • the nervous system-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 36.
  • the nervous system-specific promoter comprises the sequence SEQ ID NO: 36.
  • the nervous system-specific promoter consists of the sequence SEQ ID NO: 36.
  • the liver-specific/nervous system- specific tandem promoter comprises a hAAT promoter and a human synapsin promoter.
  • the intestine-specific promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs.
  • the intestine-specific promoter is selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti-CMV promoter, MUC2 promoter, villin promoter, and T3 b promoter.
  • the intestine-specific promoter is a villin promoter.
  • the villin promoter promoter is a human villin promoter.
  • the intestine-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37.
  • the liver-specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter. In one embodiment, the liver-specific/intestine-specific tandem promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37.
  • the vector is used as a naked DNA.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated virus vector, an adenovirus vector, a retrovirus vector (e.g., a lentivirus vector), or a hepatotropic virus vector (e.g., a hepatitis B virus (HBV) vector).
  • a retrovirus vector e.g., a lentivirus vector
  • a hepatotropic virus vector e.g., a hepatitis B virus (HBV) vector.
  • the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
  • the DNase is DNase I or a mutant or derivative thereof.
  • the DNase I is human DNase I or a mutant or derivative thereof.
  • the DNase I mutant comprises one or more mutations in an actin binding site.
  • the one or more mutations in the actin-binding site are selected from a mutation at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combinations thereof.
  • one of the mutations in the actin-binding site is a mutation at Ala-1 14.
  • the DNase I mutant comprises one or more mutations increasing DNase activity.
  • one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof. In one embodiment, one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, N74K, and A114F.
  • the DNase I mutant comprises 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
  • the DNase I mutant is a long acting form of DNase.
  • the DNase I comprises the sequence SEQ ID NO: 4.
  • the DNase I comprises the sequence SEQ ID NO: 1.
  • the DNase I mutant comprises the sequence SEQ ID NO: 5.
  • the DNase I mutant comprises the sequence SEQ ID NO: 2.
  • the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme into the hepatic porto-sinusoidal circulation upon administration of the vector to the subject.
  • the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, b-glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence.
  • the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to MRGMKLLGALL AL AALLQGAV S (SEQ ID NO: 6) or a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the nucleic acid further comprises one or more hepatic control region (HCR) enhancers located upstream or downstream of the promoter.
  • HCR hepatic control region
  • the HCR enhancer comprises a sequence having at least 80% or at least 855 or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 17.
  • the HCR enhancer comprises the sequence SEQ ID NO: 17.
  • the HCR enhancer consists of the sequence SEQ ID NO: 17.
  • the nucleic acid further comprises a Kozak sequence.
  • the Kozak sequence comprises the sequence 5'-GCCGCCACC-3' (SEQ ID NO: 33).
  • the nucleic acid further comprises a post-transcriptional regulatory element, wherein the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE) that does not encode a functional X protein.
  • the nucleic acid comprises a sequence having at least 80% or at least 855 or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 19.
  • the nucleic acid comprises the sequence SEQ ID NO: 19.
  • the nucleic acid consists of the sequence SEQ ID NO: 19.
  • the administration of the nucleic acid results in the expression of the enzyme and its secretion into the hepatic porto-sinusoidal circulation of the subject.
  • the nucleic acid is administered in a dose and regimen which is sufficient to decrease the level of the cell free DNA (cfDNA) in the porto-sinusoidal circulation of said subject.
  • the method further comprises selecting a subject with an elevated level of cell-free DNA (cfDNA) in the bloodstream as compared to a level of cfDNA in the bloodstream of a normal healthy subject.
  • cfDNA cell-free DNA
  • the method further comprises administering a deoxyribonuclease (DNase) enzyme to the subject.
  • DNase is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
  • the DNase is DNase I or a mutant or derivative thereof.
  • the subject is human.
  • the invention provides a recombinant adeno-associated virus
  • rAAV rAAV expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
  • DNase deoxyribonuclease
  • the liver-specific promoter mediates a substantially increased expression of the enzyme in the liver as compared to other tissues and organs.
  • the invention provides a recombinant adeno-associated virus expression vector (rAAV) comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the capsid protein mediates efficient and/or preferential targeting of the vector to the liver when administered in vivo.
  • the capsid protein is VP3.
  • the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver as compared to the corresponding wild-type capsid protein.
  • the improved efficiency and/or specificity of the delivery of the vector to the liver results in a substantially increased expression of the enzyme in the liver as compared to other tissues and organs.
  • Non-limiting examples of enzymes having DNase activity which can be used in the vectors of the invention include, e.g., DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, or mutants or derivatives thereof.
  • the enzyme which has a DNase activity is a DNase I (e.g., human DNase I) or a mutant or derivative thereof.
  • the nucleic acid comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.
  • the DNase I mutant comprises one or more mutations in an 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 combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence).
  • one of the mutations in the actin-binding site is a mutation at Ala- 114.
  • the DNase I mutant comprises one or more mutations increasing DNase activity (e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence).
  • the DNase I mutant comprises one or more mutations are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combinations thereof.
  • the DNase I mutant comprises the mutation Q9R.
  • the DNase I mutant comprises the mutation E13R.
  • the DNase I mutant comprises the mutation N74K.
  • the DNase I mutant comprises the mutation A114F.
  • the DNase I mutant comprises a sequence having at least 80%,
  • the DNasel mutant comprises the mutations Q9R, E13R N74K and A114F.
  • the DNase I mutant comprises the sequence of SEQ ID NO: 5.
  • the DNase I mutant comprises a sequence having at least 80%,
  • the DNasel mutant comprises the mutations Q9R, E13R N74K and A114F.
  • the DNase I mutant comprises the sequence of SEQ ID NO: 2.
  • the DNase I mutant consists of the sequence of SEQ ID NO: 2 or SEQ ID NO: 5.
  • the DNase I mutant comprises 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, H44 A : Y65 A : E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70S, S68N
  • the nucleotide sequence encodes a DNase I comprising the sequence SEQ ID NO: 4.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 23.
  • the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 23.
  • the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 23.
  • the nucleic acid comprises the nucleotide sequence SEQ ID NO: 23.
  • the nucleotide sequence encodes a DNase I comprising the sequence SEQ ID NO: 1.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 22. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 22. In one embodiment, the nucleic acid comprises a nucleotide sequence which 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 which is at least 85% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 32.
  • the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 32. In one embodiment, the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 24. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 29.
  • the nucleic acid comprises the nucleotide sequence SEQ ID NO: 29. In one embodiment, the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 26. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 28.
  • the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 5.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 21.
  • the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 21.
  • the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 21.
  • the nucleic acid comprises the nucleotide sequence SEQ ID NO: 21.
  • the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 2.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 19.
  • the enzyme which has a DNase activity is a fusion protein comprising (i) a DNase enzyme or a fragment thereof linked to (ii) an albumin or an Fc polypeptide or a fragment thereof.
  • the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme into the hepatic sinusoidal system upon expression of the vector in the liver.
  • Non-limiting examples of useful secretory signal sequences include, e.g., DNase I secretory signal sequence, IL2 secretory signal sequence, the albumin secretory signal sequence, the b-glucuronidase secretory signal sequence, the alkaline protease secretory signal sequence, and the fibronectin secretory signal sequence.
  • the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MRGMKLLGALL AL AALLQGAV S (SEQ ID NO: 6).
  • the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one specific embodiment, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one specific embodiment, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence M YRMQLL S CIAL SL AL VTN S (SEQ ID NO: 7).
  • the secretory signal sequence comprises the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one specific embodiment, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence SEQ ID NO: 20.
  • the secretory signal sequence comprises the sequence
  • the secretory signal sequence consists of the sequence MRYT GLMGTLLTL VNLLQL AGT (SEQ ID NO: 25). In one specific embodiment, the secretory signal sequence comprises a sequence having at least 80% sequence identity to sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 27. In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 27.
  • sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 27. In one specific embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence of SEQ ID NO: 27.
  • Non-limiting examples of promoters which can be used in the vectors of the invention include, e.g., an albumin promoter, an a 1 -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyr
  • the promoter is an albumin promoter.
  • the albumin promoter comprises the sequence of SEQ ID NO: 8.
  • the albumin promoter consists of the sequence of SEQ ID NO: 8. See also Frain et ah, Mol. Cell Biol., 1990, 10(3):991-999.
  • the promoter is an al -anti -trypsin (AAT) promoter.
  • the promoter is a human al -anti -trypsin (hAAT) promoter.
  • the anti-trypsin promoter comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 15.
  • the AAT promoter comprises the sequence of SEQ ID NO: 15.
  • the AAT promoter consists of the sequence of SEQ ID NO: 15.
  • the capsid protein comprises one or more mutations selected from the group consisting of S279A, S671 A, K137R, T252A, and any combinations thereof.
  • the one or more mutations in the capsid protein include mutation K137R.
  • the capsid protein comprises the sequence SEQ ID NO:3 [Anc80] In one embodiment, the capsid protein consists of the sequence SEQ ID NO:3 [Anc80] In one embodiment, the capsid protein comprises the sequence SEQ ID NO:9 [Anc80] In one embodiment, the capsid protein consists of the sequence SEQ ID NO:9 [Anc80] In one embodiment, the capsid protein comprises the sequence SEQ ID NO:34 [Anc80L65] In one embodiment, the capsid protein consists of the sequence SEQ ID NO:34 [Anc80L65] In one embodiment, the capsid protein comprises the sequence SEQ ID NO:35 [Anc80L65 variant].
  • the capsid protein consists of the sequence SEQ ID NO:35 [Anc80L65 variant].
  • the capsid protein is a mutant AAV8 capsid protein such as, e.g., AAV3G1, AAVT20 or AAVTR1, or another mutant capsid protein disclosed in Int. Pat. Appl. Pub. No.
  • WO2017/180854 (e.g., comprising VP3 mutations in amino acids 263-267 [e.g., 263NGTSG267->SGTH ("NGTSG” disclosed as SEQ ID NO: 43 and “SGTH” disclosed as SEQ ID NO: 44) or 263NGTSG267->SDTH ("NGTSG” disclosed as SEQ ID NO: 43 and “SDTH” disclosed as SEQ ID NO: 45)] and/or amino acids 457-459 [e.g., 457TAN459->SRP], and/or amino acids 455-459 [e.g., 455GGTAN459 ->DGSGL ("GGTAN” disclosed as SEQ ID NO: 46 and “DGSGL” disclosed as SEQ ID NO: 47)] and/or amino acids 583-597).
  • amino acids 263-267 e.g., 263NGTSG267->SGTH (“NGTSG” disclosed as SEQ ID NO: 43 and “SGTH” disclosed as SEQ ID NO: 44) or 263NGTSG267->
  • Non-limiting examples of AAVs which can be used in the vectors of the invention include, e.g., serotype 1 (AAV1), AAV2, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV 12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64Rl, and Anc 80.
  • the AAV is from serotype 8 or Anc 80.
  • the nucleic acid of the AAV vector further comprises two AAV inverted terminal repeats (ITRs), wherein the ITRs flank the nucleotide sequence encoding the enzyme which has a DNase activity.
  • ITRs AAV inverted terminal repeats
  • the nucleic acid of the AAV vector further comprises one or more enhancers located upstream or downstream of the promoter.
  • the enhancer may be located immediately upstream with the promoter, e.g., where the 3' end of an enhancer sequence is fused directly to the 5' end of the promoter sequence.
  • Non-limiting examples of useful enhancers include, e.g., an apolipoprotein E (ApoE) enhancer (e.g., an ApoE hepatic control region- 1 (HCR-1) enhancer or an ApoE HCR-2 enhancer), an alpha fetoprotein enhancer, a TTR enhancer, an LSP enhancer, an al-microglobulin/bikunin enhancer, an albumin gene enhancer (Ealb), a nPE2 enhancer, a Gal4 enhancer, a foxP2 enhancer, a Mef2 enhancer, a CMV enhancer, and any combination thereof.
  • the enhancer is an ApoE enhancer.
  • the enhancer is an ApoE enhancer located upstream of the promoter. In one specific embodiment, the enhancer is an ApoE enhancer fused to the 5' end of the promoter. In one specific embodiment, the ApoE enhancer is a hepatic control region (HCR) enhancer. In one specific embodiment, the enhancer comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 17. In one specific embodiment, the enhancer comprises the sequence of SEQ ID NO: 17. In one specific embodiment, the enhancer consists of the sequence of SEQ ID NO: 17.
  • the nucleic acid of the AAV vector further comprises a polyadenylation signal operably linked to the nucleotide sequence encoding the enzyme which has a DNase activity.
  • the nucleic acid further comprises a Kozak sequence.
  • the Kozak sequence comprises the sequence of 5'-GCCGCCACC-3' (SEQ ID NO: 33).
  • the nucleic acid comprises a nucleotide sequence which is at least 80% identical to SEQ ID NO: 30.
  • the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 30.
  • the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 30.
  • the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 30.
  • the nucleic acid comprises the nucleotide sequence SEQ ID NO: 30.
  • the nucleic acid further comprises a post-transcriptional regulatory element.
  • the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE).
  • WPRE woodchuck hepatitis post-transcriptional regulatory element
  • the WPRE does not encode a functional X protein.
  • the post- transcriptional regulatory element comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 16.
  • the post-transcriptional regulatory element comprises the sequence of SEQ ID NO: 16.
  • the post-transcriptional regulatory element consists of the sequence of SEQ ID NO: 16.
  • the nucleic acid of rAAV vector of the invention comprises a sequence having 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% identity to the sequence of SEQ ID NO: 19.
  • the nucleic acid comprises the sequence of SEQ ID NO: 19.
  • the nucleic acid consists of the sequence of SEQ ID NO: 19.
  • the nucleic acid has a map shown in Figure 10.
  • the invention provides a recombinant adeno-associated virus
  • rAAV rAAV expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 4 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
  • DNase deoxyribonuclease
  • the invention provides a recombinant adeno-associated virus
  • rAAV rAAV expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 5 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
  • DNase deoxyribonuclease
  • the invention provides a recombinant adeno-associated virus
  • rAAV rAAV expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 1 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
  • DNase deoxyribonuclease
  • the invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 2 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
  • DNase deoxyribonuclease
  • the invention provides a recombinant adeno-associated virus
  • (rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 30.
  • the invention provides a recombinant adeno-associated virus
  • (rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 31.
  • the invention provides pharmaceutical compositions and dosage forms comprising any of the rAAV vectors of the invention and a pharmaceutically acceptable carrier and/or excipient.
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver in a subject in need thereof, comprising administering to the subject any of the rAAV vectors or pharmaceutical compositions described above.
  • DNase deoxyribonuclease
  • the invention provides a method for treating a disease or condition (e.g., a neurodegenerative, neurodevelopmental, psychiatric, or autoimmune disease, or an infection, or a delay ed-type hypersensitivity reaction) in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of cell free DNA (cfDNA) in the hepatic porto-sinusoidal circulation of the subject, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above.
  • a disease or condition e.g., a neurodegenerative, neurodevelopmental, psychiatric, or autoimmune disease, or an infection, or a delay ed-type hypersensitivity reaction
  • cfDNA cell free DNA
  • the invention provides a method for treating a cancer in a subj ect 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.
  • the cancer originates in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein.
  • the method is effective to inhibit metastasis.
  • Non limiting examples of cancers treatable by the methods of the invention include, e.g., a peritoneal carcinomatosis, a lymphoma, a stomach cancer, a colon cancer, an intestinal cancer, a colorectal cancer, a pancreatic cancer, a liver cancer, a cancer of the bile duct, a cancer of the gall bladder, a sarcoma, an esophageal cancer, and a liver metastatic disease of any origin.
  • the invention provides a method for 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.
  • neurodegenerative diseases treatable by the methods of the invention include, e.g., Alzheimer’s disease, Mild Cognitive Impairment (MCI), CADASIL syndrome, Parkinson’s disease, Amyotrophic Lateral Sclerosis, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), agyrophilic grain disease (AGD), Pick’s disease (PiD), Huntington’s disease (HD), and Frontotemporal dementia with parkinsonism- 17 (FTDP-17).
  • the neuro egenerative disease is associated with the formation of a misfolded protein due to the presence of DNA.
  • the DNA is human DNA, microbial DNA, cell-free DNA, or intracellular DNA.
  • the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SI .! ⁇ ), gout, metabolic syndrome, an amyloidosis, asthma, or prion disease.
  • the amyloidosis is an amyloidosis with a hereditary cerebral hemorrhage, a primary systemic amyloidosis, a secondary systemic amyloidosis, a serum amyloidosis, a senile systemic amyloidosis, a hemodialysis-related amyloidosis, a Finnish hereditary systemic amyloidosis, an Atrial amyloidosis, a Lysozyme systemic amyloidosis, an Insulin-related amyloidosis, or a Fibrinogen a-chain amyloidosis.
  • the invention provides a method for 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.
  • the delayed-type hypersensitivity reaction is a graft-versus-host disease (GVHD).
  • the invention provides a method for treating an 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.
  • the invention provides a method for treating Antiphospholipid Syndrome and/or its complications 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.
  • the invention provides a method for treating an anemia 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.
  • the anemia is a sickle cell anemia.
  • the anemia is athalassemia.
  • the methods of the invention can be used as anti-sickling or anti-adhesive sickle cell therapy.
  • the methods of the invention can be used for the treatment and/or prevention of aplastic, acute sequestration, hyper-haemolytic, vaso- occlusive crises and their consequences in patients with sickle cell anemia.
  • the invention provides a method for preventing or ameliorating one or more side effects associated with a chemotherapy or a 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.
  • said one or more side effects of the chemotherapy are selected from the group consisting of body weight loss, bone marrow toxicity, catabolic changes in blood biochemistry, cardiotoxicity (e.g., myocardial necrosis), gastrointestinal toxicity, suppression of immunity, and neutropenia.
  • the chemotherapy comprises administration of one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule targeting agents, topoisomerase inhibitors, alkaloids, and targeted therapeutics.
  • the chemotherapy comprises administration of one or more compounds selected from the group consisting of anthracycline, doxorubicin, 5-fluorouracil (5-FU), etoposide, taxane, and cyclophosphamide.
  • said one or more side effects of the radiation therapy are selected from the group consisting of body weight loss, skin irritation, skin damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, and a second cancer.
  • the radiation therapy is external beam radiation therapy or systemic radioisotope therapy.
  • the rAAV vector or the vector composition is administered during a cycle of the chemotherapy or radiation therapy.
  • the rAAV vector or the vector composition is administered after a cycle of the chemotherapy or radiation therapy.
  • chemotherapy or radiotherapy is used for treatment of a cancer selected from the group consisting of a peritoneal carcinomatosis, a lymphoma, a stomach cancer, a colon cancer, an intestinal cancer, a colorectal cancer, a pancreatic cancer, a liver cancer, a cancer of the bile duct, a cancer of the gall bladder, a sarcoma, and a liver metastatic disease of any origin.
  • the administration of the rAAV vector or the vector composition results in the expression of the enzyme which has a DNase activity and its secretion into the hepatic porto-sinusoidal circulation of the subject.
  • the rAAV vector or the vector composition is administered in a dose and regimen which is sufficient to decrease the level of the cell free DNA (cfDNA) in the porto-sinusoidal circulation of said subject.
  • the subject is human.
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver cells when administered in vivo.
  • DNase deoxyribonuclease
  • the invention provides a method for treating a disease or condition in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of cell free DNA (cfDNA) in the hepatic porto-sinusoidal circulation of the subject, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
  • the disease is selected from the group consisting of a cancer (including a liver metastatic disease), a neurodegenerative disease, an atherosclerosis, and a delayed-type hypersensitivity reaction.
  • the invention provides a method for treating a disease or condition in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of cell free DNA (cfDNA) in the hepatic porto-sinusoidal circulation of the subject, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
  • the disease is selected from the group consisting of a cancer (including a liver metastatic disease), a neurodegenerative disease, an atherosclerosis, and a delayed-type hypersensitivity reaction.
  • the invention provides a method for treating a cancer in a subj ect in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
  • the cancer originates in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein.
  • the invention provides a method for treating a cancer in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
  • the cancer originates in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein.
  • the invention provides a method for treating a cancer in a subj ect in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver of said subject and wherein the cancer originates in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein.
  • DNase deoxyribonuclease
  • Non-limiting examples of cancers treatable by any of the above methods for treating cancer include, e.g., a peritoneal carcinomatosis, a lymphoma, a stomach cancer, a colon cancer, an intestinal cancer, a colorectal cancer, a pancreatic cancer, a liver cancer, a cancer of the bile duct, a cancer of the gall bladder, a sarcoma, and a liver metastatic disease of any origin.
  • the method is effective to inhibit metastasis.
  • the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
  • DNase deoxyribonuclease
  • the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
  • DNase deoxyribonuclease
  • the 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 encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
  • DNase deoxyribonuclease
  • Non-limiting examples of neurodegenerative diseases treatable by any of the above methods for treating neurodegenerative diseases include, e.g., Alzheimer’s disease, Mild Cognitive Impairment (MCI), CADASIL syndrome, Parkinson’s disease, Amyotrophic Lateral Sclerosis, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), agyrophilic grain disease (AGD), Pick’s disease (PiD), Huntington’s disease (HD), and Frontotemporal dementia with parkinsonism- 17 (FTDP-17).
  • the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, an amyloidosis, asthma, or prion disease.
  • the amyloidosis is an amyloidosis with a hereditary cerebral hemorrhage, a primary systemic amyloidosis, a secondary systemic amyloidosis, a serum amyloidosis, a senile systemic amyloidosis, a hemodialysis-related amyloidosis, a Finnish hereditary systemic amyloidosis, an Atrial amyloidosis, a Lysozyme systemic amyloidosis, an Insulin-related amyloidosis, or a Fibrinogen a-chain amyloidosis.
  • the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
  • the delayed-type hypersensitivity reaction is a graft-versus-host disease (GVHD).
  • the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
  • the delayed-type hypersensitivity reaction is a graft-versus-host disease (GVHD).
  • the invention provides a method for treating an atherosclerosis in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver- specific promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for treating an atherosclerosis in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
  • DNase deoxyribonuclease
  • the invention provides a method for preventing or ameliorating one or more side effects associated with a chemotherapy or a radiation therapy in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
  • DNase deoxyribonuclease
  • the invention provides a method for preventing or ameliorating one or more side effects associated with a chemotherapy or a radiation therapy in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
  • DNase deoxyribonuclease
  • said one or more side effects of the chemotherapy are selected from the group consisting of body weight loss, bone marrow toxicity, catabolic changes in blood biochemistry, cardiotoxicity (e.g., myocardial necrosis), gastrointestinal toxicity, suppression of immunity, and neutropenia.
  • the chemotherapy comprises administration of one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule-targeting agents, topoisomerase inhibitors, alkaloids, and targeted therapeutics.
  • the chemotherapy comprises administration of one or more compounds selected from the group consisting of anthracycline, doxorubicin, 5- fluorouracil (5-FU), etoposide, taxane, and cyclophosphamide.
  • said one or more side effects of the radiation therapy are selected from the group consisting of body weight loss, skin irritation, skin damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, and a second cancer.
  • the radiation therapy is external beam radiation therapy or systemic radioisotope therapy.
  • the vector is administered during a cycle of the chemotherapy or radiation therapy.
  • the vector is administered after a cycle of the chemotherapy or radiation therapy.
  • the vector further comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
  • the promoter is a liver-specific promoter.
  • the vector is packaged in a liposome or a nanoparticle.
  • the vector is used as a naked DNA.
  • the vector is a viral vector.
  • useful viral vectors include, e.g., adeno-associated virus vectors, adenovirus vectors, retrovirus vectors (e.g., lentivirus vectors), and hepatotropic virus vectors (e.g., hepatitis B virus (HBV) vectors).
  • the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNaselL3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
  • the DNase is DNase I (e.g., human DNase I) or a mutant or derivative thereof.
  • the DNase I mutant comprises one or more mutations in an 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 combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence).
  • one of the mutations in the actin-binding site is a mutation at Ala-114.
  • the DNase I mutant comprises one or more mutations increasing DNase activity (e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence).
  • the DNase I mutant comprises one or more mutations are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combinations thereof.
  • the DNase I mutant comprises the mutation Q9R.
  • the DNase I mutant comprises the mutation E13R.
  • the DNase I mutant comprises the mutation N74K.
  • the DNase I mutant comprises the mutation A114F.
  • the DNase I mutant comprises 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, H44 A : Y65 A : E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70S, S68N
  • the DNase I mutant is a long acting form of DNase.
  • the DNase I comprises the sequence SEQ ID NO: 4.
  • the DNase I comprises the sequence SEQ ID NO: 1.
  • the DNase I mutant comprises the sequence SEQ ID NO: 5.
  • the DNase I mutant comprises the sequence SEQ ID NO: 2.
  • the sequence encoding the enzyme which has a DNase activity comprises a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme into the hepatic porto- sinusoidal circulation upon administration of the vector to the subject.
  • the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, b-glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence.
  • the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence
  • the secretory signal sequence comprises 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 specific embodiment, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the secretory signal sequence comprises the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one specific embodiment, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
  • the liver-specific promoter mediates a substantially increased expression of the enzyme in the liver as compared to other tissues and organs.
  • liver-specific promoters useful in the methods and vectors of the present invention include, e.g., an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (A) albumin promoter, an al -anti -trypsin (
  • the liver-specific promoter is an albumin promoter.
  • the albumin promoter comprises the sequence of SEQ ID NO: 8.
  • the albumin promoter consists of the sequence of SEQ ID NO: 8. See also Frain et ak, Mol. Cell Biol., 1990, 10(3):991-999.
  • the liver-specific promoter is al -anti -trypsin (AAT) promoter.
  • the anti -trypsin promoter is a human al -anti-trypsin (AAT) promoter.
  • the AAT promoter comprises the sequence of SEQ ID NO: 15.
  • the AAT promoter comprises the sequence of SEQ ID NO: 15.
  • the AAT promoter consists of the sequence of SEQ ID NO: 15.
  • the vector comprises an enhancer.
  • the enhancer may be a hepatic control region enhancer, such as HCR-1 or HCR-2 associated with the ApoE gene, e.g., the human ApoE gene.
  • the enhancer comprises the sequence of SEQ ID NO: 17.
  • the enhancer consists of the sequence of SEQ ID NO: 17.
  • the vector comprises a Kozak sequence upstream of the DNase coding sequence.
  • the Kozak sequence may have the sequence of 5'-GCCGCCACC-3' (SEQ ID NO: 33).
  • the vector comprises a post-transcriptional regulatory element, e.g., the WPRE that does not encode a functional X protein.
  • the post-transcriptional regulatory element comprises the sequence of SEQ ID NO: 16.
  • the post-transcriptional regulatory element consists of the sequence of SEQ ID NO: 16.
  • the vector comprises a sequence having 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% identity to the sequence of SEQ ID NO: 19.
  • the nucleic acid comprises the sequence SEQ ID NO: 19.
  • the nucleic acid consists of the sequence SEQ ID NO: 19.
  • the nucleic acid has a map shown in Figure 10.
  • the administration of the vector results in the expression of the enzyme and its secretion into the hepatic porto-sinusoidal circulation of the subject.
  • the vector is administered in a dose and regimen which is sufficient to decrease the level of the cell free DNA (cfDNA) in the porto-sinusoidal circulation of said subject.
  • cfDNA cell free DNA
  • the subject is human.
  • the method further comprises selecting a subject with an elevated level of cell-free DNA (cfDNA) in the bloodstream as compared to a level of cfDNA in the bloodstream of a normal healthy subject.
  • cfDNA cell-free DNA
  • the method further comprises administering a deoxyribonuclease (DNase) enzyme to the subject (e.g., DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II (including DNase Ila and DNase IIb), Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof).
  • DNase deoxyribonuclease
  • the DNase is DNase I or a mutant or derivative thereof.
  • Figure 1 shows the organs and tissues drained by the hepatic portal vein.
  • the portal vein drains blood from the lower part of esophagus, stomach, pancreas, gall bladder, spleen, small intestine, large intestine, upper part of the bladder and liver.
  • Figures 2A-2I show patient summaries and individual blood electrophoretic data from nine patients with advanced metastatic disease who were treated with intravenous bovine DNase I.
  • A rectal carcinoma
  • B lung carcinoma
  • C lung carcinoma
  • D melanoma
  • E breast cancer
  • F adenocarcinoma
  • G renal cancer
  • H rectal carcinoma
  • I breast cancer.
  • Figure 3 shows a gamma-immunoscintigraphic image of a tumor-bearing mouse obtained at Day 15 one hour following injection of 99mTc labeled anti-DNA antibodies, with substantially more radioactivity observed in the liver zone (b) in the middle/right than in the tumor zone (a) in the lower left.
  • Figure 4 shows IHC microphotographs of liver tissue from different groups listed in Table 3 and treated as described in Example 4.
  • a large amount of cfDNA was seen in the liver of a mouse treated with gemcitabine (Group 7).
  • Comparatively little cfDNA was seen in the liver of a mouse treated with DNase I mAFP/Alb AAV8, an AAV8 vector targeting a DNase I gene to liver cells for expression under an albumin promoter (Group 2).
  • Figure 5A shows a map of the mAFP/Alb AAV8 vector 4658_pAAV-AFP-Alb- huDNasel mut.
  • the vector comprises (i) elements for propagation in bacteria, i.e., an AmpR marker, a colEl, FI and M13 origins of replication, and (ii) the hDNAsel mut sequence (SEQ ID NO: 2; hyperactive actin resistant DNase I mutant) operably linked to a mouse minimal albumin promoter, a mouse a-fetoprotein (AFP) enhancer II (with L-ITR), an hGH polyA signal, flanked by AAV8 L-ITR and R-ITR, an Ml 3 origin of replication and an FI origin of replication.
  • Figure 5B shows a map of ADV-207186 vector based on human adenovirus type 5 (dEl/E3) with human DNase I expressed under the control of the CMV promoter.
  • Figure 6 shows bioluminescence data reflective of tumor size.
  • the tumors of both mice with an orthotopic MIA PaCa-2 tumor implant and mice with a non-orthotopic MIA PaCa-2 tumor implant decreased in size when treated with hDNasel mut (SEQ ID NO: 5; hyperactive actin resistant DNase I mutant) mAFP/Alb AAV8.
  • hDNasel mut SEQ ID NO: 5; hyperactive actin resistant DNase I mutant
  • Figure 7 shows liver histology data indicating that animals treated with DNase I mAFP/Alb AAV8 have much lower inflammatory cell density, no necrosis, and much less nucleiphilic material at the vascular interface when treated with DNase I mAFP/Alb AAV8 expression vector instead of DNase I protein.
  • Figure 8 shows a significant reduction in the total numbers of donor-derived T cells, donor-derived CD4+ and CD8+ T cells in the peripheral lymph nodes, mesenteric lymph nodes and Peyer’s patches of DNase I mAFP/Alb AAV8-treated mice as compared with recombinant DNase I treated mice.
  • Figure 9 shows a restriction map of the lentiviral vector pLV2-IL2ss-DNaseI mut used to deliver DNase I in Example 7.
  • the vector comprises an AmpR marker, and origins of replication for FI and SV40.
  • the vector comprises an EF-la promoter upstream of an IL-2 leader sequence upstream of DNAse I.
  • Figure 10A shows the complete sequence (SEQ ID NO: 30) of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader-WPRE Xinact, or ApoE-HCR enhancenhAAT promoter>human DNAse I hyperactive variant with correct leader sequence (secretory sequence): Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE) that does not encode functional protein X.
  • Figure 10B shows a vector map of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader-WPRE Xinact.
  • Figure IOC shows the complete sequence (SEQ ID NO: 31) of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (wild-type)-WPRE Xinact.
  • Figure 10D shows a vector map of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (wild-type)-WPRE Xinact.
  • Figure 11 shows a map of the pAAV-MCS Promoterless expression vector used for the preparation of ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader- WPRE Xinact.
  • Figure 12 shows the results of SDS-PAGE analysis of purified Anc80 VR- 18013AD AAV vector. Three bands of 60, 72, and 90 kDa were observed in a ratio of about 1 : 1 : 10, which corresponds to the VP 1-3 proteins.
  • Figures 13A and 13B illustrate data where ApoEHCR enhancer-hAAT promoter- hDNasel (hyperactive)correct leader-WPRE Xinact and ApoEHCR enhancer-hAAT promoter- hDNasel (wild type)- WPRE Xinact vectors efficiently transduce cells of hepatocyte origin only and trigger high yield expression and secretion of biologically enhanced DNase I enzyme or wild type DNase I enzyme from transduced G2 cells.
  • Figure 14 shows the results of a Y Maze behavioral test.
  • Figure 15 shows the results of a contextual fear condition test.
  • Figure 16 shows that microglia activation was significantly reduced in VR- 18013 AD treated 3xTg-AD mice as compared with 3xTg-AD control mice, especially in cortical area of brain.
  • Figure 17 shows data indicating a significant reduction of microglia activation in the cortical brain area of VR-18013AD treated 3xTg-AD mice vs 3xTg-AD control mice.
  • Figure 18 shows data indicating that treatment with VR- 18013 AD significantly reduces amyloid deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice.
  • FIG. 19 shows that treatment with VR- 18013 AD significantly reduces hyperphosphorylated Tau deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice.
  • Figure 20 shows data indicating a significant reduction of p-tau reduction in hippocampal region after VR- 18013 AD treated treatment.
  • Figure 21 shows a typical morphometric image of control (single IV injection of PBS) and VR-18014AD (single IV injection of VR-18014AD at 1.0 xlO 11 GC/per mouse dose) treated mice.
  • FIGS 22A-F show that AAV-mediated DNase I liver gene transfer reduces the growth of colorectal liver metastases.
  • A A schematic showing experimental design in vivo.
  • D Liver metastasis model using MC38 cells (l x lO 6 ) injected through the portal vein, showing smaller tumors harvested 3 weeks post inoculation in AAV-DNase I treated mice compared with AAV-null control.
  • FIGS 23A-E show that AAV-mediated DNase I liver gene transfer reduces neutrophil recruitment and NET formation in CRC liver metastasis.
  • A Representative images of H&E staining of the liver section exhibit increased immune cells in mice with AAV-DNase I treatment compared with AAV-null treated group. Cell counts were averaged in the four fields. Magnification, x20; magnification, x40. HPF, high-power field.
  • C and D The expression of CitFB by western blot analysis and quantification of serum MPO-DNA by ELISA were assessed at 21 days.
  • n 3-4 mice/group (E) Immunofluorescent staining of tumor tissue shows NETs formation 21 days after tumor cell injection in AAV-DNase I and AAV-null treated mice. Magnification, x60. Nucleus, black; Ly6G, red; CitH3, dark grey, actin, light grey. All data represents at least three independent experiments. Data represent mean ⁇ SD, *p ⁇ 0.05, **p ⁇ 0.01.
  • Figures 24A-D show that AAV-mediated DNase I liver gene transfer recruits CD8 + T cells to CRC liver metastasis.
  • a and B The ratios of CD8 + and CD4 + T cells in livers.
  • the present invention is based on an unexpected discovery that during the course of diseases and conditions characterized by elevation of circulating cfDNA level (e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.) a significant quantity of cfDNA accumulates in the liver, mainly in the periendothelial space and space of Disse. Even high quantities of a systemically administered DNase protein show limited efficacy to digest liver cfDNA. This cfDNA is continuously sourced from liver depot to hepatic portal vein and further to systemic blood circulation.
  • diseases and conditions characterized by elevation of circulating cfDNA level e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.
  • transgenic expression of a DNase I in the liver leads to almost complete clearance of cfDNA accumulated in the liver and in the porto-sinusoidal circulation when DNase is secreted to sinusoidal space of the liver.
  • Such transgenic expression of DNase in the liver leads to significant antitumor effects (especially in tumors in organs and tissues drained by the portal vein), reduction of toxicity of anticancer chemotherapy, a slowdown in autoimmune and neurodegenerative disease progression, etc.
  • Such transgenic expression of DNase in the liver when DNase is secreted to sinusoidal space provides even more benefits when tumor is draining by portal vein system and cfDNA generated in the tumor growth area are subject to“first pass” through porto-sinusoidal circulation.
  • Figure 1 illustrates the organs and systems drained by the portal vein.
  • the present invention is based on an unexpected discovery that during the course of diseases and conditions characterized by elevation of circulating cfDNA level (e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.) a significant quantity of cfDNA accumulates in the liver, mainly in the periendothelial space and space of Disse. Even high quantities of a systemically administered DNase protein show limited efficacy to digest liver cfDNA. This cfDNA is continuously sourced from liver depot to hepatic portal vein and further to systemic blood circulation.
  • diseases and conditions characterized by elevation of circulating cfDNA level e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.
  • transgenic expression of a DNase I in the liver leads to almost complete clearance of cfDNA accumulated in the liver and in the porto-sinusoidal circulation when DNase is secreted to sinusoidal space of the liver.
  • Such transgenic expression of DNase in the liver leads to significant antitumor effects (especially in tumors in organs and tissues drained by the portal vein), reduction of toxicity of anticancer chemotherapy, a slowdown in autoimmune and neurodegenerative disease progression, etc.
  • Such transgenic expression of DNase in the liver when DNase is secreted to sinusoidal space provides even more benefits when tumor is draining by portal vein system and cfDNA generated in the tumor growth area are subject to“first pass” through porto-sinusoidal circulation.
  • Figure 1 illustrates the organs and systems drained by the portal vein.
  • both intestinal sub-mucus gel layer zone and extracellular nervous tissue space comprise privileged compartments with limited penetration by vast majority of regular components of human blood which are isolated from blood circulation by so called intestinal and blood-brain barriers.
  • An additional aspect of the present invention is based on the realization that an efficient enzymatic cleavage of cfDNA in such privileged compartments benefits from provision of high local concentration of a DNase enzyme in addition to the liver-specific DNase expression (e.g., using viral expression vectors).
  • the present invention further provides DNase gene transfer vectors with liver tandem promoters designed to provide DNase expression in (i) the liver and (ii) the nervous system (e.g., CNS and/or ENS) and/or intestine.
  • the present invention provides various vectors for delivery of the DNase to the liver and/or nervous system and/or intestine.
  • vectors include AAV vectors, adenovirus vectors, retrovirus vectors (e.g., lentivirus vectors), hepatotropic virus vectors (e.g., hepatitis B virus (HBV) vectors), nanoparticles (e.g., phosphoramidite nanoparticles), liposomes (e.g., cationic liposomes such as N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethyl ammonium chloride (DOTMA)), lactoferrin, poly L-lysine, polyethyleneimine, chitosan, etc.).
  • AAV vectors e.g., lentivirus vectors
  • hepatotropic virus vectors e.g., hepatitis B virus (HBV) vectors
  • nanoparticles e.g.
  • liver-specific promoters and control elements include, e.g., an albumin promoter; human alpha- 1 anti -trypsin (hAAT) promoter; thyroxine binding globulin (TBG) promoter; apolipoprotein E hepatic control region promoter; apolipoprotein A-II (APOA2) promoter; serpin peptidase inhibitor, clade A, member 1 (SERPINA1) promoter; cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter; microRNA 122 (MIR122) promoter; liver-specific IGF-II promoter PI; murine transthyretin (MTTR) promoter; and the alpha- fetoprotein (AFP) promoter.
  • the promoter may be upstream or
  • an enzyme which has a DNase activity may be expressed under the control of a liver-specific/nervous system-specific tandem promoter.
  • the liver- specific/nervous system-specific tandem promoter may comprise a liver-specific promoter and a nervous system-specific promoter.
  • the liver-specific promoter and the nervous system-specific promoter may be positioned in any order.
  • the liver-specific promoter may be positioned upstream of the nervous system-specific promoter.
  • the nervous system- specific promoter may be positioned upstream of the liver-specific promoter.
  • nervous system-specific promoters and control elements include, microglia-specific promoters (e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, and Ibal promoters), myeloid- specific promoters (e.g., TTR, CDl lb, and c-fes promoters), neuron-specific promoters (e.g., CMV, NSE, synapsin [e.g., Synl, Synll], CamKII , a-CaMKIL and VGLUT1 promoters), and other neural and glial cell (e.g., oligodendrocytes astrocytes) type-specific promoters (e.g., glial fibrillary acidic protein [GFAP] promoter).
  • microglia-specific promoters e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, and Ibal promoters
  • myeloid-specific promoters
  • the nervous system-specific promoter is a central nervous system (CNS)-specific promoter. In other embodiments, the nervous system-specific promoter is the enteric nervous system (ENS)-specific promoter. In one specific embodiment, the nervous system-specific promoter comprises a human synapsin promoter. In one specific embodiment, the liver-specific/nervous system-specific tandem promoter comprises a hAAT promoter and a human synapsin promoter. The promoter may be upstream or downstream of an enhancer. The promoter sequence may be directly fused to an enhancer sequence. [00186] In some embodiments, an enzyme which has a DNase activity may be expressed under the control of a liver-specific/intestine-specific tandem promoter.
  • the liver- specific/intestine tandem promoter may comprise a liver-specific promoter and an intestine- specific promoter.
  • the liver-specific promoter and the intestine-specific promoter may be positioned in any order.
  • the liver-specific promoter may be positioned upstream of the intestine-specific promoter.
  • the intestine-specific promoter may be positioned upstream of the liver-specific promoter.
  • Specific non-limiting examples of intestine-specific promoters and control elements include, e.g., CB/CMV, GFAP, miCMV, CMV+I, tetO-CMV, b- aeti-CMV, MUC2, Villin, and T3 b promoter.
  • the intestine-specific promoter comprises a human villin promoter.
  • the liver- specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter.
  • the promoter may be upstream or downstream of an enhancer.
  • the promoter sequence may be directly fused to an enhancer sequence.
  • an enzyme which has a DNase activity may be expressed under the control of a liver-specific/ nervous system-specific/ intestine-specific tandem promoter.
  • the liver-specific/nervous system-specific/intestine-specific tandem promoter may comprise a liver-specific promoter, a nervous system-specific promoter and an intestine-specific promoter.
  • the three promoters may be positioned in any order. Specific non-limiting examples of useful nervous system-specific and intestine-specific promoters are provided in the two preceding paragraphs.
  • enzymes which have a DNase activity include DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II (e.g., DNase Ila, DNase IIb), caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
  • DNase I DNase I
  • DNase X DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II (e.g., DNase Ila, DNase IIb), caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
  • DNase II e.g., DNase
  • the enzyme which has a DNase activity is DNase I
  • various mutants weakening actin-binding may be used.
  • residues in wild-type recombinant human DNase I (SEQ ID NO: 4) that can be mutated include, e.g., Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, and Ala-114.
  • the Ala- 114 mutation is used.
  • the Ala-114 residue is mutated.
  • Complementary residues in other DNases may also be mutated.
  • mutations in wild-type human recombinant 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, H44 A : Y65 A : E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S68N:
  • Various DNase mutants for increasing DNase activity may be used.
  • Specific non limiting examples of mutations in wild-type human recombinant DNAse I include, e.g., 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 for increasing the activity of wild-type human recombinant DNase I include Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and A114F.
  • a combination of the Q9R, E13R, N74K and A114F mutations may be used, with such combination found at least in the hyperactive DNase I mutant comprising the sequence of SEQ ID NO: 5.
  • AAV vectors When used for DNase expression, they can be derived from any serotype, e.g., from serotype 1 (AAV1), AAV2, AAV3 (e.g., AAV3A, AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO (as disclosed, e.g., in U.S. Patent No. 9,790,472, Int. Pat. Appl. Pub. Nos. WO2017/180857 and W02017/180861), AAVLK03, AAVLK06, AAVLK12 (as disclosed, e.g., in Wang et al., Mol.
  • AAVhu37 (as disclosed, e.g., in Int. Pat. Appl. Pub. No. WO2017180857)
  • AAVrh64Rl (as disclosed, e.g., in Int. Pat. Appl. Pub. No. WO2017180857)
  • AAV-KP1 (as disclosed, e.g., in Int. Pat. Appl. Pub. No. W02019191701A1)
  • Anc80 Zainn et al., Cell Rep., 2015, 12(67): 1056-1068).
  • Point mutations can be made to the capsid protein (e.g., VP3) to improve the efficiency and/or specificity of liver-specific delivery.
  • capsid protein e.g., VP3
  • Specific non-limiting examples of such point mutations in the AAV8 VP3 capsid protein include, e.g., S279A, S671A, K137R, and T252A, as well as AAV8 capsid mutations disclosed in Int. Pat. Appl. Pub. No.
  • WO2017/180854 e.g., AAV3G1, AAVT20 or AAVTR1, VP3 mutations in amino acids 263-267 [e.g., 263NGTSG267- >SGTH ("NGTSG” disclosed as SEQ ID NO: 43 and “SGTH” disclosed as SEQ ID NO: 44) or 263NGTSG267->SDTH (“NGTSG” disclosed as SEQ ID NO: 43 and “SDTH” disclosed as SEQ ID NO: 45)] and/or amino acids 457-459 [e.g., 457TAN459->SRP], and/or amino acids 455-459 [e.g., 455GGTAN459 ->DGSGL ("GGTAN” disclosed as SEQ ID NO: 46 and “DGSGL” disclosed as SEQ ID NO: 47)] and/or amino acids 583-597). Additionally, mutations may be made to the capsid protein (e.g., VP3) to improve the efficiency and/or specificity of nervous system- and/or intestine-specific delivery.
  • the vectors and compositions of the invention can be targeted to the liver, nervous system and/or intestine using various administration routes.
  • routes of administration to the liver include intrahepatic injection, intravenous injection, and intra arterial injection.
  • routes of administration to the nervous system include intravenous injection, intramuscular injection, intracranial injection, intraspinal injection, intracerebral delivery, oral administration, rectal administration, parenteral administration by nasogastric tube, administration by colonoscopy, and oral administration with or without enhancers to transport therapeutics across the blood-brain barrier (BBB).
  • routes of administration to the intestine include intravenous injection, intramuscular inection, rectal administration, parenteral administration by nasogastric tube, administration by colonoscopy, and oral administration.
  • SEQ ID NO: 1 human DNase I, wild-type (WT), precursor; Genbank Accession No.
  • SEQ ID NO: 2 human DNAse I mutant, precursor; the mutated residues as compared to SEQ ID NO: 1 are in bold and underlined; the secretory signal sequence is underlined:
  • SEQ ID NO: 3 - Anc80 VP1 capsid protein SEQ ID NO: 3 - Anc80 VP1 capsid protein:
  • 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
  • SEQ ID NO: 5 mature human DNAse I mutant (without secretory signal sequence); the mutated residues as compared to SEQ ID NO: 4 are in bold and underlined:
  • SEQ ID NO: 8 human albumin promoter
  • 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;
  • SEQ ID NO: 10 - human beta globin primer SEQ ID NO: 10 - human beta globin primer:
  • SEQ ID NO: 16 Woodchuck hepatitis virus post-transcriptional regulatory element that does not encode functional protein X:
  • SEQ ID NO: 18 - a polynucleotide encoding human DNase I hyperactive variant of SEQ ID NO: 5:
  • SEQ ID NO: 19 a polynucleotide encoding human DNAse I mutant precursor of SEQ ID NO: 2 (secretory signal sequence underlined):
  • SEQ ID NO: 20 - a polynucleotide encoding the secretory signal sequence (SEQ ID NO: 6) of human DNase I:
  • SEQ ID NO: 21 - a polynucleotide encoding the mature human DNAse I mutant (without secretory signal sequence) of SEQ ID NO: 5:
  • SEQ ID NO: 22 - a polynucleotide encoding human DNase I, wild-type (WT), precursor of SEQ ID NO: 1:
  • SEQ ID NO: 23 - a polynucleotide encoding the mature wild-type (WT) human DNase I of SEQ ID NO: 4:
  • SEQ ID NO: 26 mature wild-type (WT) Mus musculus wild type DNase I
  • SEQ ID NO: 27 - a polynucleotide encoding the secretory signal sequence of Mus musculus wild type DNase I
  • SEQ ID NO: 28 - a polynucleotide encoding the mature wild-type (WT) Mus musculus wild type DNase I
  • SEQ ID NO: 29 - a polynucleotide encoding the Mus musculus wild type DNase I, precursor
  • SEQ ID NO: 30 Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader- WPRE Xinact (VR-18013AD)
  • SEQ ID NO: 31 Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNasel wild type-WPRE Xinact (VR-18014AD)
  • SEQ ID NO: 32 - a polynucleotide encoding human DNase I, wild-type (WT), precursor of SEQ ID NO: 1 with stop codon:
  • DNase deoxyribonuclease
  • deoxyribonuclease and“DNase” are used to refer to any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone.
  • DNase a wide variety of deoxyribonucleases is known and can be used in the methods of the present invention.
  • Non-limiting examples of DNases useful in the methods of the present invention include, e.g., DNase I (e.g., recombinant human DNase I (rhDNase I) or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, and DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, and acetylcholinesterase.
  • DNase I e.g., recombinant human DNase I (rhDNase I) or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, and DNAS1L2
  • DNase II e.g., DNase II-alpha, DNase II-beta
  • phosphodiesterase I lactoferr
  • DNase enzymes which have an extended half-life (e.g., albumin and/or Fc fusions, or protected from binding to actin by modification of actin binding-site; see, e.g., 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 of recombinant human DNase I (SEQ ID NO: 4).
  • the Ala-114 residue is mutated.
  • Exemplary 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, A114C, 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
  • DNase I with increased DNase I activity.
  • Non-limiting examples of such mutations are, e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and Al 14Fof recombinant human DNase I (SEQ ID NO: 4).
  • Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and Al 14Fof recombinant human DNase I SEQ ID NO: 4
  • a combination of the Q9R, E13R, N74K and A114F mutations is found in the hyperactive DNase I comprising the sequence of SEQ ID NO: 5.
  • DNase I cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminaied polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides.
  • DNase I acts on single-stranded DNA, double-stranded DNA, and chromatin.
  • the term“about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within an acceptable standard deviation, per the practice in the art.
  • “about” can mean a range of up to ⁇ 20%, up to ⁇ 10%, up to ⁇ 5%, and up to ⁇ 1% of a given value.
  • the term can mean within an order of magnitude, such as within 2-fold, of a value.
  • extracellular DNA e.g., of eukaryotic, viral, archaeal, prokaryotic intracellular or extracellular parasites origin
  • extracellular DNA e.g., of eukaryotic, viral, archaeal, prokaryotic intracellular or extracellular parasites origin
  • DNA in extracellular vesicles e.g., exosomes and microvesicles
  • the cfDNA relevant for the present invention can be found in blood, lymph, liver, nervous tissues, cerebrospinal fluid (CSF), and/or intestine, including DNA in extracellular vesicles (e.g., exosomes and microvesicles) found in these boldily fluids and tissues.
  • CSF cerebrospinal fluid
  • the terms“treat” or“treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • 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, etc.) and experimental animal models of diseases (e.g., mice, rats).
  • the term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. 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 employed, the mode of administration, and the like.
  • the term“therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof.
  • a combination of active ingredients e.g., a combination of DNase and another compound
  • the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
  • 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 with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent 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 sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • A“tissue specific” promoter may be preferentially active in specific types of tissues or cells.
  • liver-specific expression refers to a predominant or exclusive expression in the liver, i.e., expression to a substantially greater extent than in other tissues and organs.
  • liver-specific promoter is used herein to refer to a promoter which is predominantly or exclusively active in a liver cell (e.g., hepatocyte) and directs/initiates transcription in the liver to a substantially greater extent than in other tissues and organs.
  • the term“predominantly” means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (such as 100% of said promoter expression) occurs in liver cells.
  • the ratio of liver expression to non-liver expression can vary between different liver-specific promoters.
  • a liver-specific promoter may preferentially direct/initiate transcription in a particular liver cell type (e.g., hepatocytes, Kupffer cells, endothelial cells, etc.).
  • a particular liver cell type e.g., hepatocytes, Kupffer cells, endothelial cells, etc.
  • Some liver-specific promoters useful in the expression cassettes of the invention include at least one, typically several, hepatic nuclear factor binding sites. Liver- specific promoters useful in the expression cassettes of the invention can be constitutive or inducible promoters.
  • hepatic promoters useful in the expression cassettes of the invention include: albumin promoter (Alb), human alpha-1 anti-trypsin (hAAT) promoter, thyroxine binding globulin (TBG), Apolipoprotein E hepatic control region promoter, Apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3 A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter PI, murine transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, lecithin-cholesterol acyl transferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, and mouse prealbumin gene promoter.
  • albumin promoter Alb
  • liver-specific promoters include, e.g., albumin promoter (Alb), human alpha-1 anti-trypsin (hAAT) promoter, thyroxine binding globulin (TBG) promoter, Apolipoprotein E hepatic control region promoter, Apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINAl) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, Liver- specific IGF-II promoter PI, murine transthyretin (MTTR) promoter, the alpha-fetoprotein (AFP) promoter, a thyroid hormone-binding globulin promoter, an alcohol dehydrogenase promoter, the factor VIII (FVIII) promoter, a HBV basic core promoter (BCP) and Pre
  • the term“nervous system-specific promoter” is used herein to refer to a promoter which is predominantly or exclusively active in a nervous system cell and directs/initiates transcription in the nervous system (e.g., central nervous system (CNS), including brain, and/or enteric nervous system (ENS)) to a substantially greater extent than in other tissues and organs.
  • the term“predominantly” means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (such as 100% of said promoter expression) occurs in cells of the nervous system.
  • the ratio of nervous system expression to non- nervous system expression can vary between different nervous system-specific promoters.
  • a nervous system-specific promoter may preferentially direct/initiate transcription in a particular CNS and/or ENS cell type (e.g., neurons, glial cells [e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, satellite cells], enteric neurons, intrinsic primary afferent neurons, interneurons, motor neurons, etc.).
  • ENS cell type e.g., neurons, glial cells [e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, satellite cells], enteric neurons, intrinsic primary afferent neurons, interneurons, motor neurons, etc.).
  • Nervous system- specific promoters useful in the expression cassettes of the invention can be constitutive or inducible promoters.
  • nervous system promoters useful in the expression cassettes of the invention include microglia-specific promoters (e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, and Ibal promoters), myeloid-specific promoters (e.g., TTR, CD1 lb, and c-fes promoters), neuron-specific promoters (e.g., CMV, NSE, synapsin [Synl, Synll], CamKII , a-CaMKII, and VGLUT1 promoters), and other neural and glial cell (e.g., oligodendrocytes, astrocytes) type-specific promoters (e.g., glial fibrillary acidic protein [GFAP] promoter).
  • microglia-specific promoters e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, and Ibal promoters
  • intestine-specific promoter is used herein to refer to a promoter which is predominantly or exclusively active in an intestinal cell and directs/initiates transcription in the intestine to a substantially greater extent than in other tissues and organs.
  • the term “predominantly” means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (such as 100% of said promoter expression) occurs in intestine cells.
  • the ratio of intestine expression to non- intestine expression can vary between different intestine-specific promoters.
  • an intestine-specific promoter may preferentially direct/initiate transcription in a particular intestinal cell type (e.g., enterocytes, goblet cells, enteroendocrine cells, etc.).
  • Intestine-specific promoters useful in the expression cassettes of the invention can be constitutive or inducible promoters.
  • Some non-limiting examples of intestine promoters useful in the expression cassettes of the invention include CB/CMV, GFAP, mi CMV, CMV+I, tetO-CMV, b-acti-CMV, MUC2, Villin, and T3 b promoters.
  • a nucleic acid encoding an enzyme which has a DNase activity can be operably linked to a promoter that allows for efficient systemic expression (e.g., CMV promoter, chicken b-actin promoter (CBA), or EFla promoter).
  • a promoter that allows for efficient systemic expression (e.g., CMV promoter, chicken b-actin promoter (CBA), or EFla promoter).
  • the term“portal vein cancer” is used herein to refer to a cancer (e.g., carcinoma, sarcoma, or lymphoma) that has originated in, or metastasized to, an organ or tissue that drains to the portal vein.
  • a cancer e.g., carcinoma, sarcoma, or lymphoma
  • Non-limiting examples of such organs and tissues are shown in Figure 1 and include, e.g., liver, colon, small and large intestine, stomach, spleen, pancreas, and gall bladder.
  • neurodegeneration is used herein to refer to a separate clinical pathological condition with progressive loss of structure and/or function of neurons, including death of neurons.
  • the neurodegeneration can be primary or secondary.
  • diseases involving primary neurodegeneration include, e.g., Alzheimer's disease (AD), Mild Cognitive Impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion-caused diseases, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism- 17 (FTDP- 17), Lewy body dementia, vascular dementias, Amyotrophic Later Sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), agyrophilic grain disease (AGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear pals
  • AD Alzheimer's disease
  • Secondary neurodegeneration is caused primarily by necrosis.
  • conditions which may result in secondary neurodegeneration, include destruction of neurons by neoplasm, edema, hemorrhage, stroke, trauma, immune attack, hypoxia, poisoning, metabolic defects, and infections.
  • compositions of the invention refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal such as a human).
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
  • cytostatic and/or cytotoxic chemotherapy” and“chemotherapy” are used interchangeably herein to refer to a therapy involving administering of a cytostatic and/or cytotoxic agent.
  • anti-cancer agent and“anti-cancer chemotherapeutic agent” are used herein to refer to any chemical compound, which is used to treat cancer.
  • Anti-cancer chemotherapeutic agents are well known in the art (see, e.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12: 1202-1263 (1990)). Specific examples of chemotherapeutic agents are provided throughout the specification.
  • side effect of a chemotherapy refers to an undesirable and unintended, although not necessarily unexpected, result of a chemotherapy.
  • the terms“radiotherapy”,“radiation therapy”, and“RT” are used interchangeably to refer to the medical use of ionizing radiation as part of a cancer treatment to damage the DNA of malignant cells, either directly or by creating charged particles within the affected cells that damage the DNA.
  • Commonly used types of radiation therapy encompassed by the present invention include, e.g., external beam radiation therapy (EBRT or XRT), brachytherapy/sealed source radiation therapy, and systemic radioisotope therapy/unsealed source radiotherapy.
  • side effect of a radiotherapy or“side effect of a radiation therapy” as used herein refer to an undesirable and unintended, although not necessarily unexpected, result of a radiation therapy. Which side effects develop depend on the area of the body being treated, the dose given per day, the total dose given, the patient's general medical condition, and other treatments given at the same time, and may include, e.g., skin irritation or damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, or a second cancer.
  • catabolic state refers to a condition characterized by a rapid weight loss and loss of fat and skeletal muscle mass, which may occur in a background of chemotherapy or chemoradiation therapy. Associated clinical events include, for example, immunosuppression, muscle weakness, predisposition to pulmonary embolism, thrombophlebitis, and altered stress response.
  • the terms“viral vector” and“viral construct” refer to a recombinant viral construct that comprises one or more heterologous nucleotide sequences (e.g., a nucleotide sequence encoding an enzyme which has a DNase activity).
  • the viral vector is replication deficient.
  • viral structural and non-structural coding sequences are not present in the viral vector and are provided during viral vector production in trans by a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell line.
  • a viral vector can be packaged within a capsid (e.g., an AAV vector) and/or a lipid envelope (e.g., a lentiviral vector).
  • polyadenylation signal relates to a nucleic acid sequence that mediates the attachment of a polyadenine stretch to the 3 ' terminus of the mRNA.
  • Non-limiting examples of polyadenylation signals which can be used on the expression constructs of the invention include, e.g., the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 Elb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like.
  • the inventors have unexpectedly found that targeting expression of an enzyme which has a DNase activity to the liver can bring about an enhanced clearance of disease-associated cfDNA from the entire circulation, and especially from the porto-sinusoidal circulation.
  • cfDNA can accumulate in the liver, particularly in the periendothelial space and the space of Disse. Degradation of such reservoir of cfDNA via localized expression of an enzyme which has DNase activity can lower the overall level of cfDNA in the circulation.
  • an enzyme which has DNase activity can be efficiently expressed in liver cells through the use of viral vectors (e.g., adeno-associated viral (AAV) vectors, retroviral vectors (e.g., lentiviral vectors), or adenoviral vectors), liposomes, nanoparticles carrying a DNase transgene, or naked DNA.
  • viral vectors e.g., adeno-associated viral (AAV) vectors, retroviral vectors (e.g., lentiviral vectors), or adenoviral vectors)
  • liposomes e.g., adeno-associated viral (AAV) vectors, retroviral vectors (e.g., lentiviral vectors), or adenoviral vectors)
  • nanoparticles carrying a DNase transgene e.g., adeno-associated viral (AAV) vectors, retroviral vectors (e.g., lentiviral vectors), or adenoviral vector
  • Such hepatocytes can be transfected by the vector particles and go on to produce DNase.
  • the produced DNase is secreted into the periendothelial space and the space of Disse, where it can efficiently degrade the concentrated pool of cfDNA.
  • liver cells to express an enzyme which has DNase activity provides a surprisingly greater reduction in cfDNA, along with substantial and surprising improvement in diseases and conditions associated with high levels of cfDNA in the bloodstream.
  • expression of DNase enzyme in the liver is accomplished by use of nucleic acid expression cassettes that are predominantly expressed in the liver.
  • expression cassettes can comprise one or more of the following elements: (a) a hepatic locus control element; (b) a hepatic promoter located 3' to the hepatic locus control element; (c) a coding sequence located 3' to the hepatic promoter, said coding sequence encoding a polypeptide, e.g.
  • the expression cassettes of the invention direct expression of a therapeutic amount of a polypeptide in liver cells for a period of at least 100 days (such as at least 200 days, at least 300 days, at least 400 days, or at least 500 days).
  • the polypeptide is a DNase.
  • the DNase may be any DNase described herein, e.g., DNase I.
  • the polyadenylation signal may be operably linked to the nucleic acid encoding DNase.
  • polyadenylation signals include, e.g., the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 Elb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like.
  • the invention provides vectors that comprise a nucleic acid expression cassette that is predominantly expressed in the mammalian liver.
  • Such expression cassette can comprise, e.g. : (a) a hepatic locus control element; (b) an hepatic promoter located 3' to the hepatic locus control element; (c) a coding sequence located 3' to the hepatic promoter, said coding sequence encoding a polypeptide, e.g. a deoxyribonuclease (DNase); (d) a polyadenylation signal located 3' to the coding sequence; and (e) an intron located 3' to the hepatic promoter and 5' to the polyadenylation signal.
  • Elements (a), (b), (c), (d) and (e) are operably linked to express the polypeptide encoded by the coding sequence.
  • Some vectors of the invention are episomal vectors, and some vectors of the inventions are integrating vectors such as integrating viral vectors.
  • the polypeptide is a deoxyribonuclease (DNase).
  • the DNase may be any DNase described herein, e.g., DNase I.
  • the present invention provides methods for treating a disease or condition associated with increased levels of cfDNA.
  • the disease or condition may be any disease or condition described herein or in any of U.S. Patent Nos. 7,612,032; 8,388,951; 8,431,123; 8,535,663; 8,710,012; 8,796,004; 8,871,200; 8,916, 151; 9,072,733; 9,248, 166; 9,770,492; U.S. Pat.
  • the method comprises the steps of: (1) introducing into the liver of a subject a vector comprising a nucleic acid expression cassette, said expression cassette comprising: (a) a liver- specific promoter; (b) a coding sequence located 3' to the liver-specific promoter, said coding sequence encoding an enzyme which has a DNase activity (e.g., any DNAs described herein); (c) a polyadenylation signal located 3' to the coding sequence; and optionally (d) an intron located 3' to the hepatic promoter and 5' to the polyadenylation signal.
  • a vector comprising a nucleic acid expression cassette, said expression cassette comprising: (a) a liver- specific promoter; (b) a coding sequence located 3' to the liver-specific promoter, said coding sequence encoding an enzyme which has a DNase activity (e.g., any DNAs described herein); (c) a polyadenylation signal located 3' to the coding sequence; and optionally (d
  • 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.
  • a therapeutic amount of the polypeptide is expressed for at least 100 days (such as at least 200 days, at least 300 days, at least 400 days, and at least 500 days).
  • the nucleic acid expression vectors and cassettes of the invention which comprise just the liver-specific promoter are predominantly expressed in the liver, e.g., at least 50% of the expression of the encoded polypeptide occurs within the liver. More typically, at least 90% of the expression occurs within the liver. Some of such expression vectors and cassettes of this aspect of the invention are exclusively expressed within the liver.
  • the nucleic acid expression vectors and cassettes of the invention which comprise a liver-specific/nervous system-specific tandem promoter or a liver-specific/intestine-specific tandem promoter or a liver-specific/nervous system- specific/intestine-specific tandem promoter are expressed both (i) in the liver and (ii) in the nervous system and/or intestine.
  • Hepatic, nervous system or intestinal locus control elements may be incorporated into the expression cassettes of the invention that are capable of enhancing the expression of nucleic acid molecules in liver cells, nervous system cells or intestinal cells and confer copy number dependent, position independent expression on the expression cassette.
  • Some hepatic locus control regions useful in the expression cassettes of the invention include, e.g., a matrix attachment region and/or a liver-specific enhancer element.
  • an expression cassette and/or a vector may contain one or more additional transcription initiation, termination, and/or enhancer sequences; RNA processing signals (such as splicing and polyadenylation (poly A) signals); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence, such as 5'-GCCGCCACC-3' [SEQ ID NO: 33]); sequences that enhance protein stability; and sequences that enhance secretion of the encoded polypeptide.
  • suitable polyA sequences include, e.g., SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic poly As.
  • the expression cassette comprises one or more expression enhancers.
  • the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another.
  • an enhancer may include an Alpha mic/bik enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences.
  • the expression cassette further contains an intron (e.g., the Promega intron, a truncated chimeric intron (T-chimeric intron), introns described in Int. Pat. Appl. Pub. No. WO 2011/126808).
  • an intron e.g., the Promega intron, a truncated chimeric intron (T-chimeric intron), introns described in Int. Pat. Appl. Pub. No. WO 2011/126808).
  • one or more sequences may be selected to stabilize mRNA.
  • An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence (see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605-619).
  • the enhancer may be an ApoE enhancer element of approximately 155 bp derived from apolipoprotein E or ApoE.
  • ApoE is an apolipoprotein that mediates binding, internalization and catabolism of lipoprotein particles and is a ligand for the low-density lipoprotein (ApoB/E) receptor and for the ApoE receptor of hepatic tissues.
  • the genetic enhancer associated with the ApoE gene is a eukaryotic control element that can increase transcription of a nucleic acid specifically in the liver.
  • the enhancer may include an element from a hepatic control region (e.g., HCR-1 or HCR-2) associated with the ApoE gene.
  • a hepatic control region e.g., HCR-1 or HCR-2
  • sequence from HCR-1 is found in the sequence of SEQ ID NO: 17.
  • the expression cassette and/or vector further contains a post-transcriptional regulatory element (PRE).
  • An exemplary post-transcriptional regulatory element is the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
  • WPRE Woodchuck hepatitis virus post-transcriptional regulatory element
  • SEQ ID NO: 16 a sequence from the WPRE is shown in SEQ ID NO: 16.
  • the WPRE may be effective to increase expression of the protein (e.g., DNase).
  • the WPRE may be modified so as not to produce an active form of a truncated X protein that is encoded in the WPRE.
  • One such modified WPRE is provided in at least the sequence of SEQ ID NO: 16 and in the ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader-WPRE Xinact (bases 1576-2212 of SEQ ID NO: 30; see also Figures 10A and 10B).
  • the cassettes of the invention direct the expression of a therapeutic amount of the polypeptide encoded by the coding sequence for an extended period, typically greater than 200 days, and in some instances greater than 500 days.
  • Expression of the polypeptide encoded by the coding sequence can be measured by any art-recognized means, such as, e.g., by antibody-based assays, such as a Western Blot or an ELISA assay.
  • expression of the polypeptide encoded by the coding sequence within the expression cassette can be measured in a bioassay that detects an enzymatic or biological activity of the polypeptide.
  • the vectors can remain episomal (i.e., do not integrate into the genome of a host cell), or can integrate into the host cell genome.
  • episomal vectors include adenoviral vectors
  • vector that integrate into the host cell genome include retroviral vectors.
  • the expression vectors comprising a nucleotide sequence encoding an enzyme which has 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- diolcyloxy propyl -3 -trimethyl ammonium bromide) and DOPE
  • DOTMA 1,2- diolcyloxy propyl -3 -trimethyl ammonium bromide
  • cationic liposomes containing DOTMA are effective to target expression vectors of the invention to the liver.
  • lactoferrin and/or poly L-lysine and/or poly ethyl enemine and/or chitosan is conjugated to an expression vector, with the conjugate effectively targeted to the liver after direct introduction into a subject.
  • the liposomes have dimensions smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver.
  • the expression vector is packaged into a nanovector or is encapsulated by a nanovector.
  • the expression vector is packaged into phosphoramidite nanoparticles.
  • nucleic acids of the invention encoding enzymes which has DNase activity
  • molecular conjugates consist of protein or synthetic ligands to which a nucleic acid-binding agent has been attached for the specific targeting of nucleic acids to cells
  • This gene delivery system has been shown to be capable of targeted delivery to many cell types through the use of different ligands (R. J. Cristiano et al., Proc. Natl. Acad. Sci. USA 90: 11548-52 (1993)).
  • lipofection and malaria circumsporozoite protein have been used for the liver-specific delivery of genes (Z. Ding et al., J. Biol. Chem. 270:3667-76 (1995)).
  • liver delivery of the expression vectors of the invention are intrahepatic, intravenous, and intra-arterial administration.
  • routes of administration allowing liver targeting can be also employed such as, e.g., enteral (e.g., oral), intramuscular, intraperitoneal, etc.
  • the expression vectors of the invention can be introduced directly into the liver by any art-recognized means, such as by direct injection into the liver, portal vein injection, or tail vein injection.
  • the expression vectors of the invention may be delivered by (1) creating an access site in an artery of a mammal, (2) introducing a guidewire into the artery, (3) introducing a sheath over the guidewire into the artery, (4) advancing a catheter through the sheath into the liver, (5) infusing the expression vector, e.g., rAAV virions, through the catheter into the liver, (6) removing the catheter and the sheath from the artery, and (7) repairing the access site.
  • the catheter may be introduced into the target organ through an appropriate vein or artery.
  • the liver may be accessed via either the portal vein or the hepatic artery.
  • An appropriate vein or artery may be accessed using techniques known in the art, such as the Seldinger technique. See, e.g., Conahan et al., (1977) JAMA 237:446-447, herein incorporated by reference. Other methods of accessing veins and arteries are also known. See, e.g., U.S. Pat. No. 5,944,695 herein incorporated by reference.
  • occluding one or more of the blood vessels delivering blood to, or emptying blood from, the liver is conducted.
  • the occluding device can be, for example, a balloon attached to the tip of a catheter. All of the arteries and veins entering or leaving the liver may be occluded so that the expression vector is delivered by way of asanguinous hepatic perfusion, whereby the liver is selectively perfused after excluding normal blood flow from the liver. This may be accomplished by clamping the hepatic artery, portal vein, suprahepatic vena cava, right suprarenal vein and infrahepatic vena cava to achieve the total vascular exclusion of the liver.
  • the portal vein is then cannulated with a catheter of appropriate gauge, while an incision is made in the anterior wall of the infrahepatic vena cava, with a suction cannula placed into the vena cava to collect outflow.
  • Liver perfusion preferably single pass perfusion, is then performed using a pump.
  • the flow rate may range 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.
  • the portal and caval vein incisions are sutured and the liver is revascularized. Cardoso, et al., Human Gene Therapy 4:411-418 at 412 (1993).
  • expression vectors are delivered into the liver by injection into the hepatic artery.
  • the parenchymal cells include hepatocytes, Kupffer cells and the epithelial cells that line the biliary tract and bile ductules.
  • the major constituent of the liver parenchyma are polyhedral hepatocytes (also known as hepatic cells) that presents at least one side to a hepatic sinusoid and apposed sides to a bile canaliculus.
  • Liver cells that are not parenchymal cells include cells within the blood vessels such as the endothelial cells or fibroblast cells.
  • the hepatic vein is an efferent blood vessel since it normally carries blood away from the liver into the inferior vena cava. Also in the liver, the portal vein and hepatic arteries are afferent blood vessels in relation to the liver since they normally carry blood towards the liver.
  • a liver blood vessel includes the portal venous system which transports blood from the gastrointestinal tract and other internal organs (e.g. spleen, pancreas and gall bladder) to the liver.
  • Another liver blood vessel is the hepatic vein.
  • the hepatic vein may also be reached via the inferior vena cava or another blood vessel that ultimately connects to the liver.
  • a needle or catheter may be used to inject the polynucleotide into the vascular system.
  • the injection can be performed under direct observation following an incision and visualization of the tissues blood vessels.
  • a catheter can be inserted at a distant site and threaded so that it resides in the vascular system that connects with the target tissue.
  • deep injection into the vena cava is performed.
  • the injection could be performed by using a needle that traverses the intact skin and enters a vessel that supplies or drains from the target tissue.
  • the delivery is performed as follows.
  • the liver and portal vein are visualized through a ventral midline incision.
  • a vector/liposome/naked DNA in 1 ml of various solutions containing heparin to prevent clotting is injected into the portal vein using a needle over approximately 30 sec.
  • the efficiency of liver delivery, expression and secretion of the enzyme which has DNase activity can be determined, e.g., by assaying the level of the enzyme in the blood (e.g., blood obtained from the retro-orbital venous sinus), e.g., using immunological assays (e.g., assays with antibodies recognizing the enzyme such as., e.g., a radioimmune assay (RIA) (HGH-TGES 100T kit from Nichols Institute, San Juan Capistrano, Calif., USA).
  • RIA radioimmune assay
  • the efficiency of liver delivery, expression and secretion of the enzyme which has DNase activity can be determined, e.g., by sacrificing the animals by cervical dislocation and the livers (average weight of 1.5 g) divided into six sections composed of two pieces of median lobe, two pieces of left lateral lobe, the right lateral lobe, and the caudal lobe plus a small piece of right lateral lobe.
  • Each of the six sections may be placed separately into a homogenizing buffer.
  • the homogenates may be centrifuged and the supernatant analyzed for the foreign gene product (i.e., enzyme which has DNase activity).
  • Liver injections can be directed into the inferior cava which was clamped in two locations: proximal and distal to the entry of the hepatic vein into the inferior vena cava.
  • the downstream inferior vena cava clamp can be placed between the diaphragm and the entry point of the hepatic vein.
  • the upstream inferior vena cava clamp can be placed just upstream of the entry point of the renal veins. Since the veins of other organs such as the renal veins enter the inferior vena cava at this location, not all of the injection fluid goes into the liver.
  • the hepatic artery, mesenteric artery, and portal vein can be clamped (occluded).
  • the present invention provides compositions and methods for achieving DNase expression in (i) the liver and (ii) in the nervous system and/or intestine.
  • DNase expression is achieved using vectors that comprise a nucleic acid expression cassette that is expressed (i) in the mammalian liver and also (ii) in the nervous system and/or intestine.
  • Such expression cassette can comprise a liver-specific/nervous system-specific tandem promoter or a liver-specific/intestine-specific tandem promoter or a liver-specific/nervous system-specific/intestine-specific tandem promoter.
  • DNase expression (using tandem promoters) in the nervous system and/or intestine, in addition to its expression in the liver, can lead to superior therapeutic efficacy of DNase-mediated treatment.
  • a DNase enzyme is targeted for expression in both the liver and the nervous system. Delivery of the DNase enzyme to the nervous system may be accomplished by use of nucleic acid expression cassettes wherein DNase is expressed in the nervous system under the control of a nervous system-specific promoter. In some embodiments, delivery of the DNase enzyme to the nervous system is accomplished by the use of nucleic acid expression cassettes that are expressed in both the liver and nervous system due to the use of a tandem promoter comprising a liver-specific promoter and a nervous system-specific promoter. In some embodiments, the nervous system-specific promoter is a central nervous system (CNS)- specific promoter.
  • CNS central nervous system
  • the nervous system-specific promoter is the enteric nervous system (ENS)-specific promoter.
  • the nervous system-specific promoter is human synapsin promoter.
  • the human synapsin promoter comprises a sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the sequence SEQ ID NO: 36.
  • the human synapsin promoter comprises the sequence SEQ ID NO: 36.
  • the human synapsin promoter consists of the sequence SEQ ID NO: 36.
  • such expression cassettes comprise a liver-specific/nervous system-specific tandem promoter.
  • the liver-specific/nervous system-specific tandem promoter comprises a hAAT promoter and a human synapsin promoter.
  • a DNase enzyme is targeted for expression in both the liver and the intestine.
  • Delivery of the DNase enzyme to the intestine may be accomplished by use of nucleic acid expression cassettes wherein DNase is expressed in the intestine under the control of an intestine-specific promoter.
  • delivery of the DNase enzyme to the intestine is accomplished by use of nucleic acid expression cassettes that are expressed in both the liver and the intestine due to the use of a tandem promoter comprising a liver-specific promoter and an intestine-specific promoter.
  • the intestine-specific promoter is human villin promoter.
  • the intestine-specific promoter comprises a sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the sequence SEQ ID NO: 37 or a fragment thereof.
  • the liver- specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter.
  • a DNase enzyme is targeted for expression in the liver, nervous system and intestine. Delivery of the DNase enzyme to nervous system and the intestine may be accomplished by use of nucleic acid expression cassettes that are expressed in both the nervous system and the intestine. In some embodiments, delivery of the DNase enzyme to the nervous system and the intestine is accomplished by use of nucleic acid expression cassettes that are expressed in the liver, nervous system and intestine. In some embodiments, such expression cassettes comprise a nervous system-specific promoter and an intestine-specific promoter. In some embodiments, such expression cassettes comprise DNase-coding sequence under the control of a liver-specific/nervous system-specific/intestine-specific tandem promoter.
  • the liver-specific/nervous system-specific/intestine-specific tandem promoter may comprise a liver-specific promoter, a nervous system-specific promoter and an intestine-specific promoter.
  • the three promoters may be positioned in any order.
  • the liver-specific/nervous system- specific/intestine-specific tandem promoter comprises a hAAT promoter, a human synapsin promoter, and a human villin promoter.
  • the vector used in the invention is a parvovirus vector, such as an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • parvovirus encompasses the family Parvoviridae, including autonomously-replicating parvoviruses and dependoviruses.
  • the autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus.
  • Autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, HI parvovirus, muscovy duck parvovirus, B 19 virus, and any other autonomous parvovirus now known or later discovered.
  • Other autonomous parvoviruses are known to those skilled in the art. See, e.g., Bernard N. Fields et ah, Virology, Vol. 2, Chapter 69 (4th ed., Lippi ncott-Raven Publishers).
  • the parvovirus vector is a single-stranded parvovirus vector, such as an AAV vector.
  • AAV gene Dependovirus
  • humans e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6
  • primates e.g., serotypes 1 and 4
  • other warm-blooded animals e.g., bovine, canine, equine, and ovine AAVs.
  • parvoviruses and other members of the Parvoviridae is provided, e.g., in Kenneth I. Berns,“Parvoviridae: The Viruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed. 1996).
  • AAV vectors disclosed herein may be derived from any AAV serotype, including combinations of serotypes (e.g.,“pseudotyped” AAV) or from various genomes (e.g., single- stranded or self-complementary).
  • serotype e.g.,“pseudotyped” AAV
  • A“serotype” is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences).
  • Non-limiting examples of AAV serotypes which can be used to develop the AAV expression vectors of the invention include, e.g., AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO (as disclosed, e.g., in U.S. Patent No. 9,790,472, Int. Pat. Appl. Pub. No. WO2017180857 and W02017/180861), AAVLK03, AAVLK06, AAVLK12 (as disclosed, e.g., in Wang et al., Mol.
  • AAV-KPl (as disclosed, e.g., in Int. Pat. Appl. Pub. No. W02019191701A1)
  • AAVhu37 (as disclosed, e.g., in Int. Pat. Appl. Pub. No. WO2017180857)
  • AAVrh64Rl (as disclosed, e.g., in Int. Pat. Appl. Pub. No.
  • Anc80 (based on a predicted ancestor of serotypes AAV1, AAV2, AAV8 and AAV9; 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 now known or later discovered. See, e.g., Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • the genomic organization of all known AAV serotypes is very similar.
  • the genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length.
  • Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins.
  • the VP proteins (VPl, VP2 and VP3) form the capsid.
  • the terminal 145 nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex.
  • the Rep genes i.e. Rep78 and Rep52
  • both Rep proteins have a function in the replication of the viral genome.
  • recombinant AAV (rAAV) vectors comprise one or more nucleotide sequences of interest that are flanked by at least one parvoviral or AAV inverted terminal repeat sequence (ITR).
  • ITR parvoviral or AAV inverted terminal repeat sequence
  • Such rAAV vectors can be replicated and packaged into viral particles when produced in a packaging cell that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins).
  • AAV Cap protein or“AAV capsid protein”, as used herein, refer to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VPl, VP2, VP3).
  • Cap proteins examples include the ability to induce formation of a capsid, facilitate accumulation of single-stranded DNA, facilitate AAV DNA packaging into capsids (i.e., encapsidation), bind to cellular receptors, and facilitate entry of the virion into host cells.
  • the AAV vectors may comprise desired proteins or protein variants.
  • A“variant” as used herein refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have“conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have“nonconservative” changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • AAV vectors are packaged into AAV viral capsids.
  • the sequence of an AAV viral capsid protein defines numerous features of a particular AAV vector.
  • the capsid protein affects capsid structure and assembly, interactions with AAV nonstructural proteins such as Rep and AAP proteins, interactions with host body fluids and extracellular matrix, clearance of the virus from the blood, vascular permeability, antigenicity, reactivity to neutralizing antibodies, tissue/organ/cell type tropism, efficiency of cell attachment and internalization, intracellular trafficking routes, virion uncoating rates, among others.
  • the sequence of a capsid protein (e.g., VP3) may be altered to enhance delivery to the liver.
  • AAV constructs may further comprise a sequence encoding one or more capsid proteins (VP1 and/or VP2, and/or VP3 capsid proteins, preferably just VP3 capsid protein) which package the above-mentioned polynucleotide sequence.
  • the sequences coding for the capsid protein(s) for use in the context of the present invention may be taken from any of the known 42 serotypes, such as, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80, or newly developed AAV-like particles obtained by, e.g., capsid shuffling techniques and/or use of AAV capsid libraries.
  • capsid protein sequences see, e.g., U.S. Patent Nos. 9,790,472; 9,677,089; 7,282,199; Int. Pat. Appl. Publ. Nos. WO 2015/054653, WO2017/180857 (AAV8, AAV9, AAVrhlO, AAVhu37, AAVrh64Rl), W02017/180861 (AAVrhlO), WO2017/180854 (AAV8 mutants), and Wang et ah, Mol. Ther., 2015, 23(12): 1877-1887 (AAV8, AAVrhlO, AAV3B, and AAVLK03).
  • the AAV construct is known as a“hybrid” parvovirus genome (i.e., in which the AAV capsid and the AAV terminal repeat(s) are from different AAV) as described in Int. Pat. Appl. Publ. No. WO 00/28004 and Chao et ah, (2000) Molecular Therapy 2:619.
  • the capsid protein(s) mediates efficient targeting of the AAV vector to the liver. In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector to the liver.
  • Some capsid proteins e.g., VP3 of Anc80, AAV8 and AAV3B
  • the invention also encompasses the use of AAV capsid mutants which enhance liver targeting and/or liver specificity.
  • Non-limiting examples of such point mutations to the AAV8 capsid sequence include, e.g., S279A, S671A, K137R, and T252A, as well as AAV8 capsid mutations disclosed in Int. Pat. Appl. Pub. No.
  • WO2017/180854 e.g., AAV3G1, AAVT20 or AAVTR1, VP3 mutations in amino acids 263-267 [e.g., 263NGTSG267->SGTH ("NGTSG” disclosed as SEQ ID NO: 43 and “SGTH” disclosed as SEQ ID NO: 44) or 263NGTSG267- >SDTH ("NGTSG” disclosed as SEQ ID NO: 43 and “SDTH” disclosed as SEQ ID NO: 45)] and/or amino acids 457-459 [e.g., 457TAN459->SRP], and/or amino acids 455-459 [e.g., 455GGTAN459 ->DGSGL ("GGTAN” disclosed as SEQ ID NO: 46 and “DGSGL” disclosed as SEQ ID NO: 47)] and/or amino acids 583-597).
  • the capsid protein(s) mediates efficient targeting of the AAV vector to the nervous system. In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector both to the liver and to the nervous system. Some capsid proteins would naturally target both the liver and the nervous system.
  • the invention also encompasses the use of AAV capsid mutants which enhance targeting to the liver and/or the nervous system.
  • the capsid protein(s) mediates efficient targeting of the AAV vector to the intestine. In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector both to the liver and to the intestine. Some capsid proteins would naturally target both the liver and the intestine.
  • the invention also encompasses the use of AAV capsid mutants which enhance targeting to the liver and/or the intestine.
  • the capsid protein(s) mediate preferential targeting of the AAV vector to the liver, nervous system and intestine. Some capsid proteins would naturally target the liver, nervous system and intestine.
  • the invention also encompasses the use of AAV capsid mutants which enhance targeting to the liver and/or the nervous system and/or intestine.
  • the AAV vectors disclosed herein include a nucleic acid encoding an enzyme which has a DNase activity, such as, e.g., DNase I.
  • the nucleic acid also may include one or more regulatory sequences allowing expression and secretion of the encoded enzyme, such as e.g., a promoter, enhancer, polyadenylation signal, an internal ribosome entry site (IRES), a sequence encoding a protein transduction domain (PTD), a secretory signal sequence, and the like.
  • the nucleic acid may comprise a promoter region operably linked to the coding sequence to cause or improve expression of the protein of interest in transfected cells.
  • Such a promoter may be ubiquitous, cell- or tissue-specific, strong, weak, regulated, chimeric, etc., for example to allow efficient and stable production of the protein in the liver, nervous system and/or intestine.
  • the promoter may be homologous to the encoded protein, or heterologous, although generally promoters of use in the disclosed methods are functional in human cells.
  • Examples of regulated promoters include, without limitation, Tet on/off element- containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters.
  • Other promoters that may be used include promoters that are tissue specific, e.g., for liver, nervous system and/or intestine.
  • liver-specific promoters include, e.g., the albumin promoter (Alb), human alpha- 1 anti -trypsin (hAAT) promoter, thyroxine binding globulin (TBG), apolipoprotein E hepatic control region promoter, apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3 A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter PI, murine transthyretin (MTTR) promoter, and the alpha-fetoprotein (AFP) promoter.
  • Alb albumin promoter
  • hAAT human alpha- 1 anti -trypsin
  • TBG thyroxine binding globulin
  • APOA2 apolipoprotein E hepatic control
  • Non-limiting examples of nervous system-specific promoters include, e.g., microglia-specific promoters (e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, Ibal), myeloid-specific promoters (e.g., TTR, CD1 lb, c-fes), neuron specific promoters (e.g., CMV, NSE, synapsin [Synl, Synll], CamKII , a- CaMKII, VGLUT 1 ), and other neural and glial cell (e.g., oligodendrocytes astrocytes) type- specific promoters (e.g., GFAP).
  • microglia-specific promoters e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, Ibal
  • myeloid-specific promoters e.g., TTR, CD1 lb, c-fes
  • Non-limiting examples of intestine-specific promoters include, MUC2, Villin, T3b, CB/CMV, GFAP, miCMV, CMV+I, tetO-CMV, and b-acti-CMV.
  • Non limiting examples of ubiquitous promoters include, e.g., viral promoters such as the CMV promoter, the RSV promoter, the SV40 promoter, etc., and cellular promoters such as the phosphoglycerate kinase (PGK) promoter, EF la promoter, CM VE/C AG promoter system, and the b-actin promoter.
  • PGK phosphoglycerate kinase
  • the AAV constructs of the invention may also contain non-resolvable terminal repeats.
  • the expression“non-resolvable terminal repeat”, as used herein, relates to terminal repeats which are not recognized by and resolved (i.e.,“nicked”) by the AAV Rep proteins, such that resolution of the terminal repeat is substantially reduced (e.g., by at least about 50%, 60%, 70%, 80%. 90%, 95%, 98% or greater as compared with a resolvable terminal repeat) or eliminated.
  • non-resolvable terminal repeats may be naturally-occurring terminal repeat sequences (including altered forms thereof) and, for example, can be derived from a parvovirus, including an AAV, or can be from another virus or, as a further alternative, can be partially or completely synthetic.
  • the non-resolvable terminal repeat may be a non-AAV viral sequence that is not recognized by the AAV Rep proteins, or it can be an AAV terminal repeat that has been modified (e.g., by insertion, substitution and/or deletion) so that it is no longer recognized by the AAV Rep proteins.
  • a non-resolvable terminal repeat can be any terminal repeat that is non-resolvable under the conditions used to produce the virus vector.
  • an AAV terminal repeat can be modified so that resolution by the AAV Rep proteins is substantially reduced or eliminated.
  • the non-resolvable terminal repeat can be any inverted repeat sequence that forms a hairpin structure and cannot be nicked by the AAV Rep proteins.
  • the inverted terminal repeats are typically present in at least two copies in the AAV vector, typically flanking the expression cassette containing the nucleotide sequence(s) encoding an enzyme which has a DNase activity.
  • the ITRs typically will be at the 5' and 3' ends of the nucleotide sequence(s) encoding an enzyme which has a DNase activity but need not be contiguous thereto.
  • the ITRs can be the same or different from each other.
  • the term“terminal repeat” includes any viral terminal repeat and/or partially or completely synthetic sequences that form hairpin structures and function as an inverted terminal repeat, such as the“double-D sequence” as described in U.S. Pat. No. 5,478,745 to Samulski et al.
  • An“AAV terminal repeat” may be 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 now known or later discovered.
  • the AAV terminal repeat need not have a wild-type sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, nicking, virus packaging, integration, and/or provirus rescue, and the like.
  • the vector construct can comprise one or more (e.g., two) AAV terminal repeats, which may be the same or different.
  • the one or more AAV terminal repeats can be from the same AAV serotype as the AAV capsid, or can be different.
  • the vector construct comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, and/or Anc80 terminal repeat.
  • Parvoviral ITR nucleotide sequences are typically palindromic sequences, comprising mostly complementary, symmetrically arranged sequences also referred to as“A,”“B,” and“C” regions.
  • the ITR functions as an origin of replication, a site having a“cis” role in replication, i.e., being a recognition site for trans acting replication proteins such as e.g. Rep 78 (or Rep68) which recognize the palindrome and specific sequences internal to the palindrome.
  • Rep 78 or Rep68
  • One exception to the symmetry of the ITR sequence is the“D” region of the ITR. It is unique (not having a complement within one ITR). Nicking of single-stranded DNA occurs at the junction between the A and D regions.
  • a parvovirus replicating in a mammalian cell typically has two ITR sequences. It is, however, possible to engineer an ITR so that binding sites are on both strands of the A regions and D regions are located symmetrically, one on each side of the palindrome.
  • the Rep78- or Rep68-assisted nucleic acid replication then proceeds in both directions and a single ITR suffices for parvoviral replication of a circular vector.
  • one ITR nucleotide sequence can be used in the context of the present invention. Two or another even number of regular ITRs can be used.
  • the ITR sequences are necessary for the replication, rescue and packaging of AAV virions.
  • the ITR sequences may be wild type sequences or may have at least 80%, 85%, 90%, 95%, or 100% sequence identity with wild type sequences.
  • the ITR sequences may be altered by for example in insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional.
  • functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be transduced or target cell.
  • the AAV vector can comprise single stranded or double stranded (self complementary) DNA.
  • the single stranded nucleic acid molecule is either sense or antisense strand, as both polarities are equally capable of gene expression.
  • the AAV vector may further comprise a marker or reporter gene, such as a gene 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.
  • a marker or reporter gene such as a gene 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.
  • AAV expression vectors may comprise a nucleic acid that may include a secretory signal sequence allowing secretion of the encoded enzyme which has a DNase activity from the transduced cell.
  • secretory signal sequences include, e.g., DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, b- glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence.
  • an AAV8-based or an Anc80-based vector is used as the expression vector.
  • the AAV8 and Anc80 vectors are particularly suited for liver targeting and expression.
  • both AAV8 and Anc80 vectors can transfect liver cells with greater efficiency as compared to an AAV2 vector.
  • Both AAV8 and Anc80 also induce lower amounts of neutralizing antibodies than some of the other AAV vectors.
  • An AAV8 vector or an Anc80 vector comprising a nucleotide encoding for an enzyme which has a DNase activity can be administered intrahepatically (e.g., via direct organ injection) or systemically, e.g., by intravenous injection, with the AAV8 or Anc80 vector effective to transfect liver cells and mediate effective production of the encoded enzyme which has a DNase activity and its secretion in the periendothelial space and the space of Disse.
  • an Anc80 capsid protein (e.g., Anc80 VP1 capsid protein comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 9, or an Anc80L65 VP1 capsid protein comprising the sequence of SEQ ID NO:34, or a variant Anc80L65 VP1 capsid protein comprising the sequence of SEQ ID NO: 35) is encoded by a nucleotide sequence in the expression vector.
  • an AAV8 capsid protein (e.g., AAV8 VP1 or AAV8 VP3) is encoded by a nucleotide sequence in the expression vector.
  • Peripheral administration of the AAV vectors of the invention may include systemic injections, such as, e.g., intramuscular, intravenous, intraperitoneal, and intra-arterial injections.
  • an AAV3-based, or an AAV-LK03, or an AAV-KP1 vector is used as the expression vector.
  • the AAV3, AAV-LK03 and AAV-KP1 vectors are particularly suited for liver targeting and expression.
  • AAV3, AAV-LK03 and AAV-KP1 vectors can transfect liver cells with greater efficiency as compared to an AAV2 vector.
  • Both AAV-LK03 and AAV-KPl also induce lower amounts of neutralizing antibodies than some of the other AAV vectors.
  • the desired doses of the DNase enzyme encoding AAV vectors of the invention may be easily adapted by the skilled artisan, e.g., depending on the disease condition, the subject, the treatment schedule, etc.
  • from 10e5 to 101e4 recombinant viral particles are administered per dose, for example, from 10e6 to lOel l, from 10e7 to lOel l, or from 108 to 1014.
  • exemplary doses for achieving therapeutic effects may include titers of at least about 10e5, 10e6, 10e7, 10e8, 10e9, lOe 10 or lOel l recombinant viral particles or more.
  • the exogenous targeting sequence(s) may replace or substitute part or all of a maj or capsid subunit (e.g., VP3).
  • a maj or capsid subunit e.g., VP3
  • more than one exogenous targeting sequence e.g., two, three, four, five or more sequences, may be introduced into the virion capsid.
  • insertions and substitutions within the minor capsid subunits e.g., VPl and VP2
  • insertions or substitutions in VP2 or VP3 may be undertaken.
  • the native virion tropism may be reduced or abolished by insertion or substitution of the amino acid sequence.
  • the insertion or substitution of the exogenous amino acid sequence may target the virion to a particular 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.
  • the targeting peptide or protein may be naturally occurring or, alternately, completely or partially synthetic.
  • Exemplary peptides and proteins include ligands and other peptides that bind to cell surface receptors present in liver cells include ligands capable of binding the Sr-Bl receptor for apoliprotein E, galactose- and lactose-specific lectins, low density lipoprotein receptor ligands, asialoglycoprotein (galactose-terminal) ligands and the like.
  • the exogenous targeting sequence may be an antibody or an antigen recognizing moiety thereof.
  • 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 species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. Also encompassed by the term“antibody” are bispecific or“bridging” antibodies as known by 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 the corresponding fragments obtained from antibodies other than IgG. Such fragments may be produced by known techniques.
  • the exogenous amino acid sequence inserted into the virion capsid may be one that facilitates purification or detection of the virion.
  • the exogenous amino acid sequence may include a poly-histidine sequence that is useful for purifying the virion over a nickel column, as is known to those skilled in the art or an antigenic peptide or protein that may be employed to purify the virion by standard immunopurification techniques.
  • the amino acid sequence may encode a receptor ligand or any other peptide or protein that may be used to purify the modified virion by affinity purification or any other techniques known in the art (e.g., purification techniques based on differential size, density, charge, or isoelectric point, ion- exchange chromatography, or peptide chromatography).
  • the exogenous targeting sequence may be an antibody or an antigen recognizing moiety thereof.
  • 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 species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies.
  • antibody also encompassed by the term “antibody” are bispecific or “bridging” antibodies as known by 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 the corresponding fragments obtained from antibodies other than IgG. Such fragments may be produced by known techniques.
  • the exogenous amino acid sequence inserted into the virion capsid 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 alters the virion of the modified parvovirus.
  • the exogenous amino acid sequence may include a poly-histidine sequence that is useful for purifying the virion over a nickel column, as is known to those skilled in the art or an antigenic peptide or protein that may be employed to purify the virion by standard immunopurification techniques.
  • the amino acid sequence may encode a receptor ligand or any other peptide or protein that may be used to purify the modified virion by affinity purification or any other techniques known in the art (e.g., purification techniques based on differential size, density, charge, or isoelectric point, ion-exchange chromatography, or peptide chromatography) .
  • Adenovirus genome is a linear, 36-Kb double-stranded DNA containing multiple, heavily spliced transcripts. At either end of the genome are inverted terminal repeats (ITRs). Genes are divided into early (E1-E4) and late (L1-L5) transcripts.
  • recombinant adenovirus vectors have two genes deleted: El and E3 (dEl/E3). Deletion of El makes the viral vector replication-deficient. El can be supplied by the adenovirus packaging cell lines (e.g., T 293 or 911). E3 is involved in evading host immunity and is not essential for virus production. Deletion of El and E3 results in a transgene packaging capacity of >8 Kb. In some embodiments, Constructs contain left and right arms to facilitate homologous recombination of the transgene into the adenoviral plasmid.
  • any of the accepted human adenovirus types may be used, such as, e.g., an adenoviral vector based on Ad5.
  • Ad5-based vectors use the Coxsackie- Adenovirus Receptor (CAR) to enter cells.
  • human adenovirus Type 5 (dEl/E3) is used as a vector.
  • Most adenovirus vectors are engineered such that a transgene replaces the Ad Ela, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans.
  • Adenovirus vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, nervous system and intestine.
  • Conventional Ad vectors have a large carrying capacity.
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)).
  • Additional examples of the use of adenovirus 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.
  • an expression vector comprising a sequence encoding an enzyme which has a DNase activity is administered to the subject as a naked DNA.
  • naked DNA can be delivered, e.g., into a liver blood vessel at distal or proximal points.
  • such naked DNA can be delivered, e.g., intravenously (iv), intramuscularly (im), per os (po), intraspinally, intracranially, or intraventricularly.
  • intravenously (iv), intramuscularly (im), per os (po), or rectally e.g., intravenously (iv), intramuscularly (im), per os (po), or rectally.
  • an enveloped viral particle can be used for liver, nervous system and/or intestinal delivery of a nucleic acid encoding an enzyme which has a DNase activity.
  • the viral particle described herein is derived from a virus of the family Retroviridae.
  • the viral particle described herein is a retroviral particle.
  • the viral particle described herein is a lentiviral particle.
  • lentiviral and retroviral vectors offer a wide range of advantages, including their ability to transduce a variety of cell types, to stably integrate transferred genetic material into the genome of the targeted host cell, and to express the transduced gene at significant levels.
  • Vectors derived from the gamma-retroviruses for example, the murine leukemia virus (MLV) have been used in clinical gene therapy trials (Ross et al., Hum. Gen Ther. 7: 1781-1790, 1996).
  • MMV murine leukemia virus
  • the at least one viral element associated with a nucleotide of interest is a retroviral element. In another specific embodiment, the at least one viral element associated with a nucleotide of interest is a lentiviral element. In one specific embodiment, the at least one viral element associated with a nucleotide of interest is a Psi (y) packaging signal. [00299] In one specific embodiment, the lentiviral particle does not contain gpl20 surface envelope protein and/or gp41 transmembrane envelope protein. In another specific embodiment, the lentiviral particle contains a mutant gpl20 surface envelope protein and/or a mutant gp41 transmembrane envelope protein.
  • the viral particles described herein are replication deficient and only contain an incomplete genome of the virus from which they are derived.
  • the lentiviral and retroviral particles do not comprise the genetic information of the gag, env, or pol genes (which may be involved in the assembly of the viral particle), which is a known minimal requirement for successful replication of a lentivirus or retrovirus.
  • the minimal set of viral proteins needed to assemble the vector particle are provided in trans by means of a packaging cell line.
  • env, tat, vif, vpu and nef genes are lacking in lentiviral particles derived from HIV-1 and are provided in trans or are made inactive by the use of frame shift mutation(s).
  • the RNA molecule incorporated into the lentiviral or retroviral particles comprises the psi packaging signal and LTRs.
  • the psi packaging signal and LTRs are usually placed under the control of various promoters discussed above.
  • the RNA molecule together with the gag and pol encoded proteins, provided in trans by the packaging cell line are then assembled into the vector particles, which then infect cells, reverse-transcribe the RNA molecule that comprises a nucleotide sequence encoding an enzyme which has a DNase activity under the control of a promoter, and either integrate said genetic information into the genome of the target cells or remain episomal (if one or more of the components required for integration are disrupted).
  • the vector particles are replication deficient, i.e., no new generation of said vector particles will thus be generated by the transduced cell, thus ensuring safety in clinical applications.
  • the one or more viral elements encodes 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), Murine Stem Cell Virus (MSCV) component(s), Murine Leukemia Virus (MLV) component(s), Avian leukosis virus (ALV) component(s), Feline leukemia virus (FLV) component(s), Bovine leukemia virus (BLV) component(s), Human T-lymphotropic virus (HTLV) component(s), feline sarcoma virus component(s), avian reticuloendotheliosis virus component(s), caprine arthritis encephalitis virus (CAEV) component s), and/or Visna
  • HCV Human Immunodeficiency Virus
  • viral particles comprising a selectable marker (e.g., neomycin resistance) and/or a reporter (e.g., GFP or eGFP) as a nucleotide sequence of interest allowing target cells to be selected using, e.g., selection compound exposure or fluorescent activated cell sorting (FACS).
  • a selectable marker e.g., neomycin resistance
  • a reporter e.g., GFP or eGFP
  • FACS fluorescent activated cell sorting
  • the retroviral vectors described herein are derived from murine leukemia virus (MLV). Retroviral vectors encoding MLV are widely available to those skilled in the art, such as PINCO (Grignani et ah, 1998) or the pBabe vector series (Morgenstern and Land, 1990).
  • the lentiviral particles described herein are derived from a lentivirus such as human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Suitable vectors encoding HIV and other useful viruses can be readily identified and/or prepared by the skilled person.
  • the transfer vector comprises: a cytomegalovirus (CMV) enhancer/promoter sequence; the R and U5 sequences from the HIV 5' LTR; the HIV-1 flap signal; an internal enhancer; an internal promoter; a nucleotide sequence of interest; the woodchuck hepatitis virus responsive element; a tRNA amber suppressor sequence; a U3 element with a deletion of its enhancer sequence; the chicken b-globin insulator; and the R and U5 sequences of the 3' HIV LTR.
  • CMV cytomegalovirus
  • the invention also contemplates enveloped viral vectors comprising a heterologous targeting sequence or molecule inserted or substituted into the native envelope.
  • the heterologous targeting sequence or molecule may confer an altered tropism towards liver, nervous system and/or intestine or increase the efficiency of liver, nervous system and/or intestinal delivery of the vector.
  • expression vectors comprising a nucleic acid encoding and enzyme which has a DNase activity can be administered to a patient at one time or over a series of treatments; once or several times per day.
  • the effective amount of 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 determined readily by one of ordinary skill in the art. Analysis of the blood (serum, plasma) or tissue levels of the vector-encoded enzyme which has a DNase activity and comparison to the initial level prior to administration can determine whether the amount being administered is too low, within the right range or too high. Suitable regimes for initial and subsequent administrations are also variable, but are typified by an initial administration followed by subsequent administrations if necessary. Subsequent administrations may be administered at variable intervals, ranging from daily to weekly to monthly to annually to every several years.
  • Doses of an expression vector encoding for an enzyme which has a DNase activity depend on the type of condition to be treated, the severity and course of these side effects, the patient’s clinical history, discretion of the attending physician, and response to a prior treatment such as, e.g., chemotherapy, or radiation therapy, and DNase protein therapy.
  • the effective amount of expression vector is the amount which results in DNase protein level in hepatic portal vein which is from 0.5 to 100 mg/L or from 1000 to 200000 Kunitz units (KU)/L, from 0.5 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.
  • an expression vector e.g., AAV- or viral-vector, encoding for DNase enzyme, and/or DNase enzyme, according to the methods of the invention can be performed by any suitable route, including, e.g., intravenous, intra-arterial and intrahepatic administration.
  • a method for treating or ameliorating cancer in a subject suffering from a cancer comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase Il-alpha, DNase Il-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, or DNAS1L2
  • DNase II e.g., DNase Il-alpha
  • the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition).
  • the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/nervous system- specific tandem promoter and/or an enhancer element.
  • an enhancer element e.g., a liver-specific enhancer element.
  • the DNase enzyme is expressed under the control of a liver-specific/intestine-specific tandem promoter and/or an enhancer element (e.g., CMV enhancer). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
  • an enhancer element e.g., CMV enhancer
  • the amount and manner of administration of the expression vectors of the invention should be effective to treat a cancer, e.g., by one or more of reducing tumor size, reducing the rate of tumor growth, reducing the rate of metastasis, enhancing the therapeutic effects of chemotherapy and/or radiotherapy, prolonging lifespan, reducing the rate of cancer- or treatment- associated weight loss, or increasing the rate of weight gain.
  • the administration of an expression vector encoding an enzyme which has a DNase activity can be combined with administration of the same enzyme as protein (e.g., intravenously, intra-arterially, intrahepatically, intramuscularly, intraperitoneally, enterally, etc.).
  • the enzyme can be, e.g., any one of DNase enzymes (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase Il-alpha, DNase Il-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • DNase I e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, or DNAS1L2
  • DNase II e.g., DNase Il-alpha, DNase Il-beta
  • phosphodiesterase I e.g., lactoferrin, or acetyl
  • the methods of treatment using the vectors of the invention can be used in subjects suffering from a broad range of cancers.
  • the present invention is particularly useful for treating “portal vein cancers”, which include any carcinoma, sarcoma, melanoma, or lymphoma originating in or having metastasized to organs, tissues and structures that drain to the portal vein (see Figure 1).
  • portal vein cancers include, e.g., peritoneal carcinomatosis, lymphomas (e.g., B cell lymphoma, T cell lymphoma, non-Hodgkins lymphomas, Hodgkins lymphoma), stomach cancers, colon cancers, intestinal cancers, colorectal cancers, pancreatic cancers, liver cancers, splenic cancers, cancers of the bile duct, cancers of the gall bladder, sarcomas in a tissue draining to the portal vein (e.g., an angiosarcoma, endotheliosarcoma, hemangiosarcoma, leiomyosarcoma, other sarcomas, and epithelial carcinoma in such tissue), and liver metastatic diseases of any origin.
  • lymphomas e.g., B cell lymphoma, T cell lymphoma, non-Hodgkins lymphomas, Hodgkins lymphoma
  • stomach cancers colon cancers
  • Non-limiting examples of other cancers which can be treated by the methods of the invention include, e.g., breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g., non-small cell lung cancer (NSCLC)), renal cancer, thyroid cancer, leukemia (e.g., 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 cancer, bone cancer (including both primary bone cancers (e.g., osteosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, malignant fibrous histiocytoma,
  • lung cancer
  • an expression vector e.g., AAV vector or lentiviral vector or adenoviral vector encoding an enzyme which has a DNase activity can be administered before, with, or after a chemotherapeutic agent (or a radiation therapy).
  • the methods of the invention are used to prevent a cancer in a patient, e.g. a patient with a high risk of predisposition to cancer.
  • the methods of the invention are used to prevent a cancer following a positive result from a blood-based DNA screening test (e.g., liquid biopsy) for the detection of predisposition to a cancer.
  • a blood-based DNA screening test e.g., liquid biopsy
  • the methods of the invention are used to prevent a cancer following the evaluation of a tumor susceptibility gene.
  • tumor susceptibility genes include, but are not limited to, ABL1 (ABL), ABL2(ABLL, ARG), AKAP13 (HT31, LBC.
  • BRX ARAFl, ARHGEF5 (TIM), ATF1, AXL, BCL2, BRAF (BRAFl, RAFB l), BRCA1, BRCA2(FANCD1), BRIP1, CBL (CBL2), CSF1R (CSF-1, FMS, MCSF), DAPKl (DAPK), DEK (D6S23 IE), DUSP6(MKP3,PYST1), EGF, EGFR (ERBB, ERBBl), ERBB3 (HER3), ERG, ETS1, ETS2, EWSR1 (EWS, ES, PNE), FES (FPS), FGF4 (HSTF1,KFGF), FGFR1, FGFR10P (FOP), FLCN, FOS (c-fos), FRAPl, FUS (TLS), HRAS, GLI1, GLI2, GPC3, HER2 (ERBB2, TKR1, NEU), HGF (SF), IRF4 (LSIRF, MUM1), JUNB, K
  • the susceptibility gene can be evaluated, e.g., by one or more of next-generation sequencing (NGS), whole genome sequencing (WGS), exome sequencing, an ELISA-based method, and PCR amplification. Other methods can be used to evaluate the susceptibility gene as well.
  • the invention provides a method for treating or ameliorating a cancer in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and/or nervous system and/or intestine of said subject.
  • the cancer is accompanied by an increase in the total level or relative content of cell free DNA (cfDNA) of microbial origin and/or is accompanied by an increase in the total level of the cfDNA originating from host cells in blood of said subject.
  • the method comprises detecting the level of cfDNA of microbial origin or the ratio of the cfDNA of microbial origin to the total level of cfDNA in blood of the subject.
  • methods of detecting the cfDNA of microbial origin include, e.g., PCR, RT-PCR, next-generation sequencing (NGS) and whole genome sequencing (WGS).
  • cfDNA of microbial origin can be detected, e.g., using PCR or RT-PCR of 16S ribosomal RNA (rRNA), 16S ribosomal DNA (rDNA) or Intergenic Spacer Region.
  • determining the total level or relative content of cfDNA of microbial origin includes comparing the level of cfDNA of microbial origin or the ratio of the cfDNA of microbial origin to the total level of cfDNA in blood of the subject to a control level or ratio (e.g., a predetermined standard or a corresponding level or ratio determined using age-matched cancer-free subjects).
  • a control level or ratio e.g., a predetermined standard or a corresponding level or ratio determined using age-matched cancer-free subjects.
  • the subject is human.
  • the invention provides a method for preventing or ameliorating a toxicity, or a condition, associated with a cytostatic and/or cytotoxic chemotherapy in a subject suffering from a cancer and receiving or deemed to receive said chemotherapy, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding for an enzyme which has a DNase activity (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • DNase I e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., D
  • the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition).
  • the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system- specific/intestine-specific tandem promoter and/or an enhancer element.
  • an enhancer element e.g., a liver-specific enhancer element
  • the amount and manner of administration of the expression vector is effective to ameliorate at least one side effect of said chemotherapy.
  • the condition associated with a cytostatic and/or cytotoxic chemotherapy can include, e.g., (i) a catabolic state leading to body weight loss, (ii) bone marrow toxicity and/or catabolic changes in blood biochemistry, (iii) cardiotoxicity (e.g., myocardial necrosis), (iv) gastrointestinal toxicity, (v) suppression of immunity, (vi) neutropenia, and (vii) body weight loss.
  • the cancer is associated with an increased level of cfDNA in blood or cerebrospinal fluid (CSF) or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or CSF or intestine of a healthy age-matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age- matched individuals).
  • CSF cerebrospinal fluid
  • intestine of the control level e.g., the level of cfDNA in blood or CSF or intestine of a healthy age-matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age- matched individuals.
  • the effects of chemotherapy are associated with an increased level of cfDNA in blood or CSF, ascitic fluid, or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of an age- matched individual with a similar cancer profile who does not receive chemotherapy, or an average level of cfDNA in blood or CSF or intestine of several age-matched individuals who have cancer but do not receive chemotherapy).
  • the control level e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of an age- matched individual with a similar cancer profile who does not receive chemotherapy, or an average level of cfDNA in blood or CSF or intestine of several age-matched individuals who have cancer but do not receive chemotherapy.
  • the invention provides a method for increasing the efficacy of a cytostatic and/or cytotoxic chemotherapy in a subject suffering from a cancer and receiving or deemed to receive said chemotherapy, which method comprises administering to the subject a therapeutically effective amount of an expression vector (e.g., Anc80 or AAV8 AAV vector or lentiviral vector or adenoviral vector) encoding an enzyme which has a DNase activity (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS 1L2), DNase II (DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • an expression vector e.g., Anc80 or AAV8 AAV vector or lentiviral vector or adenoviral vector
  • the amount and manner of administration of the expression vector is effective to prevent or ameliorate at least one side effect of said chemotherapy, and to prevent or ameliorate toxicity associated with said chemotherapy.
  • any one of DNase enzyme e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase) is also administered, e.g., intravenously.
  • DNase I e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, or DNAS1L2
  • DNase II e.
  • the expression vectors of the invention can be used to ameliorate toxicity and/or increase efficacy of a wide range of different chemotherapeutic agents.
  • agents include anti-metabolites such as pyrimidine analogs (e.g., 5-fluorouracil [5-FU], floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (e.g., paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epi
  • pyrimidine analogs
  • Non-limiting examples of side effects of cytostatic and/or cytotoxic chemotherapy which can be prevented or ameliorated by administering an expression vector according to the methods of the invention include, for example, bone marrow toxicity, neutropenia, myelopathy (e.g., leukopenia, granulocytopenia, lymphopenia, thrombocytopenia, erythropenia); hematopathy (e.g., plasma fibrinogenopenia); catabolic changes in blood biochemistry; gastrointestinal disorders (e.g., nausea, vomiting, anorexia, body weight loss, heavy feeling of stomach, diarrhea, constipation, stomatitis, esophagitis); pulmonary insufficiency (e.g., chronic pneumonia, lung fibrosis, ARDS, ALS, lung emboli); dermatopathy (e.g., keratinization, pachymenia, chromatosis, epilation, rash, nail alternation, cancer-induced alopecia); nervous system disorders
  • the expression vectors of the invention can be also used to ameliorate toxicity and increase 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/unsealed source radiotherapy.
  • EBRT or XRT external beam radiation therapy
  • brachytherapy/sealed source radiation therapy brachytherapy/sealed source radiation therapy
  • systemic radioisotope therapy/unsealed source radiotherapy Non-limiting examples of side effects of radiotherapy which can be prevented or ameliorated by administering an expression vector according to the methods of the invention include, for example, skin irritation or damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, and a second cancer.
  • the expression vectors of the invention can be used to increase efficacy of cancer immunotherapy, including, for example, antibodies (naked monoclonal antibodies, conjugated monoclonal antibodies, chemolabeled antibodies, bispecific monoclonal antibodies), cancer vaccines, oncolytic viruses, Immune checkpoint inhibitors (Drugs that target PD-1 or PD-L1, CTLA-4), Non-specific cancer immunotherapies and adjuvants, CAR-T therapies.
  • antibodies naked monoclonal antibodies, conjugated monoclonal antibodies, chemolabeled antibodies, bispecific monoclonal antibodies
  • cancer vaccines oncolytic viruses
  • Immune checkpoint inhibitors Drugs that target PD-1 or PD-L1, CTLA-4
  • Non-specific cancer immunotherapies and adjuvants CAR-T therapies.
  • the invention provides a method for treating or ameliorating neurodegeneration.
  • neurodegeneration is used herein to refer to a separate clinical pathological condition with progressive loss of structure and/or function of neurons, including death of neurons.
  • the neurodegeneration can be primary or secondary.
  • the neurodegeneration is associated with an increased level of cfDNA (e.g., microbial cfDNA or cfDNA originating from host cells) in blood or cerebrospinal fluid (CSF) or intestine of a patient, which level is higher than the control level (e.g., the level of cfDNA in blood or CSF or intestine of a healthy age-matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age-matched individuals).
  • the neurodegeneration is associated with a neurodegenerative disorder.
  • Non-limiting examples of encompassed neurodegenerative disorders include, e.g., Alzheimer's disease (e.g., late-onset Alzheimer's disease), Mild Cognitive Impairment (MCI), Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), and Huntington's disease (HD), prion-caused diseases, progressive supranuclear palsy (PSP), progressive supranuclear palsy (Steel-Richardson- Olszewski), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), agyrophilic grain disease (AGD), Pick disease (PiD), frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism- 17 (FTDP-17), multiple sclerosis, CADASIL Syndrome, ankylosing spondylitis, Dentatorubro-pallido-Luysian atrophy, Kennedy disease, Friedreich’s ataxia, cerebellar ataxia, spinocere
  • the neurodegeneration is associated with a nervous system dysfunction such as, e.g., 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).
  • the neurodegenerative disorder is caused by, secondary to, or associated with, amyloidosis.
  • the neurodegenerative disorder is a protein-misfolding associated disease.
  • the neurodegenerative disorder is caused by, secondary to, or associated with diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, an amyloidosis (e.g., hereditary cerebral hemorrhage with amyloidosis, primary systemic amyloidosis, secondary systemic amyloidosis, serum amyloidosis, senile systemic amyloidosis, hemodialysis-related amyloidosis, Finnish hereditary systemic amyloidosis, Atrial amyloidosis, Lysozyme systemic amyloidosis, Insulin- related amyloidosis, Fibrinogen a-chain amyloidosis), asthma, or prion disease.
  • an amyloidosis e.g., hereditary cerebral hemorrhage with amyloidosis, primary systemic amyloidosis, secondary systemic amyloido
  • the neurodegeneration is caused by, secondary to, or associated with the formation of a misfolded protein due to the presence of DNA (e.g., human, microbial, cell-free, or intracellular). In some embodiments, the neurodegeneration is caused by the formation of a misfolded protein due to the presence of DNA (e.g., human, microbial, cell-free or intracellular).
  • DNA e.g., human, microbial, cell-free, or intracellular
  • the neurodegeneration is caused by the formation of a misfolded protein due to the presence of DNA (e.g., human, microbial, cell-free or intracellular).
  • the neurodegeneration is caused by, secondary to, or associated with the formation of a misfolded protein including, but not limited to, b-amyloid, Tau protein, a-synuclein, SOD1, TDP-43, IAPP, ADan, ABri, Fused in sarcoma (FUS) protein, Notch3, Glial fibrillary acidic protein, Seipin, Transthyretin, Serpins, Apolipoproteins, Amyloid b peptide, Lactoferrin, Galectin-7 Comeodesmosin.
  • a misfolded protein including, but not limited to, b-amyloid, Tau protein, a-synuclein, SOD1, TDP-43, IAPP, ADan, ABri, Fused in sarcoma (FUS) protein, Notch3, Glial fibrillary acidic protein, Seipin, Transthyretin, Serpins, Apolipoproteins, Amyloid b peptide
  • the methods of the invention can be used to prevent development of a neurodegenerative disease or another disease associated with protein misfolding following the evaluation of the presence of, or activity of, a disease-associated susceptibility gene.
  • disease-associated susceptibility genes include, but are not limited to, AD AR1, MDA5 (IFIH1), RNAseH subunits, SamHDl, TREX, TBK1, Optineurin, P62 (sequestosome 1), Progranulin, TDP43, FUS, VCP, CHMP2B, Profilin-1, Amyloid-b, Tau, a-synuclein, PINK, Parkin, LRRK2, DJ-1, GBA, ATPA13A2, EXOSCIII, TSEN2, TBC1D23, Risk-factor alleles, PLCG2, TREM2, APOE, TOMM40, IL-33, Glucocerebrosidase, Ataxin2 , C9orf72, SOD
  • prevention or treatment of the misfolded protein aggregate formation in mammalian biological fluids and tissues is achieved by the use of expression vectors disclosed herein, including those comprising the sequence of SEQ ID NO: 30 (ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact) or SEQ ID NO: 31 (ApoEHCR enhancer-hAAT promoter-hDNasel wild type-WPRE Xinact5653).
  • 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 human DNasel hyperactive variant with natural full correct leader sequence
  • bases 1576-2212 correspond to the inactivated WPRE X protein.
  • 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 human DNasel wild type with natural full correct leader sequence
  • bases 1576-2212 correspond to the inactivated WPRE X protein.
  • cfDNA from the intestine, blood and CSF of patients suffering from neurodegeneration causes neuronal cell death and apoptosis, and treatment with recombinant DNase protein destroying such cfDNA improves the nervous system function in these patients.
  • the use of the vectors of the invention for liver-specific, nervous system-specific and/or intestine-specific expression of DNase provides a further treatment improvement as compared to DNase protein administration.
  • the assessment of treatment efficacy for neurodegeneration and neuroinflammation can be performed using any methods known in the art, e.g., according to widely accepted clinical diagnostic criteria of cognitive decline such as MMSE, PANSS, physical function, and/or functional tasks (see, e.g., Holmes et ah, (1999) The British Journal of Psychiatry, 174(1), 45-50; Os et al., (2006) Acta Psychiatrica Scandinavica, 113(2), 91-95; O'Shea et ah, (2002) Physical therapy, 82(9), 888-897; Rochester et al., Arch. Phys. Med. Rehabil. (2004) 85(10), 1578- 1585).
  • the neurodegeneration is caused by, or aggravated by, increased intestinal permeability and translocation of gut bacteria and DAMPs of microbial origin including microbial cfDNA.
  • cfDNA may trigger neuroinflammation and a spectrum of neurodegenerative diseases.
  • cfDNA originating from digestive tract microbiota are subject to“first pass” through porto-sinusoidal circulation in the liver.
  • the NLRP3 inflammasome is considered a key contributor to the development of neuroinflammation.
  • the NLRP3 inflammasome can undergo significant upregulation in the brain, e.g., brain cortical tissue. Aberrant activation of NLRP3 inflammasome signaling has been demonstrated to contribute to pathology in a broad spectrum of neurological diseases (Song, 2017).
  • the invention provides a method for treating neurodegeneration in a subject suffering from neurodegeneration, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS 1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • DNase I e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, or DNAS 1L2
  • DNase II e.g., DNase II-alpha,
  • the enzyme becomes produced by cells in one or more of these tissues and is secreted into the areas where the enzyme efficiently degrades the concentrated pool of cfDNA present therein (e.g., the periendothelial space, the space of Disse, extracellular nervous tissue space, and intestinal sub-mucus gel layer zone).
  • the concentrated pool of cfDNA present therein e.g., the periendothelial space, the space of Disse, extracellular nervous tissue space, and intestinal sub-mucus gel layer zone.
  • the use of the expression vectors of the invention is combined with the administration (e.g., intravenous, intra-arterial, intrahepatic, intramuscular, intraperitoneal, or enteral administration) of a DNase protein (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • a DNase protein e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), D
  • the systemic administration of a DNase protein in conjunction with the above described production of DNase in liver, nervous system and/or intestine using the expression vectors of the invention more quickly degrades cfDNA in both the bloodstream and in any concentrated pool of cfDNA in the liver, nervous system and/or intestine.
  • the expression vectors encoding an enzyme which has a DNase activity can be administered in combination with other treatments useful for treatment of neurodegenerative diseases or other encompassed nervous system dysfunctions (e.g., bipolar disorder, migraine, schizophrenia, epilepsy).
  • other treatments useful for treatment of neurodegenerative diseases or other encompassed nervous system dysfunctions e.g., bipolar disorder, migraine, schizophrenia, epilepsy.
  • Non-limiting examples of such additional treatments include, e.g., 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 (NMD A) receptor inhibitors (e.g., memantine), transcranial magnetic stimulation, transcranial direct current stimulation, etc.
  • NMD A N-methyl-d-aspartate
  • the invention provides a method for treating a delayed hypersensitivity reaction (e.g., graft-versus host disease (GVHD)) in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma
  • the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition).
  • the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
  • an enhancer element e.g., a liver-specific enhancer element
  • dsDNA double stranded DNA
  • NETs extracellular fibers
  • Activation of complement can lead to the symptoms observed in delayed hypersensitivity reactions.
  • the delayed hypersensitivity reaction is associated with an increased level of cfDNA in blood or CSF or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of a healthy age- matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age-matched individuals).
  • the control level e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of a healthy age- matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age-matched individuals.
  • Administration of an expression vector encoding an enzyme which has a DNase activity could inhibit NET formation and lead to less activation of complement.
  • the expression vector encoding DNase enzyme can also be administered in combination with one or more other treatments useful for treating a delayed hypersensitivity reaction such as, e.g., cyclosporine, tacrolimus (FK506), methylprednisolone, prednisone, ATG, sirolimus, mycophenolate mofetil, anti-interleukin-2 (IL-2) receptor, anti-CD5-specific immunotoxin, a pan T-cell ricin A-chain immunotoxin (XomaZyme), the addition of ex vivo cultured mesenchymal cells derived from unrelated donors to conventional steroid therapy, and any combinations thereof.
  • a delayed hypersensitivity reaction such as, e.g., cyclosporine, tacrolimus (FK506), methylprednisolone, prednisone, ATG, sirolimus, mycophenolate mofetil, anti-interleukin-2 (IL-2) receptor, anti-CD5-specific immunotoxi
  • a method for treating atherosclerosis in a subject in need thereof comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, or DNAS1L2
  • DNase II e.g., DNase II-alpha, D
  • the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition).
  • the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
  • an enhancer element e.g., a liver-specific enhancer element
  • Atherosclerosis is associated with an increased level of cfDNA in blood or CSF or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of a healthy age-matched individual or an average level of cfDNA in blood or cerebrospinal fluid or intestine of several healthy age- matched individuals).
  • control level e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of a healthy age-matched individual or an average level of cfDNA in blood or cerebrospinal fluid or intestine of several healthy age- matched individuals.
  • the expression vector e.g., Anc80 or another AAV- or lentiviral vector, encoding for DNase enzyme may be administered optionally with additional DNase enzyme.
  • any one of DNase enzyme e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase) is also administered, e.g., intravenously.
  • DNase I e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, or DNAS1L2
  • DNase II
  • the systemic administration of DNase enzyme in conjunction with the above described production of DNase in the liver, nervous system and/or intestine more quickly degrades cfDNA in both the bloodstream and in any concentrated pool of such cfDNA in the liver, nervous system and/or intestine.
  • the expression vector encoding for DNase enzyme can also be administered in combination with other treatments useful for treating atherosclerosis.
  • the invention provides a method for increasing longevity and individual life expectancy and delaying unhealthy manifestations brought by ageing in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II- beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
  • DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I
  • analogues of DNase I such as, e.g., DNase X, DNase gamma, or DNAS1L2
  • the DNase enzyme comprises the sequence set forth in SEQ ID NO: 1 (wild-type precursor sequence comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition).
  • the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
  • an enhancer element e.g., a liver-specific enhancer element
  • the AAV vectors disclosed herein may be administered in any suitable form, for instance, either as a liquid solution or suspension, as a solid form suitable for solution or suspension in liquid prior to injection.
  • the vectors may be formulated with any appropriate and pharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc.
  • a suitable carrier or diluent may be an isotonic solution, a buffer, sterile and pyrogen-free water, or, for instance, a sterile and pyrogen-free phosphate-buffered saline solution.
  • the expression vectors may be formulated in pharmaceutical compositions and dosage forms.
  • compositions and dosage forms can be formulated in any conventional manner using one or more physiologically acceptable carriers and/or excipients.
  • the vector particles may be formulated for administration by, for example, injection.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
  • Formulations also include suspensions in liquid or emulsified liquids.
  • the active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.
  • the formulations used in the methods of the 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 a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration.
  • the amount of active ingredients that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • compositions suitable for parenteral administration may further comprise one or more additional active ingredients (e.g., a DNase protein or another active compound) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • additional active ingredients e.g., a DNase protein or another active compound
  • aqueous and nonaqueous carriers examples 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 injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions can also contain preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may 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 desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
  • EXAMPLE 1 Limited ability of DNase enzyme to digest circulating cell free DNA (cfDNA) in tumor patients
  • 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 following last DNase I protein infusion. DNA was isolated from blood plasma using a classic phenol-chloroform method. Electrophoresis of blood cfDNA was performed on 1% agarose gel. DNA was visualized with ethidium bromide. The treatment efficacy was evaluated one week after the last DNase I protein infusion using a spiral CT scan based on RECIST criteria (see, e.g., RECIST 1.1, published in January 2009, www.irrecist.com). The patient summaries and individual blood electrophoretic data are presented in Figures 2A-2IJ. Overall, there was little decrease in the diameter of solid tumors according to the RECIST outcome ( Figures 2A-2H). In most patients, there was still cfDNA in circulation after Week III.
  • PVIII recurrent rectal carcinoma
  • EXAMPLE 2 Accumulation of cell free DNA (cfDNA) in the liver of tumor bearing animals
  • mice Approximately 12 x 10 6 of LS174T human colon carcinoma cells were inoculated subcutaneously into the left flank of each of twelve female nu/nu mice (IBCH RAS breed) that were 8 weeks old. The mice received a single IV injection of 99 mTc labeled anti DNA antibodies according the following schedule: three mice received the injection at D5, three mice received the injection at DIO, three mice received the injection at D15 and three mice received the injection at D25. Mice were sacrificed two hours following the injection and tissues and blood were collected for gamma counting.
  • AC-30-10 DNA binding antibodies (Genetex) were labeled with 99mTc as follows:
  • 0.024mg/ml solution of antibody in 0.3M citrate buffer (pH4.2) was mixed with lmCi of 99mTc- pertechnetate in 0.25 ml saline and mixed 10-mg/ml solution of SnC12 dihydrate in 0.1N HC1. After 5 min incubation at room temperature, 1.9 ml of saline and 825 m ⁇ of IgM solution were added.
  • the single injection doses were prepared as follows: 16.6pCi (36852000CPM approx. 0.047pg of 99mTc-IgM) of 99mTc-IgM solution was mixed with 3.3 pg of unlabeled IgM and diluted with saline up to 100 pL volume.
  • Table 1 Accumulation of anti-DNA antibodies in tissues of tumor bearing mice. (% of Injected dose /s of tissue).
  • Figure 3 shows a gamma-immunoscintigraphic image of a tumor-bearing mouse obtained at Day 15 one hour following injection of 99mTc labeled anti-DNA antibodies.
  • the liver zone (b) in the middle-right portion of the image produced a radioactive signal that is greater than that of tumor zone (a) in the lower left portion of the image.
  • the liver showed the most accumulation of circulating cfDNA.
  • mice Six 6-week-old female nude mice (IBCH RAS breed) were given subcutaneous injections with 2xl0 6 of MCF-7 cells into the left flank. At Day 20 following the injection tumor was surgically removed. At Day 25 all mice were given anesthesia and 500 pi blood samples were taken from portal vein, hepatic artery and hepatic vein of each mouse. Plasma was separated from the blood cells by centrifugation at 2000 g for 10 minutes; the clear plasma phase on top was transferred to a new tube, and centrifuged at 14000 g. The supernatant was transferred to a fresh tube for DNA extraction. cfDNA was extracted from blood plasma samples using QIAamp DNA Blood Mini Kit according to manufacturer’s instructions.
  • the human beta globin sequence in cell free DNA was quantified in each sample using RT-PCR (iCycler iQ56 Bio-Rad) with human beta globin primer CAACTTCATCCACGTTCACC (SEQ ID NO: 10).
  • RT-PCR iCycler iQ56 Bio-Rad
  • human beta globin primer CAACTTCATCCACGTTCACC SEQ ID NO: 10
  • the Ct values of human beta globin sequence in blood plasma sampled from different vessels are presented at the Table 2 below:
  • An AAV vector for liver specific transgenic expression of DNase I (4658_pAAV- AFP-Alb-huDNaseI_mut) was custom manufactured by Sirion Biotech (Martinsried, Germany).
  • a vector map is shown in Figure 5A.
  • the AAV vector is based on AAV serotype 8 which effectively targets liver.
  • the vector comprises the sequence set forth in SEQ ID NO: 2, which codes for a mutant human DNase I enzyme which comprises several mutations which make the enzyme less sensitive to actin inhibition and more catalytically active.
  • the DNase I coding sequence further comprises a secretory signal sequence.
  • the DNase I coding sequence is operably linked at the 5' end to mAFP/Alb enhancer/promoter system (liver-specific mouse alpha fetoprotein enhancer and liver-specific mouse minimal albumin promoter) and at the 3 ' end to hGH polyA signal.
  • the expression cassette is further flanked by AA8 L-ITR and R-ITR.
  • the huDNasel mut expression cassette was cloned into an AAV transfer vector and produced in 293T cells via co-transfection of a transfer vector with AAV8 packaging plasmids. Viral particles were purified and concentrated up to 1x1013 GC/ml and kept frozen in PBS.
  • rhDNasel Human recombinant DNase I protein was produced by Catalent (Madison, USA) and kept frozen at lOmg/ml stock solution in ImM CaC12 and 150mM NaCl.
  • mice 56 female B ALB/c nude mice (IBCH RAN) of 6-8 weeks age and 18-22 g weight were used in experiment. The mice were kept in individual ventilation cages at controlled temperature and humidity with four animals in each cage. Each mouse was inoculated subcutaneously at the right flank with MIA PaCa-2 pancreatic tumor cells (1 x 107) in 0.2 ml of PBS supplemented with BD Matrigel (1 : 1) for tumor development. The treatments were started on day 17 after tumor inoculation when the average tumor size reached approximately 170mm3. Each group consisted of eight tumor-bearing mice. The tested vectors and compositions were administered to the mice according to the predetermined regimen as shown in Table 3, below:
  • the animals were checked daily for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption by visual inspection only, loss or gain of body weight (measured twice weekly), eye/hair matting, and any other abnormal effects.
  • the mice were humanely sacrificed by CO2, and blood was sampled from the central and hepatic veins. Liver tissues were also sampled and immersed into formalin for fixing.
  • Circulating cfDNA was measured in plasma using a method described in H.
  • SYBR® Gold Nucleic Acid Gel Stain (Invitrogen) was diluted first at 1 : 1000 in dimethyl sulphoxide and then at 1 :8 in phosphate- buffered saline. 10 m ⁇ of plasma samples were applied to 96-well plates. 40 m ⁇ of diluted
  • SYBR® Gold was added to each well (final dilution 1 : 10,000) and fluorescence was measured with a 96 well fluorometer at an emission wavelength of 535 nm and an excitation wavelength of 485 nm.
  • Serum DNase I activity was measured using ORG590 (Orgentec) according to the manufacturer's protocol. Detection was performed using microplate photometer (Multiscan FC) at 450 nm with a correction wavelength of 620 nm. cfDNA in the liver tissue was quantified by immunohistochemistry (IHC) in liver paraffin slides using AC-30-10 mouse IgG DNA binding antibodies (Sigma) as primary antibodies and Novolink Polymer Detection Systems Novocastra (Leica Biosystems).
  • IHC immunohistochemistry
  • mice bearing MIA PaCa-2 xenografts and dosed with tested vectors and composition (as per Table 3) is shown in Table 5, below:
  • Table 5 The number of animals with body weight loss at Day 35
  • mice Twelve 8-week old SCID mice (IBCH RAS) divided into 4 experimental groups were used in the experiment.
  • Six mice received 4xl0e6 MIA PaCa-2 pancreatic cancer cells expressing luciferase subcutaneously at the right femur in 100 m ⁇ of Matrigel (non-orthotopic model).
  • Six mice were implanted in the body -tail of the pancreases with 4x10e6 MIA PaCa-2 pancreatic cancer cells expressing luciferase in 100 m ⁇ of Matrigel (orthotopic model).
  • mice with orthotopic MIA PaCa-2 implant received IV injection of mAFP/Alb AAV8 vector 4658_pAAV-AFP-Alb-huDNaseI_mut at l,00xl0el l GC/kg dose and 3 mice with orthotopic MIA PaCa-2 implant received a placebo (PBS) injection.
  • mice with non-orthotopic MIA PaCa-2 implant received an IV injection of DNasel mAFP/Alb AAV8 vector at 1.OOxlOel 1 GC/kg dose and three mice with non-orthotopic MIA PaCa-2 implant received a placebo (PBS) injection.
  • tumors were assessed by bioluminescence imaging using an AMI-1000 imaging system. The bioluminescence data are summarized in Table 7, below, with typical images presented in Figure 6.
  • transgenic delivery of DNase I enzyme to the liver completely blocks the development of tumor growth in an orthotopic fashion in an area drained by the portal vein, i.e., the pancreas.
  • Development of same pancreatic cancer xenotransplant in non-orthotopic fashion was also significantly suppressed by injection of DNase I mAFP/Alb AAV8 but not to such an extent as to eliminate tumor growth completely.
  • EXAMPLE 6 Effects of liver and pulmonary delivery of DNase transgene on growth of
  • ADV-207186 vector was purchased from Vector Biolabs (Malvern, PA).
  • a vector map is shown in Figure 5B.
  • This vector is based on human adenovirus Type 5 (dEl/E3) (which has a natural liver tropism) and contains wild-type human DNase I coding sequence which comprises a secretory signal sequence (SEQ ID NO: l).
  • the DNase I coding sequence is operably linked at the 5' end to liver non-specific CMV promoter and at the 3' end to hGH poly A signal.
  • the expression cassette is further flanked byATT-Ll and ATT-L2.
  • mice IBCH RAS breed were used for the study.
  • mice When each mouse developed at least three palpable tumors of at least 3 mm c 5 mm, which typically occurred at 8 weeks of age, six mice were injected intravenously with of 0.2xl0 u PFU per mice of ADV- 207186 and six mice were injected with 0.2x1011 PFU per mice of ADV-207186 intranasally under anaesthesia. Mice were sacrificed at 14 weeks of age. Six mice were used as control.
  • DNasel transgene provides superior breast tumor growth inhibition and clearance of blood from circulating cfDNA as compared to the delivery of DNase I transgene to bronchoalveolar system, despite approximately the same level of DNase enzymatic activity in blood.
  • cfDNA originating from gastrointestinal microbiota are subject to“first pass” through porto-sinusoidal circulation.
  • the effects of muscle and liver delivery of hyperactive actin-resistant DNase I transgene, the effects of injections of wild-type DNase I protein on the level of microbial cfDNA in brain and liver, as well as the level of neuroinflammation in CSF and brain following bacteriophage-induced leaky gut syndrome were further investigated.
  • the DNA molecule coding for the hyperactive actin-resistant DNase I mutant (SEQ ID NO: 5) with DNase I secretory signal sequence replaced by the IL2 secretory signal sequence was synthesized (GeneCust) and cloned into the pLV2 lentiviral vector (Clontech) under the control of the EFla promoter (not 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 lentiviral vector pLV2- IL2ss-DNAseI mut using Lipofectamine 2000 (Life Technology). 5 ml of supernatant was collected at 48 hours after transfection. Lentiviruses were collected by filtering through a 0.45 pm polyethersulfone membrane filter (Millipore), centrifugation during 30 m at 20,000 g. The resulting pellet was resuspended in 1 ml of PBS.
  • Leaky gut was induced in 24 rats using a bacteriophage cocktail against Enterobacteriaceae, Staphylococcaceae, and Streptococcaceae families so as to induce microbiota diseases.
  • the cocktail included a Salmonella bacteriophage cocktail 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.
  • Phages 1.5 mL, 1x106 plaque-forming units/mL of each phage cocktail were added to drinking water according to the manufacturer’s instruction and administered orally for 10 days.
  • rats were randomized into 4 groups as follows: six rats received intrahepatic injection of 1.0 c 1012 LVP in a volume of 200 pi; 6 rats received injection of 1.0 x 1012 LVP in a volume of 200 pi into a thigh muscle; six rats received twice daily intravenous injections of recombinant DNase I protein at 50mg/kg between day 7 and day 12. The remaining six rats were used as the untreated leaky gut control. An additional six healthy rats were used as a healthy control. At day 12, all rats were sacrificed and blood samples were collected for measurement of the DNase activity in blood. Brain autopsies were collected to perform NLRP3 inflammasome expression analysis and 16S universal bacterial RNA gene quantification.
  • NLRP3 inflammasome is considered a key contributor to the development of neuroinflammation. Aberrant activation of NLRP3 inflammasome signaling has been demonstrated to contribute to pathology in abroad spectrum of neurological diseases (Song, 2017). NLRP3 expression was assessed by collection of 50mg cortical tissue samples. RNA was isolated using the SV total RNA isolation system (Promega).
  • GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 14) were used to quantify presence of DNA of microbial origin in liver and brain. Gene expression analyses were done using the comparative Ct method.
  • Serum DNase I activity was measured using ORG590 (Orgentec) according to the manufacturer's protocol. Detection was performed using microplate photometer (Multiscan FC) at 450 nm with a correction wavelength of 620 nm. Results are summarized at the Table 9, below:
  • DNase enzyme activity a significant increase of microbial cfDNA presence in liver and brain cortex and significant upregulation of NLRP3 inflammasome component in brain cortical tissue.
  • Delivery of a hyperactive actin resistant DNase I lentiviral transgene to liver as compared to the delivery of the same transgene to muscle tissue or to intravenous injections of DNase I protein, almost completely prevented increase of microbial cfDNA in liver and brain and brain inflammasome activation, despite maintaining of comparable DNase enzymatic activity in the central bloodstream.
  • BMT bone marrow transplant
  • mice were treated with DNase I via IP route twice a day or by single intravenous delivery of DNase I mAFP/Alb AAV8 at 1.00x1011 GC/kg dose.
  • mice were sacrificed on day 16 post-transplant. Tissues were placed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxilin and eosin.
  • spleen SP
  • PDN peripheral lymph nodes
  • MN mesenteric lymph nodes
  • PP Peyer’s patches
  • FIG. 7 Data on liver histopathology in DNase I-treated DNase I mAFP/Alb AAV8 treated recipient mice are presented in Figure 7.
  • the left panel shows lymphoplasmacytic infiltration of portal tracts, segmental loss of bile duct epithelial cells, areas of focal necrosis of hepatocytes, and significant amount of nucleiphilic material at the vascular interface in animals treated with DNase I protein only (and not DNasel mAFP/Alb AAV8).
  • the right panel shows that the livers from the animals treated with DNasel mAFP/Alb AAV8 have much lower inflammatory cell density, no necrosis and much less nucleiphilic material at the vascular interface.
  • Figure 8 shows a significant reduction in the total numbers of donor-derived T cells, donor-derived CD4+ and CD8+ T cells in the peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Payer's patches (PP) in DNase I mAFP/Alb AAV8-treated recipients as compared with DNase I protein treated recipients.
  • PPN peripheral lymph nodes
  • MN mesenteric lymph nodes
  • PP Payer's patches
  • liver-specific transgenic expression of DNase I enzyme results in doubling of animal survival comparing with DNase I protein treatment.
  • Such survival benefit was accompanied with a significant reduction of total numbers of donor-derived T cells, donor-derived CD4+ and CD8+ T cells in the PLN, MLN and PP in DNase I mAFP/Alb AAV8 treated recipients as compared with DNase I protein treated recipients.
  • DNase I mAFP/Alb AAV8 treatment resulted in a significant reduction of lymphoplasmacytic infiltration of portal tracts, prevention of cell loss and clearance of vascular interface from nucleophilic material.
  • EXAMPLE 9 Cloning of AAV Vector: ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader- WPRE Xinact (VR-18013AD)
  • An AAV Promoterless Expression Vector pAAV-MCS-Promoterless was obtained from Cell Bio labs (Cat no. VPK-411; Figure 11). The ITR integrity was checked by performing a Smal restriction enzyme digest followed by gel purification to assess for the presence of the following expected bands: 2681 bp, 666 bp, and 112 bp. The expected 11 bp band would not be seen. If the expected bands were present, the plasmid was amplified by growing in SURE cells (Agilent Technologies) or other similar bacteria.
  • a 5' EcoRI site and a 3' Hindlll site were added to the sequence of SEQ ID NO: 30 (ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader- WPRE Xinact).
  • the polynucleotide of SEQ ID NO: 30 (with 5' EcoRI and 3' Hindlll sites) was synthesized and then digested with EcoRI-HF and Hindlll-HF (available from NEB). The 2.2 kb band was gel-extracted.
  • the pAAV-MCS Promoterless vector was digested with EcoRI-HF and Hindlll-HF, with the resulting 3.5 kb band gel extracted.
  • the digested vector and the polynucleotide of SEQ ID NO: 30 were ligated for one hour at room temperature in a 3 : 1 insert to vector molar ratio.
  • the ligated construct was transformed into SURE electrocompetent cells and plated on LB-Agar (Amp) plates. Colonies were selected and minipreps were prepared for screening.
  • a Smal digest was performed for ITR integrity to determine if the following expected bands were present: 2681 bp, 1650 bp, and 1311 bp.
  • an EcoRI-HF and Hindlll-HF digest was performed to assess if the insert was present. If the insert was present, the following bands would be seen: 3435 bp and 2218 bp.
  • a map of the VR-18013AD vector is shown in Figure 10B.
  • Clones were selected if the expected bands were present in each of the Smal digest and the EcoRI-HF and Hindlll-HF digest. For selected clones, a glycerol stock was prepared for long-term storage. The plasmid was also amplified and sequenced. Clones comprising a plasmid with the correct sequence were then cultured and purified by endo-free Gigaprep (ALTA Biotech). The purified plasmid was suitable for packaging by MEE into AAV capsids.
  • EXAMPLE 10 Cloning of AAV Vector: ApoEHCR enhancer-hAAT promoter-hDNasel [wild tvpeVWPRE Xinact iVR-18014AD)
  • An AAV Promoterless Expression Vector pAAV-MCS-Promoterless was obtained from Cell Bio labs (Cat no. VPK-411; Figure 11). The ITR integrity was checked with Smal digest and gel-purified to assess whether the plasmid produced the following expected bands: 2681 bp, 666 bp, and 112 bp. The expected 11 bp band would not be seen. If the expected bands were present, the plasmid was amplified by growing in SURE cells (Agilent Technologies) or other similar bacteria.
  • SEQ ID NO: 31 A 5' EcoRI site and a 3' Hindlll site were added to the sequence of SEQ ID NO: 31 (ApoEHCR enhancer-hAAT promoter-hDNasel (wild type)-WPRE Xinact).
  • the polynucleotide of SEQ ID NO: 31 was synthesized and then digested with EcoRI-HF and Hindlll-HF (available from NEB). The 2.2 kb band was gel-extracted.
  • the pAAV-MCS Promoterless vector was digested with EcoRI-HF and Hindlll-HF, with the resulting 3.5 kb band gel extracted.
  • the digested vector and the polynucleotide of SEQ ID NO: 31 were ligated for one hour at room temperature in a 3 : 1 insert to vector molar ratio.
  • the ligated construct was transformed into SURE electrocompetent cells and plated on LB-Agar (Amp) plates. Colonies were selected and minipreps were prepared for screening.
  • a Smal digest was performed for ITR integrity to determine if the following expected bands were present: 2681 bp, 1650 bp, and 1311 bp.
  • an EcoRI-HF and Hindlll-HF digest was performed to assess if the insert was present. If the insert was present, the following bands would be seen: 3435 bp and 2218 bp.
  • a map of the VR-18014AD vector is shown in Figure IOC.
  • Clones were selected if the expected bands were present for each of the Smal digest and the EcoRI-HF and Hindlll-HF digest. For selected clones, a glycerol stock was prepared for long-term storage. The plasmid was also amplified and sequenced. Clones comprising a plasmid with the correct sequence were then cultured and purified by endo-free Gigaprep (ALTA Biotech). The purified plasmid was suitable for packaging by MEE into AAV capsids.
  • the VR-18013AD and VR-18014AD expression plasmids were manufactured by GenScript USA Inc. (Piscataway, NJ 08854, USA).
  • the corresponding hDNAse I (hyperactive) VR-18013AD AAV vector and hDNAsel (wild-type) VR-18014AD AAV vectors were produced using large-scale polyethylenimine transfections of Anc80L65 AAV cis, AAV trans, and adenovirus helper plasmids in near-confluent monolayers of HEK293 cells with further purification on an iodixanol gradient at scale using traditional protocols.
  • 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-hDNasel (hyperactive)correct leader-WPRE Xinact (VR-18013AD) and ApoEHCR enhancer-hAAT promoter-hDNasel [wild typeVWPRE Xinact (VR-18014AD)
  • ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact and ApoEHCR enhancer-hAAT promoter-hDNasel (wild type)-WPRE Xinact.
  • HEP G2 human hepatoma derived cell line (ATCC HB-8065) and SK-HEP1 human hepatic adenocarcinoma of endothelial origin (ATCC HTB-52). Cells were seeded to 48 well plates at 5x105 cell/ml. and grown in DMEM Adv, 10% FBS media. 24 h. post seed wells were supplemented with VR-18013AD or VR-18014AD using MOI 104 GC/cell ,105 GC/cell and 106 GC/cell or blank control. Each well was in triplicate.
  • the DNase activity was analyzed using fluorescent probe (Terekhov et ah, PNAS 2017; www.pnas.org/content/pnas/114/10/2550.full.pdf) representing hairpin oligonucleotide labeled with fluorescent dye and quencher.
  • the increase in fluorescence was monitored using Variscan Flash plate reader (Thermo Scientific).
  • the culture medium was diluted from 20 to 200 folds by reaction buffer 20 mM Tris-HCl pH 7.5, 1 mM MnC12 and 0.3 mM CaC12. 0.25 mM concentration of florescent probe was used. Complete hydrolysis of florescent probe in reaction conditions resulted in 28 RFU fluorescence under assay conditions.
  • EXAMPLE 13 Use of ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact (VR-18013AD) for treatment of Alzheimer’s disease
  • the objective of the study was to quantify the effect of a single VR-18013AD IV injection on amyloid deposition and tau phosphorylation in 3xTg-AD via immunohistochemistry.
  • the impact of VR-18013AD on tau hyperphosphorylation was studied in 3xTg-AD mice using the AT8 mAh antibody specific for pSer202/Thr205 epitopes, which are among the earliest phosphorylated tau epitopes in patients with AD.
  • Total tau was also determined in treated and untreated 3xTg-AD mice.
  • Overactive microglia can induce highly-detrimental neurotoxic effects; hence the attenuation of the microglial response has been proposed as a potential therapeutic approach in AD.
  • mice received treatment (a) 13 six-month old wild type (WT) mice was injected with phosphate buffered saline (PBS); (b) 15 six-month old 3xTg-AD mice were injected with phosphate buffered saline (PBS) only; and (c) 10 six-month- old 3xTg-AD mice injected with VR-18013AD at 1.0x1011 GC/per mouse dose.
  • WT wild type mice was injected with phosphate buffered saline
  • PBS phosphate buffered saline
  • 10 six-month- old 3xTg-AD mice injected with VR-18013AD at 1.0x1011 GC/per mouse dose The data are summarized as follows.
  • the Y Maze behavioral test shows a slight improvement in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice.
  • the data of Figure 17 show that there is a significant reduction of microglia activation in the cortical brain area of VR-18013AD treated 3xTg-AD mice vs 3xTg-AD control mice.
  • the data of Figure 18 show that treatment with VR-18013AD significantly reduces amyloid deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice.
  • the data of Figure 19 show that treatment with VR-18013AD significantly reduces hyperphosphorylated Tau deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice.
  • the photographs in Figure 20 show significant reduction of p-tau in hippocampal region after VR- 18013 AD treated treatment.
  • a single IV injection of VR-18013AD shows significant therapeutic activity in an Alzheimer’s disease mouse model.
  • mice Twelve BALB/c mice (20- 22 g) of the IBCH RAS breed were housed at a controlled temperature of 20-22°C. At day 1, mice were injected intraperitoneally with 200 pi of a cell suspension comprising 5 x 10 5 CT26 NM!J induced undifferentiated colon carcinoma cells. At day 6 following tumor transplant, mice were randomly assigned to the control group (six mice, single IV injection of PBS) or the treatment group (six mice, single IV injection of VR-18014AD at 1.0 xlO 11 GC/per mouse dose). A morphometric assessment and measurement of the number of tumor nodes in each group were performed on day 16 after tumor cell inoculation following mice euthanasia.
  • mice from the control group had 14-22 tumor nodes per mouse, with part of the nodes forming agglomerates.
  • VR-18014AD treated mice had just 2-5 nodes of smaller size with no signs of agglomerate formation.
  • Typical morphometric images of control and VR- 18014AD-treated mice are shown in Figure 21.
  • EXAMPLE 15 cfDNA extracted from mice with induced leaky gut triggers Tau aggregation, and liver-specific DNase gene transfer suppresses the ability of cfDNA to induce Tau aggregation.
  • Aggregated tau protein is associated with over 20 neurological disorders, including Alzheimer's disease.
  • Leaky gut was induced in 18 rats as specified in Example 7.
  • the tested vectors and compositions were administered to the mice according to a predetermined regimen as shown in Table 12 below:
  • cfDNA extraction was performed as follows. Peripheral blood samples (5ml) were collected through cardiopuncture in cell free DNA BCT tubes. Plasma was separated from the cellular fraction with a first centrifugation at 800g at 4°C for 10 min. The plasma was further centrifuged at 13,000g at 4°C for 10 min to pellet and remove any remaining cells, and then stored in 1 ml aliquots at -80°C until DNA extraction.
  • liver-specific transgenic expression of DNase I enzyme significantly suppress the ability of cfDNA extracted from blood of animals with leaky gut to promote Tau aggregation while injections of recombinant DNasel enzyme as well as use of non-liver specific transgenic expression of DNase I enzyme are much less effective.
  • EXAMPLE 16 Development of AAV vector designed to provide DNase expression in nervous system and /or intestine [00429]
  • rAAV adeno-associated virus
  • VR- 18015 AD containing a liver-specifi c/nervous system-specific tandem promoter
  • the ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact transgene cassette (SEQ ID NO: 30) was modified by insertion of 485 bp sequence of human synapsin promoter (SEQ ID NO: 36) (P.
  • rAAV adeno-associated virus
  • hDNAse I hyperactive liver/nervous system tandem VR-18015AD and hDNAse I (hyperactive) liver/intestine tandem VR-18016AD
  • AAV vectors were produced using large-scale polyethylenimine transfections of Anc80L65 AAV cis, AAV trans, and adenovirus helper plasmids in near-confluent monolayers of HEK293 cells with further purification on an iodixanol gradient at scale using traditional protocols.
  • Vectors were reformulated in PBS supplemented with 35 mM NaCl and 0.001% PF68, and comprising 1.0 xl013 GC/ml.
  • Amyloid 1-42 (Ab1-42) was quantified in brain homogenate using Mouse Amyloid Beta Peptide 1-42 (Ab1-42) enzyme linked immuno-sorbent assay (ELISA) Kit (Cusabio). Phosphorylated tau in brain tissues (p-tau Thrl81) was measured by ELISA with the Cell Like technology kit (D9F4G, Life Technologies), according to the manufacturer instructions.
  • EXAMPLE 17 Effects of transgenic delivery of DNase in a sickle-cell animal model

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Abstract

The invention relates to the delivery and/or expression of an enzyme which has a deoxyribonuclease (DNase) activity to liver, nervous system and/or intestine resulting in enhanced clearance of cell free DNA (cfDNA) useful for treatment of various diseases and conditions, including cancer and neurodegeneration.

Description

TREATMENT OF DISEASES BY EXPRESSION OF
AN ENZYME WHICH HAS A DEOXYRIBONUCLEASE (DNase)
ACTIVITY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No.
62/873,816, filed July 12, 2019, the disclosure of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 10, 2020, is named 252732_000008_SL.txt and is 84,730 bytes in size.
FIELD OF THE INVENTION
[0003] The invention relates to the delivery and/or expression of an enzyme which has a deoxyribonuclease (DNase) activity to liver, nervous system and/or intestine resulting in enhanced clearance of cell free DNA (cfDNA) useful for treatment of various diseases and conditions, including cancer and neurodegeneration.
BACKGROUND OF THE INVENTION
[0004] Patients with malignant tumors have elevated levels of circulating cell free DNA
(cfDNA) in their plasma and serum as compared to healthy individuals (Fleischhacker, 2007). In cancer patients, circulating cfDNA originates both from dying non-tumor cells, tumor cells and neutrophils. Tumors predispose neutrophils to release extracellular DNA traps that contribute to the establishment of a pro-thrombotic state, cachexia and organ failure in cancer patients (Demmers, 2012). Tumor originated cfDNA can contribute to the development of metastasis and chemotherapy resistance (Garcia-Ol mo, 2013) The quantity of circul ating cfDNA increases as the tumor progresses (Sawyers, 2008) reaching the maximal levels in patients with advanced disease and metastatic disease (Butt, 2008). It has been shown that higher quantities of circulating cfDNA significantly correlate with poor patient survival (Schwarzenbach, 2008).
[0005] Neurodegeneration is a separate clinical pathological condition with progressive loss of structure and/or function of neurons, including death of neurons. Molecular pathways leading to neurodegeneration are highly disease-specific (e.g., accumulation of abnormally folded amyloid-beta and tau proteins in the brain in Alzheimer's disease patients; accumulation of alpha- synuclein in Parkinson's disease; accumulation of mutant Huntingtin in Huntington's disease; accumulation of TDP-43 and FUS protein aggregates in Amyelotropic Lateral Sclerosis (ALS); accumulation of mitochondrial DNA mutations and broken mitochondria division mechanics in aging) and result in neuronal cell death at advanced stages of disease progression. Programmed cell death including apoptosis seems to play a key role in the progression of neurodegeneration at late disease stage, as demonstrated by studies on animal models and cell lines (Radi E., et ah, J Alzheimers Dis. 2014; 42).
[0006] Patients with neurodegenerative diseases have increased levels of circulating cfDNA, including cfDNA of microbial origin, which significantly contribute to the progression of neurodegeneration (see, e.g., Int. Appl. Pub. No. W02016/190780) through different mechanisms including transfer through blood brain barrier (BBB) and direct neuronal damage, intracerebral neutrophil DNA traps release (Zenaro, 2015) and triggering cerebral thrombotic arteriopathies.
[0007] The present inventors have previously demonstrated that systemic administration of high doses of DNase protein into a patient’s circulation can be useful for treatment of a number of diseases and conditions associated with increased levels of cfDNA in the blood, including cancers (e.g., carcinomas, sarcomas, lymphomas, melanoma; see, e.g., U.S. Patent Nos. 7,612,032; 8,710,012; 9,248, 166), development of somatic mosaicism (see, e.g., U.S. Pat. Appl. Pub. No. US20170056482), side effects associated with a chemotherapy or a radiation therapy (see, e.g., U.S. Pat. Appl. Pub. No. US20170100463), neurodegenerative diseases (see, e.g., Int. Appl. Pub. No. WO2016/190780), infections (see, e.g., U.S. Patent Nos. 8,431,123 and 9,072,733), diabetes (see, e.g., U.S. Patent No. 8,388,951), atherosclerosis (see, e.g., U.S. Patent No. 8,388,951), stroke (see, e.g., U.S. Patent No. 8,796,004), angina (see, e.g., U.S. Patent No. 8,796,004), ischemia (see, e.g., U.S. Patent No. 8,796,004), kidney damage (see, e.g., U.S. Patent No. 9,770,492), delayed- type hypersensitivity reactions such as, e.g., graft-versus-host disease [GVHD]) (see, e.g., U.S. Patent No. 8,535,663), reduction of fertility (see, e.g., U.S. Patent No. 8,916,151), age-specific sperm motility impairment (see, e.g., U.S. Patent No. 8,871,200), and aging (see, e.g., U.S. Pat. Appl. Pub. No. US20150110769). All of these patents and applications are incorporated by reference herein in their entireties.
[0008] Others have 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% when administered daily at 0.02-2.3 mg/kg in lung carcinoma and hepatoma metastatic disease model (Patutina, 2011). Li (Li, 2015) reported successful use of DNasel to treat GVHD in mice.
[0009] While systemic administration of DNase protein appears to be useful for treating diseases and conditions associated with increased amount of circulating cfDNA, in clinical settings systemic treatment with DNase protein showed limited effects (see, e.g., Int. Appl. Pub. No. W02014/020564). Thus, there is a need for more efficient methods of reduction of circulating cfDNA levels so as to increase the effectiveness of treating diseases and conditions associated with increased levels of cfDNA in the blood such as, e.g., cancer, development of somatic mosaicism, side effects associated with a chemotherapy or a radiation therapy, neurodegenerative diseases, infections, delayed-type hypersensitivity reactions, diabetes, atherosclerosis, ischemia, stroke, angina, reduction of fertility, age-specific sperm motility impairment, aging, etc.
SUMMARY OF THE INVENTION
[0010] As discussed in the Background section, above, there is a need for more efficient methods of reduction of circulating cfDNA levels so as to increase the effectiveness of treating diseases and conditions associated with increased levels of cfDNA. The present invention addresses these and other needs by providing methods and compositions for delivery and/or expression of an enzyme which has a deoxyribonuclease (DNase) activity to liver, nervous system and/or intestine resulting in enhanced clearance of cell free DNA (cfDNA) useful for treatment of various diseases and conditions, including cancer and neurodegeneration.
[0011] In one aspect, the invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/nervous system-specific tandem promoter. In one embodiment, the liver-specific/nervous system-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system as compared to other tissues and organs. In one embodiment, the liver-specific/nervous system- specific tandem promoter comprises a liver-specific promoter selected from the group consisting of an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin- cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a- fibrinogen promoter, a b-fibrinogen promoter, an alpha 1-antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, a 1 -acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINAl) (hAAT) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a-fetoprotein (AFP) promoter. In one embodiment, the liver-specific promoter is an anti-trypsin (AAT) promoter. In one embodiment, the AAT promoter is a human a 1 -anti -trypsin (hAAT) promoter. In one embodiment, the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter is an albumin promoter. In one embodiment, the liver- specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 8. In one embodiment, the liver-specifi c/nervous system- specific tandem promoter comprises a nervous system-specific promoter selected from the group consisting of F4/80 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, CMV promoter, MEF2 promoter, FoxP2 promoter, Ibal promoter, TTR promoter, CDl lb promoter, c- fes promoter, NSE promoter, synapsin promoter, CamKII promoter, a-CaMKII promoter, VGLUT1 promoter, and glial fibrillary acidic protein (GFAP) promoter. In one embodiment, the nervous system-specific promoter is a synapsin promoter. In one embodiment, the synapsisn promoter is a human synapsin promoter. In one embodiment, the nervous system- specific promoter is a F4/80 promoter. In one embodiment, the nervous system-specific promoter is a CMV promoter. In one embodiment, the nervous system-specific promoter is a TMEM119 promoter. In one embodiment, the nervous system-specific promoter is a MEF2 promoter. In one embodiment, the nervous system-specific promoter is a FoxP2 promoter. In one embodiment, the liver-specific/nervous system-specific tandem promoter comprises a nervous-specific promoter comprising a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to any one of the sequences SEQ ID NO: 36 and 38-42. In one embodiment, the nervous system-specific promoter comprises any one of the sequences SEQ ID NO: 36 and 38-42. In one embodiment, the nervous system-specific promoter consists of any one of the sequences SEQ ID NO: 36 and 38-42.
[0012] In another aspect, the invention provides a recombinant adeno-associated virus
(rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/intestine-specific tandem promoter. In one embodiment, the liver-specific/intestine-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs. In one embodiment, the liver-specific/intestine-specific tandem promoter comprises a liver-specific promoter selected from the group consisting of an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin- cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a- fibrinogen promoter, a b-fibrinogen promoter, an alpha 1-antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, a 1 -acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINAl) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a- fetoprotein (AFP) promoter. In one embodiment, the liver-specific promoter is an anti-trypsin (AAT) promoter. In one embodiment, the AAT promoter is a human a 1 -anti -trypsin (hAAT) promoter. In one embodiment, the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter is an albumin promoter. In one embodiment, the liver- specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 8. In one embodiment, the liver-specific/intestine-specific tandem promoter comprises an intestine-specific promoter selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti-CMV promoter, MUC2 promoter, villin promoter, and T3b promoter. In one embodiment, the intestine-specific promoter is a villin promoter. In one embodiment, the villin promoter promoter is a human villin promoter. In one embodiment, the intestine-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37. In one embodiment, the liver-specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter.
[0013] In a separate embodiment, the liver-specific/nervous system-specific tandem promoter is part of the liver-specific/nervous system-specific/intestine-specific tandem promoter, which mediates a substantially increased expression of the enzyme in the liver and/or nervous system and/or intestine as compared to other tissues and organs. In one embodiment, the liver- specific/nervous system-specific/intestine-specific tandem promoter comprises an intestine- specific promoter selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti- CMV promoter, MUC2 promoter, villin promoter, and T3b promoter. In one embodiment, the intestine-specific promoter is a villin promoter. In one embodiment, the villin promoter promoter is a human villin promoter. In one embodiment, the intestine-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37. In one embodiment, the liver-specific/nervous system- specific/intestine-specific tandem promoter comprises a hAAT promoter, a human synapsin promoter and a human villin promoter.
[0014] In one embodiment of any of the above vectors, the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof. In one embodiment, the enzyme which has a DNase activity is DNase I or a mutant or derivative thereof. In one embodiment, the DNase l is a human DNase I or a mutant or derivative thereof. In one embodiment, the DNase I mutant comprises one or more mutations in an actin binding site. In one embodiment, the one or more mutations in the actin-binding site are selected from a mutation at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combinations thereof. In one embodiment, one of the mutations in the actin-binding site is a mutation at Ala-114. In one embodiment, the DNase I mutant comprises one or more mutations increasing DNase activity. In one embodiment, one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof. In one embodiment, one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combinations thereof. In one embodiment, the DNase I mutant comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 5. In one embodiment, the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 5. In one embodiment, the DNAse I mutant consists of the sequence of SEQ ID NO: 5. In one embodiment, the DNase I mutant comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 2. In one embodiment, the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 2. In one embodiment, the DNAse I mutant consists of the sequence of SEQ ID NO: 2. In one embodiment, the DNase I mutant comprises 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 combinations thereof. In one embodiment, the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 4. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 23. In one embodiment, the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 1. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or 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 which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 32. In one embodiment, the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 24. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 29. In one embodiment, the nucleic acid encodes a DNase I comprising the sequence of SEQ ID NO: 26. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 28. In one embodiment, the nucleic acid encodes a DNase I mutant comprising the sequence SEQ ID NO: 5. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 21. In one embodiment, the nucleic acid encodes a DNase I mutant comprising the sequence SEQ ID NO: 2. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 19. In one embodiment, the enzyme which has a DNase activity is a fusion protein comprising (i) a DNase enzyme or a fragment thereof linked to (ii) an albumin or an Fc or a fragment thereof. In one embodiment, the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme. In one embodiment, the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, the albumin secretory signal sequence, the b-glucuronidase secretory signal sequence, the alkaline protease secretory signal sequence, and the fibronectin secretory signal sequence. In one embodiment, the secretory signal sequence comprises the sequence MRGMKLLGALLAL AALLQGAV S (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one embodiment, the secretory signal sequence consists of the sequence MRGMKLLGALLAL AALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one embodiment, the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of MRGMKLLGALLAL AALLQGAVS (SEQ ID NO: 6) or a sequence having at least 85% or at least 90% or at least 95% sequence identity to the sequence of MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% or at least 90% or at least 95% identical to SEQ ID NO: 20. In one embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence of SEQ ID NO: 20. In one embodiment, the secretory signal sequence comprises the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one embodiment, the secretory signal sequence consists of the sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one embodiment, the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence of MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence SEQ ID NO: 27.
[0015] In a separate embodiment, the nucleic acid of the invention comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.
[0016] In one embodiment of any of the vectors described above and comprising liver- specific and nervous system-specific promoters, the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or nervous system as compared to the corresponding wild-type capsid protein. In one embodiment of any of the vectors described above and comprising liver-specific and intestine- specific promoters, the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or intestine as compared to the corresponding wild-type capsid protein. In one embodiment of any of the vectors described above and comprising liver-specific, nervous system-specific and intestine-specific promoters, the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or nervous system and/or intestine as compared to the corresponding wild-type capsid protein. In one embodiment, the improved efficiency and/or specificity of the delivery of the vector to the liver results in a substantially increased expression of the enzyme in the liver as compared to other tissues and organs. In one embodiment, the improved efficiency and/or specificity of the delivery of the vector to the nervous system results in a substantially increased expression of the enzyme in the nervous system as compared to other tissues and organs. In one embodiment, the improved efficiency and/or specificity of the delivery of the vector to the intestine results in a substantially increased expression of the enzyme in the intestine as compared to other tissues and organs. In one embodiment, the capsid protein comprisesVP3. In one embodiment, the one or more mutations in the capsid protein are selected from the group consisting of S279A, S671A, K137R, T252A, and any combinations thereof. In one embodiment, the one or more mutations in the capsid protein include mutation K137R. In one embodiment, the capsid protein comprises the sequence SEQ ID NO:34. In one embodiment, the capsid protein consists of the sequence SEQ ID NO:34
[0017] In one embodiment of any of the vectors described above, the AAV is selected from serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAVhu37, AAVrh64Rl, Anc 80, AAV-KP1, and AAV-F. In one embodiment, the AAV is from serotype 8 or Anc 80.
[0018] In one embodiment of any of the vectors described above, the nucleic acid further comprises two AAV inverted terminal repeats (ITRs), wherein the ITRs flank the nucleotide sequence encoding the enzyme which has a DNase activity.
[0019] In one embodiment of any of the vectors described above, the nucleic acid further comprises one or more enhancers located upstream or downstream of the promoter. In one embodiment, the one or more enhancers are selected from the group consisting of an apolipoprotein E (ApoE) enhancer, an alpha fetoprotein enhancer, a TTR enhancer, an LSP enhancer, an al-microglobulin/bikunin enhancer, an albumin gene enhancer (Ealb), a nPE2 enhancer, a Gal4 enhancer, a foxP2 enhancer, aMefZ enhancer, a CMV enhancer, and any combination thereof. In one embodiment, the enhancer is an apolipoprotein E (ApoE) enhancer. In one embodiment, the enhancer is a hepatic control region (HCR) enhancer. In one embodiment, the enhancer comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to SEQ ID NO: 17. In one embodiment, the enhancer comprises the sequence SEQ ID NO: 17. In one embodiment, the enhancer consists of the sequence SEQ ID NO: 17.
[0020] In one embodiment of any of the vectors described above, the nucleic acid further comprises a polyadenylation signal operably linked to the nucleotide sequence encoding the enzyme which has a DNase activity.
[0021] In one embodiment of any of the vectors described above, the nucleic acid further comprises a Kozak sequence. In one embodiment, the Kozak sequence comprises the sequence 5 '-GCCGCC ACC-3 ' (SEQ ID NO: 33).
[0022] In one embodiment of any of the vectors described above, the nucleic acid further comprises a post-transcriptional regulatory element. In one embodiment, the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE). In one embodiment, the WPRE does not encode a functional X protein. In one embodiment, the post- transcriptional regulatory element comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to SEQ ID NO: 16. In one embodiment, the post- transcriptional regulatory element comprises the sequence SEQ ID NO: 16. In one embodiment, the post-transcriptional regulatory element consists of the sequence SEQ ID NO: 16.
[0023] In another embodiment, the invention provides pharmaceutical compositions comprising any of the rAAV vectors described above and a pharmaceutically acceptable carrier and/or excipient.
[0024] In a separate aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and the nervous system in a subject in need thereof, comprising administering to the subject any of the above-described rAAV vectors comprising liver-specific and nervous system-specific promoters or pharmaceutical compositions comprising such vectors.
[0025] In another aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject any of the above-described rAAV vectors comprising liver-specific and intestine-specific promoters or pharmaceutical compositions comprising such vectors.
[0026] In another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of any of the above-described rAAV vectors or pharmaceutical compositions comprising such vectors.
[0027] In yet another aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and nervous system in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific/nervous system-specific tandem promoter.
[0028] In a further aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and nervous system in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver and nervous system cells when administered in vivo.
[0029] In another aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver- specific/intestine-specific tandem promoter.
[0030] In yet another aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver and intestine cells when administered in vivo.
[0031] In another aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver, nervous system and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific/nervous system-specific/intestine-specific tandem promoter.
[0032] In a further aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver, nervous system and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver, nervous system and intestine cells when administered in vivo.
[0033] In another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specifi c/nervous system-specific tandem promoter.
[0034] In another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver and nervous system cells.
[0035] In another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specifi c/intestine-specific tandem promoter.
[0036] In yet another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver and intestinal cells.
[0037] In a further aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specifi c/nervous system-specific/intestine-specific tandem promoter.
[0038] In another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver, nervous system and intestinal cells.
[0039] In yet another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and nervous system of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
[0040] In another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and instestine of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
[0041] In yet another aspect, the invention provides a method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver, nervous system and intestine of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
[0042] Non-limiting examples of the neurodegenerative disease treatable by the methods of the invention include, e.g., Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, CADASIL Syndrome, Friedreich’s ataxia, cerebellar ataxia, spinocerebellar ataxia, dementia (e.g., fronto-temporal dementia, frontotemporal dementia with parkinsonism- 17 (FTDP-17), familial Danish dementia, and familial British dementia), prion- caused diseases, Lewy body diseases, an amyloidosis (e.g., hereditary cerebral hemorrhage with amyloidosis, senile systemic amyloidosis, an amyloidosis with a hereditary cerebral hemorrhage, a primary systemic amyloidosis, a secondary systemic amyloidosis, a serum amyloidosis, a senile systemic amyloidosis, a hemodialysis-related amyloidosis, a Finnish hereditary systemic amyloidosis, an Atrial amyloidosis, a Lysozyme systemic amyloidosis, a Traumatic brain injury, an Insulin-related amyloidosi s, or a Fibrinogen a-ehain amyloidosis), Spinal muscular atrophy, a bipolar disorder, schizophrenia, depressive disorder, autism, autism spectrum disorders, Chronic Fatigue Syndrome, Obsessive-Compulsive Disorder, generalized anxiety disorder (GAD), major depressive disorder (MDD), social anxiety disorder (SAD), attention-deficit/hyperactivity disorder (ADHD), Huntington's disease, Spinal muscular atrophy, Mild Cognitive Impairment (MCI), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), agyrophilic grain disease (AGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel-Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, progressive familial myoclonic epilepsy, striatonigral degeneration, torsion dystonia (e.g., torsion spasm or dystonia musculorum deformans), spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration (e.g., Friedreich's ataxia), Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot-Marie-Tooth), and hypertrophic interstitial polyneuropathy (Dejerine-Sottas). In one embodiment, the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, an amyloidosis, asthma, or prion disease.
[0043] Non-limiting examples of neurodevelopmental disease treatable by the methods of the invention include, e.g., autism, neural tube defects, attention deficit hyperactivity disorder, Dawn syndrome, cerebral palsy, Rett syndrome, Landau-Kleffner syndrome, Alexander disease, Alpers' disease, alternating hemiplegia, Angelman syndrome, ataxias and cerebellar or spinocerebellar degeneration, ataxia telangiectasia, attention deficit-hyperactivity disorder, autism spectrum disorders, Asperger syndrome, Batten disease, Canavan disease, Tourette syndrome, and impairments in vision and/or hearing.
[0044] Non-limiting examples of onclological diseases treatable by the methods of the invention include, e.g., astrocytoma, gliomas, glioblastoma, ependymoma, medulloblastoma, central nervous system (CNS) lymphoma, craniopharyngioma, glioblastoma, meningioma, pituitary carcinomas, pituitary adenomas, neurofibromatosis, embryonal tumors, tumors of the pineal region, tumors of meninges, choroid plexus tumors, neuronal and mixed neuronal-glial tumors, germ cell tumors, anaplastic astrocytoma, central neurocytoma, choroid plexus carcinoma, choroid plexus papilloma, dysembryoplastic neuroepithelial tumor, giant-cell glioblastoma, gliosarcoma, hemangiopericytoma, medulloepithelioma, neuroblastoma, neurocytoma, oligoastrocytoma, oligodendroglioma, optic nerve sheath meningioma, pilocytic astrocytoma, pinealoblastoma, pineocytoma, pleomorphic anaplastic neuroblastoma, pleomorphic xanthoastrocytoma, sphenoid wing meningioma, subependymal giant cell astrocytoma, subependymoma, trilateral retinoblastoma, and nervous system metastasis of any origin.
[0045] Non-limiting examples of autoimmune diseases treatable by the methods of the invention include, e.g., autoimmune encephalitis, autoimmune-related epilepsy, CNS vasculitis, Hashimoto’s encephalopathy, neurosarcoidosis, Neuro-Behcet’s disease, cerebral lupus, diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, asthma, celiac disease, ankylosing spondylitis, vasculitis, pemphigus vulgaris, inflammatory bowel disease (IBD), Crohn's disease, and ulcerative colitis.
[0046] Non-limiting examples of psychiatric diseases treatable by the methods of the invention include, e.g., anxiety disorders, psychotic disorders, schizophrenia, bipolar disorder, depression, and post-traumatic stress disorder.
[0047] Non-limiting examples of infections treatable by the methods of the invention include, e.g., adenoviral infections, hepatitis B, hepatitis G, poxvirus infections, herpesvirus infections, papillomavirus infections, HIV infections, meningitis (bacterial, fungal, or viral), bacterial persistence in brain, fungal persistence in brain, pancreatitis, and peritonitis.
[0048] In one embodiment of any of the above methods, the vector further comprises one or more molecules capable of targeting of the nucleic acid to liver and/or nervous system and/or intestinal cells.
[0049] In one embodiment of any of the above methods, the liver-specific/nervous system- specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system as compared to other tissues and organs.
[0050] In one embodiment of any of the above methods, the liver-specific/intestine- specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs.
[0051] In one embodiment of any of the above methods, the liver-specific/nervous system- specific/intestine-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system and/or intestine as compared to other tissues and organs.
[0052] In one embodiment of any of the above methods, the liver-specific promoter is selected from the group consisting of an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a-fibrinogen promoter, a b-fibrinogen promoter, an alpha 1-antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, al-acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINA1) (hAAT) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a-fetoprotein (AFP) promoter. In one embodiment, the liver-specific promoter is an anti-trypsin (AAT) promoter. In one embodiment, the AAT promoter is a human al -anti -trypsin (hAAT) promoter. In one embodiment, the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 15. In one embodiment, the liver-specific promoter is an albumin promoter. In one embodiment, the liver-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter comprises the sequence SEQ ID NO: 8. In one embodiment, the liver-specific promoter consists of the sequence SEQ ID NO: 8.
[0053] In one embodiment of any of the above methods, the nervous system-specific promoter is selected from the group consisting of F4/80 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, CMV promoter, MEF2 promoter, FoxP2 promoter, Ibal promoter, TTR promoter, CDl lb promoter, c-fes promoter, NSE promoter, synapsin promoter, CamKII promoter, a-CaMKII promoter, VGLUT1 promoter, and glial fibrillary acidic protein (GFAP) promoter. In one embodiment, the nervous system-specific promoter is a synapsin promoter. In one embodiment, the synapsisn promoter is a human synapsin promoter. In one embodiment, the nervous system-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 36. In one embodiment, the nervous system-specific promoter comprises the sequence SEQ ID NO: 36. In one embodiment, the nervous system-specific promoter consists of the sequence SEQ ID NO: 36.
[0054] In one embodiment of any of the above methods, the liver-specific/nervous system- specific tandem promoter comprises a hAAT promoter and a human synapsin promoter.
[0055] In one embodiment of any of the above methods, the intestine-specific promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs. [0056] In one embodiment of any of the above methods, the intestine-specific promoter is selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti-CMV promoter, MUC2 promoter, villin promoter, and T3b promoter. In one embodiment, the intestine-specific promoter is a villin promoter. In one embodiment the villin promoter promoter is a human villin promoter. In one embodiment, the intestine-specific promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37.
[0057] In one embodiment of any of the above methods, the liver-specific/intestine- specific tandem promoter comprises a hAAT promoter and a human villin promoter. In one embodiment, the liver-specific/intestine-specific tandem promoter comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 37.
[0058] In one embodiment of any of the above methods, the vector is used as a naked DNA.
[0059] In one embodiment of any of the above methods, the vector is a viral vector. In one embodiment, the viral vector is an adeno-associated virus vector, an adenovirus vector, a retrovirus vector (e.g., a lentivirus vector), or a hepatotropic virus vector (e.g., a hepatitis B virus (HBV) vector).
[0060] In one embodiment of any of the above methods, the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof. In one embodiment, the DNase is DNase I or a mutant or derivative thereof. In one embodiment, the DNase I is human DNase I or a mutant or derivative thereof. In one embodiment, the DNase I mutant comprises one or more mutations in an actin binding site. In one embodiment, the one or more mutations in the actin-binding site are selected from a mutation at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combinations thereof. In one embodiment, one of the mutations in the actin-binding site is a mutation at Ala-1 14. In one embodiment, the DNase I mutant comprises one or more mutations increasing DNase activity. In one embodiment, one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof. In one embodiment, one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, N74K, and A114F. In one embodiment, the DNase I mutant comprises 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 combinations thereof. In one embodiment, the DNase I mutant is a long acting form of DNase. In one embodiment, the DNase I comprises the sequence SEQ ID NO: 4. In one embodiment, the DNase I comprises the sequence SEQ ID NO: 1. In one embodiment, the DNase I mutant comprises the sequence SEQ ID NO: 5. In one embodiment, the DNase I mutant comprises the sequence SEQ ID NO: 2.
[0061] In one embodiment, the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme into the hepatic porto-sinusoidal circulation upon administration of the vector to the subject. In one embodiment, the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, b-glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence. In one embodiment, the secretory signal sequence comprises a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to MRGMKLLGALL AL AALLQGAV S (SEQ ID NO: 6) or a sequence having at least 80% or at least 85% or at least 90% or at least 95% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one embodiment, the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one embodiment, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
[0062] In one embodiment, the nucleic acid further comprises one or more hepatic control region (HCR) enhancers located upstream or downstream of the promoter. In one embodiment, the HCR enhancer comprises a sequence having at least 80% or at least 855 or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 17. In one embodiment, the HCR enhancer comprises the sequence SEQ ID NO: 17. In one embodiment, the HCR enhancer consists of the sequence SEQ ID NO: 17.
[0063] In one embodiment, the nucleic acid further comprises a Kozak sequence. In one embodiment, the Kozak sequence comprises the sequence 5'-GCCGCCACC-3' (SEQ ID NO: 33).
[0064] In one embodiment, the nucleic acid further comprises a post-transcriptional regulatory element, wherein the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE) that does not encode a functional X protein. In one embodiment, the nucleic acid comprises a sequence having at least 80% or at least 855 or at least 90% or at least 95% sequence identity to the sequence SEQ ID NO: 19. In one embodiment, the nucleic acid comprises the sequence SEQ ID NO: 19. In one embodiment, the nucleic acid consists of the sequence SEQ ID NO: 19.
[0065] In one embodiment of any of the above methods, the administration of the nucleic acid results in the expression of the enzyme and its secretion into the hepatic porto-sinusoidal circulation of the subject.
[0066] In one embodiment of any of the above methods, the nucleic acid is administered in a dose and regimen which is sufficient to decrease the level of the cell free DNA (cfDNA) in the porto-sinusoidal circulation of said subject.
[0067] In one embodiment of any of the above methods, the method further comprises selecting a subject with an elevated level of cell-free DNA (cfDNA) in the bloodstream as compared to a level of cfDNA in the bloodstream of a normal healthy subject.
[0068] In one embodiment of any of the above methods, the method further comprises administering a deoxyribonuclease (DNase) enzyme to the subject. In one embodiment, the DNase is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof. In one embodiment, the DNase is DNase I or a mutant or derivative thereof.
[0069] In one embodiment of any of the above methods, the subject is human.
[0070] In a separate aspect, the invention provides a recombinant adeno-associated virus
(rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter. In one embodiment, the liver-specific promoter mediates a substantially increased expression of the enzyme in the liver as compared to other tissues and organs.
[0071] In another aspect, the invention provides a recombinant adeno-associated virus expression vector (rAAV) comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the capsid protein mediates 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 comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver as compared to the corresponding wild-type capsid protein. In one specific embodiment, the improved efficiency and/or specificity of the delivery of the vector to the liver results in a substantially increased expression of the enzyme in the liver as compared to other tissues and organs.
[0072] Non-limiting examples of enzymes having DNase activity which can be used in the vectors of the invention include, e.g., DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, or mutants or derivatives thereof. In one embodiment, the enzyme which has a DNase activity is a DNase I (e.g., 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 in an 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 combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence). In one embodiment, one of the mutations in the actin-binding site is a mutation at Ala- 114. In one embodiment, the DNase I mutant comprises one or more mutations increasing DNase activity (e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence). In one embodiment, the DNase I mutant comprises one or more mutations are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combinations thereof. In one embodiment, the DNase I mutant comprises the mutation Q9R. In one embodiment, the DNase I mutant comprises the mutation E13R. In one embodiment, the DNase I mutant comprises the mutation N74K. In one embodiment, the DNase I mutant comprises the mutation A114F.
[0073] In one embodiment, the DNase I mutant comprises a sequence having at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 5. In one embodiment, the DNasel mutant comprises the mutations Q9R, E13R N74K and A114F. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 5.
[0074] In one embodiment, the DNase I mutant comprises a sequence having at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 2. In one embodiment, the DNasel mutant comprises the mutations Q9R, E13R N74K and A114F. In one embodiment, the DNase I mutant comprises the sequence of SEQ ID NO: 2.
[0075] In one embodiment, the DNase I mutant consists of the sequence of SEQ ID NO: 2 or SEQ ID NO: 5.
[0076] In one embodiment, the DNase I mutant comprises 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, H44 A : Y65 A : 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 combinations thereof. In one embodiment, the nucleotide sequence encodes a DNase I comprising the sequence SEQ ID NO: 4. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 23. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 23. In one embodiment, the nucleotide sequence encodes a DNase I comprising the sequence SEQ ID NO: 1. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 22. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 22. In one embodiment, the nucleic acid comprises a nucleotide sequence which 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 which is at least 85% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 32. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 32. In one embodiment, the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 24. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 29. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 29. In one embodiment, the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 26. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 28. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 28. In one embodiment, the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 5. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 21. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 21. In one embodiment, the nucleotide sequence encodes a DNase I mutant comprising the sequence SEQ ID NO: 2. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 19. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 19.
[0077] In one embodiment, the enzyme which has a DNase activity is a fusion protein comprising (i) a DNase enzyme or a fragment thereof linked to (ii) an albumin or an Fc polypeptide or a fragment thereof. In one embodiment, the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme into the hepatic sinusoidal system upon expression of the vector in the liver. Non-limiting examples of useful secretory signal sequences include, e.g., DNase I secretory signal sequence, IL2 secretory signal sequence, the albumin secretory signal sequence, the b-glucuronidase secretory signal sequence, the alkaline protease secretory signal sequence, and the fibronectin secretory signal sequence. In one specific embodiment, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MRGMKLLGALL AL AALLQGAV S (SEQ ID NO: 6). In one specific embodiment, the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one specific embodiment, the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one specific embodiment, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence M YRMQLL S CIAL SL AL VTN S (SEQ ID NO: 7). In one specific embodiment, the secretory signal sequence comprises the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one specific embodiment, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 20. In one specific embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence SEQ ID NO: 20.
[0078] In one specific embodiment, the secretory signal sequence comprises the sequence
MRYT GLMGTLLTL VNLLQL AGT (SEQ ID NO: 25). In one specific embodiment, the secretory signal sequence consists of the sequence MRYT GLMGTLLTL VNLLQL AGT (SEQ ID NO: 25). In one specific embodiment, the secretory signal sequence comprises a sequence having at least 80% sequence identity to sequence MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25). In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 27. In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 27. In one specific embodiment, the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 27. In one specific embodiment, the sequence encoding the secretory signal sequence comprises the nucleotide sequence of SEQ ID NO: 27.
[0079] Non-limiting examples of promoters which can be used in the vectors of the invention include, e.g., an albumin promoter, an a 1 -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a-fibrinogen promoter, a b-fibrinogen promoter, an alpha 1- antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, al-acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINAl) (hAAT) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a-fetoprotein (AFP) promoter.
[0080] In one embodiment, the promoter is an albumin promoter. In one specific embodiment, the albumin promoter comprises the sequence of SEQ ID NO: 8. In one specific embodiment, the albumin promoter consists of the sequence of SEQ ID NO: 8. See also Frain et ah, Mol. Cell Biol., 1990, 10(3):991-999.
[0081] In one embodiment, the promoter is an al -anti -trypsin (AAT) promoter. In one embodiment, the promoter is a human al -anti -trypsin (hAAT) promoter. In one specific embodiment, the anti-trypsin promoter comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 15. In one specific embodiment, the AAT promoter comprises the sequence of SEQ ID NO: 15. In one specific embodiment, the AAT promoter consists of the sequence of SEQ ID NO: 15.
[0082] In one embodiment, the capsid protein comprises one or more mutations selected from the group consisting of S279A, S671 A, K137R, T252A, and any combinations thereof. In one embodiment, the one or more mutations in the capsid protein include mutation K137R. In one embodiment, the capsid protein comprises the sequence SEQ ID NO:3 [Anc80] In one embodiment, the capsid protein consists of the sequence SEQ ID NO:3 [Anc80] In one embodiment, the capsid protein comprises the sequence SEQ ID NO:9 [Anc80] In one embodiment, the capsid protein consists of the sequence SEQ ID NO:9 [Anc80] In one embodiment, the capsid protein comprises the sequence SEQ ID NO:34 [Anc80L65] In one embodiment, the capsid protein consists of the sequence SEQ ID NO:34 [Anc80L65] In one embodiment, the capsid protein comprises the sequence SEQ ID NO:35 [Anc80L65 variant]. In one embodiment, the capsid protein consists of the sequence SEQ ID NO:35 [Anc80L65 variant]. In one embodiment, the capsid protein is a mutant AAV8 capsid protein such as, e.g., AAV3G1, AAVT20 or AAVTR1, or another mutant capsid protein disclosed in Int. Pat. Appl. Pub. No. WO2017/180854 (e.g., comprising VP3 mutations in amino acids 263-267 [e.g., 263NGTSG267->SGTH ("NGTSG" disclosed as SEQ ID NO: 43 and "SGTH" disclosed as SEQ ID NO: 44) or 263NGTSG267->SDTH ("NGTSG" disclosed as SEQ ID NO: 43 and "SDTH" disclosed as SEQ ID NO: 45)] and/or amino acids 457-459 [e.g., 457TAN459->SRP], and/or amino acids 455-459 [e.g., 455GGTAN459 ->DGSGL ("GGTAN" disclosed as SEQ ID NO: 46 and "DGSGL" disclosed as SEQ ID NO: 47)] and/or amino acids 583-597).
[0083] Non-limiting examples of AAVs which can be used in the vectors of the invention include, e.g., serotype 1 (AAV1), AAV2, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV 12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64Rl, and Anc 80. In one specific embodiment, the AAV is from serotype 8 or Anc 80.
[0084] In one embodiment, the nucleic acid of the AAV vector further comprises two AAV inverted terminal repeats (ITRs), wherein the ITRs flank the nucleotide sequence encoding the enzyme which has a DNase activity.
[0085] In one embodiment, the nucleic acid of the AAV vector further comprises one or more enhancers located upstream or downstream of the promoter. In one embodiment, the enhancer may be located immediately upstream with the promoter, e.g., where the 3' end of an enhancer sequence is fused directly to the 5' end of the promoter sequence. Non-limiting examples of useful enhancers include, e.g., an apolipoprotein E (ApoE) enhancer (e.g., an ApoE hepatic control region- 1 (HCR-1) enhancer or an ApoE HCR-2 enhancer), an alpha fetoprotein enhancer, a TTR enhancer, an LSP enhancer, an al-microglobulin/bikunin enhancer, an albumin gene enhancer (Ealb), a nPE2 enhancer, a Gal4 enhancer, a foxP2 enhancer, a Mef2 enhancer, a CMV enhancer, and any combination thereof. In one specific embodiment, the enhancer is an ApoE enhancer. In one specific embodiment, the enhancer is an ApoE enhancer located upstream of the promoter. In one specific embodiment, the enhancer is an ApoE enhancer fused to the 5' end of the promoter. In one specific embodiment, the ApoE enhancer is a hepatic control region (HCR) enhancer. In one specific embodiment, the enhancer comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 17. In one specific embodiment, the enhancer comprises the sequence of SEQ ID NO: 17. In one specific embodiment, the enhancer consists of the sequence of SEQ ID NO: 17.
[0086] In one embodiment, the nucleic acid of the AAV vector further comprises a polyadenylation signal operably linked to the nucleotide sequence encoding the enzyme which has a DNase activity.
[0087] In one embodiment, the nucleic acid further comprises a Kozak sequence. In one specific embodiment, the Kozak sequence comprises the sequence of 5'-GCCGCCACC-3' (SEQ ID NO: 33). In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 80% identical to SEQ ID NO: 30. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 30. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 30. In one embodiment, the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 30. In one embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 30.
[0088] 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 post-transcriptional regulatory element (WPRE). In one specific embodiment, the WPRE does not encode a functional X protein. In one embodiment, the post- transcriptional regulatory element comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 16. In one specific embodiment, the post-transcriptional regulatory element comprises the sequence of SEQ ID NO: 16. In one specific embodiment, the post-transcriptional regulatory element consists of the sequence of SEQ ID NO: 16.
[0089] In one embodiment, the nucleic acid of rAAV vector of the invention comprises a sequence having 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% identity to the sequence of SEQ ID NO: 19. In one specific 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.
[0090] In one embodiment, the invention provides a recombinant adeno-associated virus
(rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 4 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
[0091] In one embodiment, the invention provides a recombinant adeno-associated virus
(rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 5 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
[0092] In one embodiment, the invention provides a recombinant adeno-associated virus
(rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 1 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
[0093] In one embodiment, the invention provides a recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising a nucleotide sequence encoding the deoxyribonuclease (DNase) enzyme comprising the sequence SEQ ID NO: 2 operably linked to an albumin promoter or an a 1 -anti -trypsin (AAT) promoter.
[0094] In one embodiment, the invention provides a recombinant adeno-associated virus
(rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 30.
[0095] In one embodiment, the invention provides a recombinant adeno-associated virus
(rAAV) expression vector comprising (i) a capsid protein comprising the sequence SEQ ID NO: 34 and (ii) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 31.
[0096] In a related aspect, the invention provides pharmaceutical compositions and dosage forms comprising any of the rAAV vectors of the invention and a pharmaceutically acceptable carrier and/or excipient.
[0097] In a related aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver in a subject in need thereof, comprising administering to the subject any of the rAAV vectors or pharmaceutical compositions described above.
[0098] In another aspect, the invention provides a method for treating a disease or condition (e.g., a neurodegenerative, neurodevelopmental, psychiatric, or autoimmune disease, or an infection, or a delay ed-type hypersensitivity reaction) in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of cell free DNA (cfDNA) in the hepatic porto-sinusoidal circulation of the subject, said method comprising administering to the subject a therapeutically effective amount of any of the rAAV vectors or pharmaceutical compositions described above.
[0099] In a further aspect, the invention provides a method for treating a cancer in a subj ect 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 in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein. In one embodiment, the method is effective to inhibit metastasis. Non limiting examples of cancers treatable by the methods of the invention include, e.g., a peritoneal carcinomatosis, a lymphoma, a stomach cancer, a colon cancer, an intestinal cancer, a colorectal cancer, a pancreatic cancer, a liver cancer, a cancer of the bile duct, a cancer of the gall bladder, a sarcoma, an esophageal cancer, and a liver metastatic disease of any origin.
[00100] In another aspect, the invention provides a method for 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 invention include, e.g., Alzheimer’s disease, Mild Cognitive Impairment (MCI), CADASIL syndrome, Parkinson’s disease, Amyotrophic Lateral Sclerosis, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), agyrophilic grain disease (AGD), Pick’s disease (PiD), Huntington’s disease (HD), and Frontotemporal dementia with parkinsonism- 17 (FTDP-17). In one embodiment, the neuro egenerative disease is associated with the formation of a misfolded protein due to the presence of DNA. In one specific embodiment, the DNA is human DNA, microbial DNA, cell-free DNA, or intracellular DNA.
[00101] In one embodiment, the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SI .!· ), gout, metabolic syndrome, an amyloidosis, asthma, or prion disease. In one embodiment, the amyloidosis is an amyloidosis with a hereditary cerebral hemorrhage, a primary systemic amyloidosis, a secondary systemic amyloidosis, a serum amyloidosis, a senile systemic amyloidosis, a hemodialysis-related amyloidosis, a Finnish hereditary systemic amyloidosis, an Atrial amyloidosis, a Lysozyme systemic amyloidosis, an Insulin-related amyloidosis, or a Fibrinogen a-chain amyloidosis.
[00102] In yet another aspect, the invention provides a method for 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 a graft-versus-host disease (GVHD).
[00103] In a further aspect, the invention provides a method for treating an 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.
[00104] In a further aspect, the invention provides a method for treating Antiphospholipid Syndrome and/or its complications 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.
[00105] In yet another aspect, the invention provides a method for treating an anemia 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 anemia is a sickle cell anemia. In another embodiment, the anemia is athalassemia. In some embodiments, the methods of the invention can be used as anti-sickling or anti-adhesive sickle cell therapy. In some embodiments, the methods of the invention can be used for the treatment and/or prevention of aplastic, acute sequestration, hyper-haemolytic, vaso- occlusive crises and their consequences in patients with sickle cell anemia.
[00106] In another aspect, the invention provides a method for preventing or ameliorating one or more side effects associated with a chemotherapy or a 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 the chemotherapy are selected from the group consisting of body weight loss, bone marrow toxicity, catabolic changes in blood biochemistry, cardiotoxicity (e.g., myocardial necrosis), gastrointestinal toxicity, suppression of immunity, and neutropenia. In one embodiment, the chemotherapy comprises administration of one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule targeting agents, topoisomerase inhibitors, alkaloids, and targeted therapeutics. In one embodiment, the chemotherapy comprises administration of 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 the radiation therapy are selected from the group consisting of body weight loss, skin irritation, skin damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, and a second cancer. In one embodiment, the radiation therapy is external beam radiation therapy or systemic radioisotope therapy. In one embodiment, the rAAV vector or the vector composition is administered during a cycle of the chemotherapy or radiation therapy. In another embodiment, the rAAV vector or the vector composition is administered after a cycle of the chemotherapy or radiation therapy. In one embodiment, chemotherapy or radiotherapy is used for treatment of a cancer selected from the group consisting of a peritoneal carcinomatosis, a lymphoma, a stomach cancer, a colon cancer, an intestinal cancer, a colorectal cancer, a pancreatic cancer, a liver cancer, a cancer of the bile duct, a cancer of the gall bladder, a sarcoma, and a liver metastatic disease of any origin.
[00107] In one embodiment of any of the above methods of the invention, the administration of the rAAV vector or the vector composition results in the expression of the enzyme which has a DNase activity and its secretion into the hepatic porto-sinusoidal circulation of the subject.
[00108] In one embodiment of any of the above methods of the invention, the rAAV vector or the vector composition is administered in a dose and regimen which is sufficient to decrease the level of the cell free DNA (cfDNA) in the porto-sinusoidal circulation of said subject.
[00109] In one embodiment of any of the above methods of the invention, the subject is human.
[00110] In another aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific promoter.
[00111] In a further aspect, the invention provides a method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver cells when administered in vivo.
[00112] In yet another aspect, the invention provides a method for treating a disease or condition in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of cell free DNA (cfDNA) in the hepatic porto-sinusoidal circulation of the subject, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter. In one embodiment, the disease is selected from the group consisting of a cancer (including a liver metastatic disease), a neurodegenerative disease, an atherosclerosis, and a delayed-type hypersensitivity reaction.
[00113] In another aspect, the invention provides a method for treating a disease or condition in a subject in need thereof, wherein the disease or condition is accompanied by accumulation/elevated levels of cell free DNA (cfDNA) in the hepatic porto-sinusoidal circulation of the subject, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells. In one embodiment, the disease is selected from the group consisting of a cancer (including a liver metastatic disease), a neurodegenerative disease, an atherosclerosis, and a delayed-type hypersensitivity reaction.
[00114] In a further aspect, the invention provides a method for treating a cancer in a subj ect in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter. In one embodiment, the cancer originates in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein.
[00115] In yet another aspect, the invention provides a method for treating a cancer in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells. In one embodiment, the cancer originates in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein.
[00116] In a further aspect, the invention provides a method for treating a cancer in a subj ect in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver of said subject and wherein the cancer originates in and/or metastasizes to organs, tissues and/or structures that drain to the portal vein.
[00117] Non-limiting examples of cancers treatable by any of the above methods for treating cancer include, e.g., a peritoneal carcinomatosis, a lymphoma, a stomach cancer, a colon cancer, an intestinal cancer, a colorectal cancer, a pancreatic cancer, a liver cancer, a cancer of the bile duct, a cancer of the gall bladder, a sarcoma, and a liver metastatic disease of any origin.
[00118] In one embodiment of any of the above methods for treating cancer, the method is effective to inhibit metastasis. [00119] In yet another aspect, the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
[00120] In a further aspect, the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
[00121] In another aspect, the 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 encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
[00122] Non-limiting examples of neurodegenerative diseases treatable by any of the above methods for treating neurodegenerative diseases include, e.g., Alzheimer’s disease, Mild Cognitive Impairment (MCI), CADASIL syndrome, Parkinson’s disease, Amyotrophic Lateral Sclerosis, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), agyrophilic grain 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, an amyloidosis, asthma, or prion disease. In one embodiment, the amyloidosis is an amyloidosis with a hereditary cerebral hemorrhage, a primary systemic amyloidosis, a secondary systemic amyloidosis, a serum amyloidosis, a senile systemic amyloidosis, a hemodialysis-related amyloidosis, a Finnish hereditary systemic amyloidosis, an Atrial amyloidosis, a Lysozyme systemic amyloidosis, an Insulin-related amyloidosis, or a Fibrinogen a-chain amyloidosis.
[00123] In another aspect, the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter. In one embodiment, the delayed-type hypersensitivity reaction is a graft-versus-host disease (GVHD).
[00124] In yet another aspect, the 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 comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells. In one embodiment, the delayed-type hypersensitivity reaction is a graft-versus-host disease (GVHD).
[00125] In a further aspect, the invention provides a method for treating an atherosclerosis in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver- specific promoter.
[00126] In yet another aspect, the invention provides a method for treating an atherosclerosis in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
[00127] In another aspect, the invention provides a method for preventing or ameliorating one or more side effects associated with a chemotherapy or a radiation therapy in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific promoter.
[00128] In a further aspect, the invention provides a method for preventing or ameliorating one or more side effects associated with a chemotherapy or a radiation therapy in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
[00129] In one embodiment of any of the above methods for preventing or ameliorating one or more side effects associated with a chemotherapy, said one or more side effects of the chemotherapy are selected from the group consisting of body weight loss, bone marrow toxicity, catabolic changes in blood biochemistry, cardiotoxicity (e.g., myocardial necrosis), gastrointestinal toxicity, suppression of immunity, and neutropenia.
[00130] In one embodiment of any of the above methods for preventing or ameliorating one or more side effects associated with a chemotherapy, the chemotherapy comprises administration of one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule-targeting agents, topoisomerase inhibitors, alkaloids, and targeted therapeutics.
[00131] In one embodiment of any of the above methods for preventing or ameliorating one or more side effects associated with a chemotherapy, the chemotherapy comprises administration of one or more compounds selected from the group consisting of anthracycline, doxorubicin, 5- fluorouracil (5-FU), etoposide, taxane, and cyclophosphamide.
[00132] In one embodiment of any of the above methods for preventing or ameliorating one or more side effects associated with a radiation therapy, said one or more side effects of the radiation therapy are selected from the group consisting of body weight loss, skin irritation, skin damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, and a second cancer.
[00133] In one embodiment of any of the above methods for preventing or ameliorating one or more side effects associated with a radiation therapy, the radiation therapy is external beam radiation therapy or systemic radioisotope therapy.
[00134] In one embodiment of any of the above methods for preventing or ameliorating one or more side effects associated with a chemotherapy or a radiation therapy, the vector is administered during a cycle of the chemotherapy or radiation therapy.
[00135] In one embodiment of any of the above methods for preventing or ameliorating one or more side effects associated with a chemotherapy or a radiation therapy, the vector is administered after a cycle of the chemotherapy or radiation therapy.
[00136] In one embodiment of any of the methods of the invention, the vector further comprises one or more molecules capable of targeting of the nucleic acid to liver cells.
[00137] In one embodiment of any of the methods of the invention, the promoter is a liver- specific promoter. [00138] In one embodiment of any of the methods of the invention, the vector is packaged in a liposome or a nanoparticle.
[00139] In one embodiment of any of the methods of the invention, the vector is used as a naked DNA.
[00140] In one embodiment of any of the methods of the invention, the vector is a viral vector. Non-limiting examples of useful viral vectors include, e.g., adeno-associated virus vectors, adenovirus vectors, retrovirus vectors (e.g., lentivirus vectors), and hepatotropic virus vectors (e.g., hepatitis B virus (HBV) vectors).
[00141] In one embodiment of any of the methods of the invention, the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNaselL3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof. In one embodiment, the DNase is DNase I (e.g., human DNase I) or a mutant or derivative thereof. In one embodiment, the DNase I mutant comprises one or more mutations in an 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 combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence). In one embodiment, one of the mutations in the actin-binding site is a mutation at Ala-114. In one embodiment, the DNase I mutant comprises one or more mutations increasing DNase activity (e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof; positions indicated in relation to a mature protein sequence lacking secretory signal sequence). In one embodiment, the DNase I mutant comprises one or more mutations are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combinations thereof. In one embodiment, the DNase I mutant comprises the mutation Q9R. In one embodiment, the DNase I mutant comprises the mutation E13R. In one embodiment, the DNase I mutant comprises the mutation N74K. In one embodiment, the DNase I mutant comprises the mutation A114F.
[00142] In one embodiment, the DNase I mutant comprises 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, H44 A : Y65 A : 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 combinations thereof. In one embodiment, the DNase I mutant is a long acting form of DNase. In one embodiment, the DNase I comprises the sequence SEQ ID NO: 4. In one embodiment, the DNase I comprises the sequence SEQ ID NO: 1. In one embodiment, the DNase I mutant comprises the sequence SEQ ID NO: 5. In one embodiment, the DNase I mutant comprises the sequence SEQ ID NO: 2.
[00143] In one embodiment of any of the methods of the invention, the sequence encoding the enzyme which has a DNase activity comprises a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme into the hepatic porto- sinusoidal circulation upon administration of the vector to the subject. In one embodiment, the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, b-glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence. In one specific embodiment, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence
MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6). In one embodiment, the secretory signal sequence comprises 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 specific embodiment, the secretory signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one embodiment, the secretory signal sequence comprises the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). In one specific embodiment, the secretory signal sequence consists of the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
[00144] In one embodiment of any of the methods of the invention, the liver-specific promoter mediates a substantially increased expression of the enzyme in the liver as compared to other tissues and organs. Non-limiting examples of liver-specific promoters useful in the methods and vectors of the present invention include, e.g., an albumin promoter, an al -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a-fibrinogen promoter, a b-fibrinogen promoter, an alpha 1-antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, al-acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINAl) (hAAT) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a-fetoprotein (AFP) promoter. In one embodiment, the liver-specific promoter is an albumin promoter. In one specific embodiment, the albumin promoter comprises the sequence of SEQ ID NO: 8. In one embodiment, the albumin promoter consists of the sequence of SEQ ID NO: 8. See also Frain et ak, Mol. Cell Biol., 1990, 10(3):991-999.
[00145] In one specific embodiment, the liver-specific promoter is al -anti -trypsin (AAT) promoter. In one specific embodiment, the anti -trypsin promoter is a human al -anti-trypsin (AAT) promoter. In one specific embodiment, the AAT promoter comprises the sequence of SEQ ID NO: 15. In one specific embodiment, the AAT promoter comprises the sequence of SEQ ID NO: 15. In one specific embodiment, the AAT promoter consists of the sequence of SEQ ID NO: 15. In various embodiments of any of the methods of the invention, the vector comprises an enhancer. The enhancer may be a hepatic control region enhancer, such as HCR-1 or HCR-2 associated with the ApoE gene, e.g., the human ApoE gene. In one specific embodiment, the enhancer comprises the sequence of SEQ ID NO: 17. In one specific embodiment, the enhancer consists of the sequence of SEQ ID NO: 17.
[00146] In various embodiments of any of the methods of the invention, the vector comprises a Kozak sequence upstream of the DNase coding sequence. The Kozak sequence may have the sequence of 5'-GCCGCCACC-3' (SEQ ID NO: 33).
[00147] In various embodiments of any of the methods of the invention, the vector comprises a post-transcriptional regulatory element, e.g., the WPRE that does not encode a functional X protein. In one specific embodiment, the post-transcriptional regulatory element comprises the sequence of SEQ ID NO: 16. In one specific embodiment, the post-transcriptional regulatory element consists of the sequence of SEQ ID NO: 16.
[00148] In various embodiments of any of the methods of the invention, the vector comprises a sequence having 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% identity to the sequence of SEQ ID NO: 19. In one specific embodiment, the nucleic acid comprises the sequence SEQ ID NO: 19. In one specific embodiment, the nucleic acid consists of the sequence SEQ ID NO: 19. In one specific embodiment, the nucleic acid has a map shown in Figure 10.
[00149] In one embodiment of any of the methods of the invention, the administration of the vector results in the expression of the enzyme and its secretion into the hepatic porto-sinusoidal circulation of the subject.
[00150] In one embodiment of any of the methods of the invention, the vector is administered in a dose and regimen which is sufficient to decrease the level of the cell free DNA (cfDNA) in the porto-sinusoidal circulation of said subject.
[00151] In one embodiment of any of the methods of the invention, the subject is human.
[00152] In one embodiment of any of the methods of the invention, the method further comprises selecting a subject with an elevated level of cell-free DNA (cfDNA) in the bloodstream as compared to a level of cfDNA in the bloodstream of a normal healthy subject.
[00153] In one embodiment of any of the methods of the invention, the method further comprises administering a deoxyribonuclease (DNase) enzyme to the subject (e.g., DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II (including DNase Ila and DNase IIb), Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof). In one specific embodiment, the DNase is DNase I or a mutant or derivative thereof.
[00154] These and other aspects of the present invention will be apparent to those of ordinary skill in the art in the following description, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[00155] Figure 1 shows the organs and tissues drained by the hepatic portal vein. For example, the portal vein drains blood from the lower part of esophagus, stomach, pancreas, gall bladder, spleen, small intestine, large intestine, upper part of the bladder and liver.
[00156] Figures 2A-2I show patient summaries and individual blood electrophoretic data from nine patients with advanced metastatic disease who were treated with intravenous bovine DNase I. (A) rectal carcinoma, (B) lung carcinoma, (C) lung carcinoma, (D) melanoma, (E) breast cancer, (F) adenocarcinoma, (G) renal cancer, (H) rectal carcinoma, (I) breast cancer.
[00157] Figure 3 shows a gamma-immunoscintigraphic image of a tumor-bearing mouse obtained at Day 15 one hour following injection of 99mTc labeled anti-DNA antibodies, with substantially more radioactivity observed in the liver zone (b) in the middle/right than in the tumor zone (a) in the lower left.
[00158] Figure 4 shows IHC microphotographs of liver tissue from different groups listed in Table 3 and treated as described in Example 4. A large amount of cfDNA was seen in the liver of a mouse treated with gemcitabine (Group 7). Comparatively little cfDNA was seen in the liver of a mouse treated with DNase I mAFP/Alb AAV8, an AAV8 vector targeting a DNase I gene to liver cells for expression under an albumin promoter (Group 2).
[00159] Figure 5A shows a map of the mAFP/Alb AAV8 vector 4658_pAAV-AFP-Alb- huDNasel mut. The vector comprises (i) elements for propagation in bacteria, i.e., an AmpR marker, a colEl, FI and M13 origins of replication, and (ii) the hDNAsel mut sequence (SEQ ID NO: 2; hyperactive actin resistant DNase I mutant) operably linked to a mouse minimal albumin promoter, a mouse a-fetoprotein (AFP) enhancer II (with L-ITR), an hGH polyA signal, flanked by AAV8 L-ITR and R-ITR, an Ml 3 origin of replication and an FI origin of replication. Figure 5B shows a map of ADV-207186 vector based on human adenovirus type 5 (dEl/E3) with human DNase I expressed under the control of the CMV promoter.
[00160] Figure 6 shows bioluminescence data reflective of tumor size. The tumors of both mice with an orthotopic MIA PaCa-2 tumor implant and mice with a non-orthotopic MIA PaCa-2 tumor implant decreased in size when treated with hDNasel mut (SEQ ID NO: 5; hyperactive actin resistant DNase I mutant) mAFP/Alb AAV8.
[00161] Figure 7 shows liver histology data indicating that animals treated with DNase I mAFP/Alb AAV8 have much lower inflammatory cell density, no necrosis, and much less nucleiphilic material at the vascular interface when treated with DNase I mAFP/Alb AAV8 expression vector instead of DNase I protein. [00162] Figure 8 shows a significant reduction in the total numbers of donor-derived T cells, donor-derived CD4+ and CD8+ T cells in the peripheral lymph nodes, mesenteric lymph nodes and Peyer’s patches of DNase I mAFP/Alb AAV8-treated mice as compared with recombinant DNase I treated mice.
[00163] Figure 9 shows a restriction map of the lentiviral vector pLV2-IL2ss-DNaseI mut used to deliver DNase I in Example 7. The vector comprises an AmpR marker, and origins of replication for FI and SV40. The vector comprises an EF-la promoter upstream of an IL-2 leader sequence upstream of DNAse I.
[00164] Figure 10A shows the complete sequence (SEQ ID NO: 30) of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader-WPRE Xinact, or ApoE-HCR enhancenhAAT promoter>human DNAse I hyperactive variant with correct leader sequence (secretory sequence): Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE) that does not encode functional protein X. Figure 10B shows a vector map of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader-WPRE Xinact. Figure IOC shows the complete sequence (SEQ ID NO: 31) of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (wild-type)-WPRE Xinact. Figure 10D shows a vector map of the construct ApoEHCR enhancenhAAT promoter>hDNaseI (wild-type)-WPRE Xinact.
[00165] Figure 11 shows a map of the pAAV-MCS Promoterless expression vector used for the preparation of ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader- WPRE Xinact.
[00166] Figure 12 shows the results of SDS-PAGE analysis of purified Anc80 VR- 18013AD AAV vector. Three bands of 60, 72, and 90 kDa were observed in a ratio of about 1 : 1 : 10, which corresponds to the VP 1-3 proteins.
[00167] Figures 13A and 13B illustrate data where ApoEHCR enhancer-hAAT promoter- hDNasel (hyperactive)correct leader-WPRE Xinact and ApoEHCR enhancer-hAAT promoter- hDNasel (wild type)- WPRE Xinact vectors efficiently transduce cells of hepatocyte origin only and trigger high yield expression and secretion of biologically enhanced DNase I enzyme or wild type DNase I enzyme from transduced G2 cells.
[00168] Figure 14 shows the results of a Y Maze behavioral test.
[00169] Figure 15 shows the results of a contextual fear condition test.
[00170] Figure 16 shows that microglia activation was significantly reduced in VR- 18013 AD treated 3xTg-AD mice as compared with 3xTg-AD control mice, especially in cortical area of brain.
[00171] Figure 17 shows data indicating a significant reduction of microglia activation in the cortical brain area of VR-18013AD treated 3xTg-AD mice vs 3xTg-AD control mice.
[00172] Figure 18 shows data indicating that treatment with VR- 18013 AD significantly reduces amyloid deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice.
[00173] Figure 19 shows that treatment with VR- 18013 AD significantly reduces hyperphosphorylated Tau deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice.
[00174] Figure 20 shows data indicating a significant reduction of p-tau reduction in hippocampal region after VR- 18013 AD treated treatment.
[00175] Figure 21 shows a typical morphometric image of control (single IV injection of PBS) and VR-18014AD (single IV injection of VR-18014AD at 1.0 xlO11 GC/per mouse dose) treated mice.
[00176] Figures 22A-F show that AAV-mediated DNase I liver gene transfer reduces the growth of colorectal liver metastases. (A) A schematic showing experimental design in vivo. (B and C) The use of luciferase-labeled MC38 cells allowed weekly in vivo tracking of tumor growth with bioluminescence imaging. In the AAV-DNase I treated group, tumor growth was significantly inhibited at days 7, 14 and 21. n=7 mice/group. (D) Liver metastasis model using MC38 cells (l x lO6) injected through the portal vein, showing smaller tumors harvested 3 weeks post inoculation in AAV-DNase I treated mice compared with AAV-null control. (E and F) Graphs showing that AAV-DNase I significantly decreased surface tumor nodules on liver and lower ratio of liver weight/body weight compared with AAV-null group. n=8 mice/group. Data represent mean ± SD, *p< 0.05, **/K 0.01.
[00177] Figures 23A-E show that AAV-mediated DNase I liver gene transfer reduces neutrophil recruitment and NET formation in CRC liver metastasis. (A) Representative images of H&E staining of the liver section exhibit increased immune cells in mice with AAV-DNase I treatment compared with AAV-null treated group. Cell counts were averaged in the four fields. Magnification, x20; magnification, x40. HPF, high-power field. (B) Flow cytometry of tumor tissue showing neutrophils infiltration. n=4 mice/group. (C and D) The expression of CitFB by western blot analysis and quantification of serum MPO-DNA by ELISA were assessed at 21 days. n=3-4 mice/group (E) Immunofluorescent staining of tumor tissue shows NETs formation 21 days after tumor cell injection in AAV-DNase I and AAV-null treated mice. Magnification, x60. Nucleus, black; Ly6G, red; CitH3, dark grey, actin, light grey. All data represents at least three independent experiments. Data represent mean ± SD, *p< 0.05, **p< 0.01.
[00178] Figures 24A-D show that AAV-mediated DNase I liver gene transfer recruits CD8+ T cells to CRC liver metastasis. (A and B) The ratios of CD8+ and CD4+ T cells in livers. (C and D) Quantification of cellular percentages in tumor tissues. Data represent mean ± SD; n=4 mice/group, *p< 0.05.
PET ATT D DESCRIPTION OF THE INVENTION
[00179] The present invention is based on an unexpected discovery that during the course of diseases and conditions characterized by elevation of circulating cfDNA level (e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.) a significant quantity of cfDNA accumulates in the liver, mainly in the periendothelial space and space of Disse. Even high quantities of a systemically administered DNase protein show limited efficacy to digest liver cfDNA. This cfDNA is continuously sourced from liver depot to hepatic portal vein and further to systemic blood circulation. As demonstrated in the Examples section, below, transgenic expression of a DNase I in the liver (e.g., using viral expression vectors) leads to almost complete clearance of cfDNA accumulated in the liver and in the porto-sinusoidal circulation when DNase is secreted to sinusoidal space of the liver. Such transgenic expression of DNase in the liver leads to significant antitumor effects (especially in tumors in organs and tissues drained by the portal vein), reduction of toxicity of anticancer chemotherapy, a slowdown in autoimmune and neurodegenerative disease progression, etc. Such transgenic expression of DNase in the liver when DNase is secreted to sinusoidal space provides even more benefits when tumor is draining by portal vein system and cfDNA generated in the tumor growth area are subject to“first pass” through porto-sinusoidal circulation. (Figure 1 illustrates the organs and systems drained by the portal vein.) The same applies to neurodegenerative and autoimmune conditions facilitated by leaky gut with microbial DNA entering systemic blood circulation having respective “first pass” through porto-sinusoidal circulation.
[00180] The present invention is based on an unexpected discovery that during the course of diseases and conditions characterized by elevation of circulating cfDNA level (e.g., tumor growth and progression, autoimmune and neurodegenerative diseases, infections, etc.) a significant quantity of cfDNA accumulates in the liver, mainly in the periendothelial space and space of Disse. Even high quantities of a systemically administered DNase protein show limited efficacy to digest liver cfDNA. This cfDNA is continuously sourced from liver depot to hepatic portal vein and further to systemic blood circulation. As demonstrated in the Examples section, below, transgenic expression of a DNase I in the liver (e.g., using viral expression vectors) leads to almost complete clearance of cfDNA accumulated in the liver and in the porto-sinusoidal circulation when DNase is secreted to sinusoidal space of the liver. Such transgenic expression of DNase in the liver leads to significant antitumor effects (especially in tumors in organs and tissues drained by the portal vein), reduction of toxicity of anticancer chemotherapy, a slowdown in autoimmune and neurodegenerative disease progression, etc. Such transgenic expression of DNase in the liver when DNase is secreted to sinusoidal space provides even more benefits when tumor is draining by portal vein system and cfDNA generated in the tumor growth area are subject to“first pass” through porto-sinusoidal circulation. (Figure 1 illustrates the organs and systems drained by the portal vein.) The same applies to neurodegenerative and autoimmune conditions facilitated by leaky gut with microbial DNA entering systemic blood circulation having respective “first pass” through porto-sinusoidal circulation.
[00181] Besides liver, both intestinal sub-mucus gel layer zone and extracellular nervous tissue space comprise privileged compartments with limited penetration by vast majority of regular components of human blood which are isolated from blood circulation by so called intestinal and blood-brain barriers.
[00182] An additional aspect of the present invention is based on the realization that an efficient enzymatic cleavage of cfDNA in such privileged compartments benefits from provision of high local concentration of a DNase enzyme in addition to the liver-specific DNase expression (e.g., using viral expression vectors). In light of this, the present invention further provides DNase gene transfer vectors with liver tandem promoters designed to provide DNase expression in (i) the liver and (ii) the nervous system (e.g., CNS and/or ENS) and/or intestine.
[00183] The present invention provides various vectors for delivery of the DNase to the liver and/or nervous system and/or intestine. Specific non-limiting examples of such vectors include AAV vectors, adenovirus vectors, retrovirus vectors (e.g., lentivirus vectors), hepatotropic virus vectors (e.g., hepatitis B virus (HBV) vectors), nanoparticles (e.g., phosphoramidite nanoparticles), liposomes (e.g., cationic liposomes such as N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethyl ammonium chloride (DOTMA)), lactoferrin, poly L-lysine, polyethyleneimine, chitosan, etc.).
[00184] An enzyme which has a DNase activity may be expressed under the control of a liver-specific promoter and/or another liver-specific control element (e.g., enhancer). Specific non-limiting examples of liver-specific promoters and control elements include, e.g., an albumin promoter; human alpha- 1 anti -trypsin (hAAT) promoter; thyroxine binding globulin (TBG) promoter; apolipoprotein E hepatic control region promoter; apolipoprotein A-II (APOA2) promoter; serpin peptidase inhibitor, clade A, member 1 (SERPINA1) promoter; cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter; microRNA 122 (MIR122) promoter; liver-specific IGF-II promoter PI; murine transthyretin (MTTR) promoter; and the alpha- fetoprotein (AFP) promoter. The promoter may be upstream or downstream of an enhancer. The promoter sequence may be directly fused to the enhancer sequence.
[00185] In some embodiments, an enzyme which has a DNase activity may be expressed under the control of a liver-specific/nervous system-specific tandem promoter. The liver- specific/nervous system-specific tandem promoter may comprise a liver-specific promoter and a nervous system-specific promoter. The liver-specific promoter and the nervous system-specific promoter may be positioned in any order. For example, the liver-specific promoter may be positioned upstream of the nervous system-specific promoter. Alternatively, the nervous system- specific promoter may be positioned upstream of the liver-specific promoter. Specific non-limiting examples of nervous system-specific promoters and control elements include, microglia-specific promoters (e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, and Ibal promoters), myeloid- specific promoters (e.g., TTR, CDl lb, and c-fes promoters), neuron-specific promoters (e.g., CMV, NSE, synapsin [e.g., Synl, Synll], CamKII , a-CaMKIL and VGLUT1 promoters), and other neural and glial cell (e.g., oligodendrocytes astrocytes) type-specific promoters (e.g., glial fibrillary acidic protein [GFAP] promoter). In some embodiments, the nervous system-specific promoter is a central nervous system (CNS)-specific promoter. In other embodiments, the nervous system-specific promoter is the enteric nervous system (ENS)-specific promoter. In one specific embodiment, the nervous system-specific promoter comprises a human synapsin promoter. In one specific embodiment, the liver-specific/nervous system-specific tandem promoter comprises a hAAT promoter and a human synapsin promoter. The promoter may be upstream or downstream of an enhancer. The promoter sequence may be directly fused to an enhancer sequence. [00186] In some embodiments, an enzyme which has a DNase activity may be expressed under the control of a liver-specific/intestine-specific tandem promoter. The liver- specific/intestine tandem promoter may comprise a liver-specific promoter and an intestine- specific promoter. The liver-specific promoter and the intestine-specific promoter may be positioned in any order. For example, the liver-specific promoter may be positioned upstream of the intestine-specific promoter. Alternatively, the intestine-specific promoter may be positioned upstream of the liver-specific promoter. Specific non-limiting examples of intestine-specific promoters and control elements include, e.g., CB/CMV, GFAP, miCMV, CMV+I, tetO-CMV, b- aeti-CMV, MUC2, Villin, and T3b promoter. In one specific embodiment, the intestine-specific promoter comprises a human villin promoter. In one specific embodiment, the liver- specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter. The promoter may be upstream or downstream of an enhancer. The promoter sequence may be directly fused to an enhancer sequence.
[00187] In some embodiments, an enzyme which has a DNase activity may be expressed under the control of a liver-specific/ nervous system-specific/ intestine-specific tandem promoter. The liver-specific/nervous system-specific/intestine-specific tandem promoter may comprise a liver-specific promoter, a nervous system-specific promoter and an intestine-specific promoter. The three promoters may be positioned in any order. Specific non-limiting examples of useful nervous system-specific and intestine-specific promoters are provided in the two preceding paragraphs.
[00188] Specific non-limiting examples of enzymes which have a DNase activity that can be used in the compositions and methods of the invention include DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II (e.g., DNase Ila, DNase IIb), caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
[00189] If the enzyme which has a DNase activity is DNase I, various mutants weakening actin-binding may be used. Specific non-limiting examples of residues in wild-type recombinant human DNase I (SEQ ID NO: 4) that can be mutated include, e.g., 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 human DNase I hyperactive mutant comprising the sequence of SEQ ID NO: 5, the Ala-114 residue is mutated. Complementary residues in other DNases may also be mutated. Specific non-limiting examples of mutations in wild-type human recombinant 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, H44 A : Y65 A : E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T.
[00190] Various DNase mutants for increasing DNase activity may be used. Specific non limiting examples of mutations in wild-type human recombinant DNAse I include, e.g., 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 for increasing the activity of wild-type human recombinant DNase I include Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and A114F. For example, a combination of the Q9R, E13R, N74K and A114F mutations may be used, with such combination found at least in the hyperactive DNase I mutant comprising the sequence of SEQ ID NO: 5.
[00191] When AAV vectors are used for DNase expression, they can be derived from any serotype, e.g., from serotype 1 (AAV1), AAV2, AAV3 (e.g., AAV3A, AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO (as disclosed, e.g., in U.S. Patent No. 9,790,472, Int. Pat. Appl. Pub. Nos. WO2017/180857 and W02017/180861), AAVLK03, AAVLK06, AAVLK12 (as disclosed, e.g., in Wang et al., Mol. Ther., 2015, 23(12): 1877-1887), AAVhu37 (as disclosed, e.g., in Int. Pat. Appl. Pub. No. WO2017180857), AAVrh64Rl (as disclosed, e.g., in Int. Pat. Appl. Pub. No. WO2017180857), AAV-KP1 (as disclosed, e.g., in Int. Pat. Appl. Pub. No. W02019191701A1), or Anc80 (Zinn et al., Cell Rep., 2015, 12(67): 1056-1068).
[00192] Point mutations can be made to the capsid protein (e.g., VP3) to improve the efficiency and/or specificity of liver-specific delivery. Specific non-limiting examples of such point mutations in the AAV8 VP3 capsid protein include, e.g., S279A, S671A, K137R, and T252A, as well as AAV8 capsid mutations disclosed in Int. Pat. Appl. Pub. No. WO2017/180854 (e.g., AAV3G1, AAVT20 or AAVTR1, VP3 mutations in amino acids 263-267 [e.g., 263NGTSG267- >SGTH ("NGTSG" disclosed as SEQ ID NO: 43 and "SGTH" disclosed as SEQ ID NO: 44) or 263NGTSG267->SDTH ("NGTSG" disclosed as SEQ ID NO: 43 and "SDTH" disclosed as SEQ ID NO: 45)] and/or amino acids 457-459 [e.g., 457TAN459->SRP], and/or amino acids 455-459 [e.g., 455GGTAN459 ->DGSGL ("GGTAN" disclosed as SEQ ID NO: 46 and "DGSGL" disclosed as SEQ ID NO: 47)] and/or amino acids 583-597). Additionally, mutations may be made to the capsid protein (e.g., VP3) to improve the efficiency and/or specificity of nervous system- and/or intestine-specific delivery.
[00193] The vectors and compositions of the invention can be targeted to the liver, nervous system and/or intestine using various administration routes. Specific non-limiting examples of routes of administration to the liver include intrahepatic injection, intravenous injection, and intra arterial injection. Specific non-limiting examples of routes of administration to the nervous system include intravenous injection, intramuscular injection, intracranial injection, intraspinal injection, intracerebral delivery, oral administration, rectal administration, parenteral administration by nasogastric tube, administration by colonoscopy, and oral administration with or without enhancers to transport therapeutics across the blood-brain barrier (BBB). Specific non-limiting examples of routes of administration to the intestine include intravenous injection, intramuscular inection, rectal administration, parenteral administration by nasogastric tube, administration by colonoscopy, and oral administration.
Sequences
SEQ ID NO: 1 - human DNase I, wild-type (WT), precursor; Genbank Accession No.
NP 005214.2; the secretory signal sequence is underlined:
MRGMKLLGALLAL AALLOGAY SLKI AAFNIOTF GETKMSNATL V S YIV OIL SRYDIAL V QEVRD SHLT AV GKLLDNLNQD APDT YHYV V SEPLGRN S YKERYLF VYRPDQ V S AVD S Y YYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDV QEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYD RIVV AGMLLRGAVVPD S ALPFNF Q AAY GL SDQL AQ AISDHYP VEVMLK
SEQ ID NO: 2 - human DNAse I mutant, precursor; the mutated residues as compared to SEQ ID NO: 1 are in bold and underlined; the secretory signal sequence is underlined:
MRGMKLLGALLAL AALLOGAY SLKIAAFNIRTF GRTKMSNATLVS YIVOILSRYDIALV QEVRD SHLT AV GKLLDNLNQD APDT YHYV V SEPLGRKS YKERYLF VYRPDQ V S AVD S YYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDV QEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYD RIVV AGMLLRGAVVPD S ALPFNF Q AAY GL SDQL AQ AISDHYP VEVMLK
SEQ ID NO: 3 - Anc80 VP1 capsid protein:
AADGYLPDWLEDNLSEGIREWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGL DKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGR AVFQ AKKRVLEPLGL VEEGAKT APGKKRP VEQ SPQEPD S S SGIGKKGQQP AX^KRLNF G QTGDSESVPDPQPLGEPPAAPSGVGSNTMX^AGGGAPMADNNEGADGVGNASGNWHC D S TWLGDRVITT S TRT ALPT YNNHL YKQIS S Q S GX S TNDNT YF GY S TP W GYFDFNRFHC HF SPRDW QRLINNNW GFRPKX^LNFKLFNIQ VKE VTTNDGTTTIANNLTST V Q VFTD SE Y QLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN NFX^FSYTFEDVPFHSSYAHSQSLDRLNPLIDQYLYYLSRTQTTSGTAGNRX LQFSQAGP S SMANQ AKNWLPGPCYRQQRV SKTX^NQNNN SNF AWTGATKYHLNGRDSLVNPGP A MATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITX EEEIKTTNPVATEX^YGTV ATNLOSX10NTAPATGTVNSOGALPGMVWOX11RDVYLOGPIWAKIPHTDGHFHPSPLM GGF GLKHPPPQILIKNTP VP ANPPTTF SP AKF ASFITQ YSTGQ V S VEIEWELQKEN SKRWN PEIQ YT SNYNK S TNVDF A VDTN GV Y SEPRPIGTRYLTRNL
X1 = K or R; X2= A or S; X3 = A or G; X4 = R or K; X5 = E or Q; X6 = T or E; X7 = A or T; X8 = S or N; X9 = Q or E; X10 = S or A and X1 1 = N or D.
SEQ ID NO: 4 - mature wild-type (WT) human DNase I (without secretory signal sequence; Genbank Accession No. 4AWN_A:
LKI A AFNIQ TF GETKM SN ATL V S YI V QIL SRYDI AL V QE VRD SHLT A V GKLLDNLN QD AP DT YHYVV SEPLGRN S YKERYLF VYRPDQ V S AVD S YYYDDGCEPCGNDTFNREP AIVRFF SRF TEVREF AIVPLH A APGD A V AEID AL YD VYLD V QEK W GLED VMLMGDFN AGC S Y V RP SQW S SIRLWT SPTF QWLIPD S ADTT ATPTHC AYDRIVVAGMLLRGAVVPD S ALPFNF QAAYGLSDQLAQAISDHYPVEVMLK
SEQ ID NO: 5 - mature human DNAse I mutant (without secretory signal sequence); the mutated residues as compared to SEQ ID NO: 4 are in bold and underlined:
LKIAAFNIRTF GRTKMSNATL V S YIVQILSRYDIAL V QEVRD SHLT AV GKLLDNLNQD AP DTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFF SRF TEVREF AIVPLH AAPGD A V AEID AL YD VYLD V QEK W GLED VMLMGDFN AGC S Y V RP SQW S SIRLWT SPTF QWLIPD S ADTT ATPTHC AYDRIVVAGMLLRGAVVPD S ALPFNF QAAYGLSDQLAQAISDHYPVEVMLK
SEQ ID NO: 6 - secretory signal sequence of human DNase I:
MRGMKLLGALL AL AALLQGAV S
SEQ ID NO: 7 - secretory signal sequence of IL2:
M YRMQLL S Cl AL SL AL VTN S
SEQ ID NO: 8 - human albumin promoter:
ACTAGTTCCAGATGGTAAATATACACAAGGGATTTAGTCAAACAATTTTTTGGCAAG AAT ATT AT GAATTTTGT AATCGGTTGGC AGCC AATGAAAT AC AAAGAT GAGTCT AGT TAATAATCTACAATTATTGGTTAAAGAAGTATATTAGTGCTAATTTCCCTCCGTTTGT CCTAGCTTTTCTCTTCTGTCAACCCCACACGCCTTTGGCACC
SEQ ID NO: 9 - Anc80 VP1 capsid protein:
AADGYLPDWLEDNLSEGIREWDLKPGAPKPKANQQKQDDGRGLVLPGYYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRA VF Q AKKRVLEPLGL VEEGAKT APGKKRP VEQ SPQEPD S S SGIGKKGQQP AX^KRLNF GQ TGDSESVPDPQPLGEPPAAPSGVGSNTMX^AGGGAPADNNEGADGVGNASGNWHCDST WLGDRVITTSTRTALPTYNNHLYKQISSQSGX STNDNTYFGYSTPWGYFDFNRFHCHFS PRD W QRLINNNW GFRPKX^LNFKLFNIQ VKE VTTNDGTTTI ANNLT S TV Q VF TD SE Y QLP YVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX
^FSYTFEDVPFHSSYAHSQSLDRLNPLIDQYLYYLSRTQTTSGTAGNRX LQFSQAGPSSA NQAKNWLPGPCYRQQRVSKTX¾QNNNSNFAWTGATKYHLNGRDSLVNPGPAMATH KDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVITXfEEEIKTTNPVATEX^YGTVATNLQS X10NTAPATGTVNSOGALPGVWOX11RDVYLOGPIWAKIPHTDGHFHPSPLMGGFGLKHP PPQILIKNTP VP ANPPTTF SP AKF ASFIT Q YSTGQ V S VEIEELQKEN SKRWNPEIQ YT SNYN K S TNVDF A VDTN GV Y SEPRPIGTRYLTRNL
X1 = K or R; X2= A or S; X3 = A or G; X4 = R or K; X5 = E or Q; X6 = T or E;
X7 = A or T; X8 = S or N; X9 = Q or E; X10 = S or A and X1 1 = N or D
SEQ ID NO: 10 - human beta globin primer:
CAACTTCATCCACGTTCACC
SEQ ID NO: 11 - forward NLRP3 primer:
GTTCTGAGCTCCAACCATTCT
SEQ ID NO: 12 - reverse NLRP3 primer:
CACTGTGGGTCCTTCATCTTT
SEQ ID NO: 13 - forward 16S universal bacterial RNA gene primer:
T C GT C GGC AGC GT C AG AT GT GT AT A AG AG AC AGC C T AC GGGN GGC W GC AG
SEQ ID NO: 14 - reverse 16S universal bacterial RNA gene primer:
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC
SEQ ID NO: 15 - human anti-trypsin promoter sequence:
GATCTT GCT ACC AGT GGAAC AGCC ACT AAGGATTCTGC AGT GAGAGC AGAGGGCC A
GCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGAC
ACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGG
AAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATC
CCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTA
ATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACG
AGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAA
T
SEQ ID NO: 16 - Woodchuck hepatitis virus post-transcriptional regulatory element that does not encode functional protein X:
AGTGGCGGCCGCTCGAGCTAGCGGCCGCTCTAGAAGATAATCAACCTCTGGATTAC
AAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTG
GATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTC
TCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCA
GGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCA
TTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCAC
GGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGG
CACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCC
TGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCA
ATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCT TCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGAC
TAG
SEQ ID NO: 17 - apolipoprotein E (ApoE) enhancer, hepatic control region (HCR):
AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAG
TTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTT
CAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACAC
ACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGG
GCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAG
AGGTT GT C CTGGCGT GGTTT AGGT AGT GT G AGAGG
SEQ ID NO: 18 - a polynucleotide encoding human DNase I hyperactive variant of SEQ ID NO: 5:
ATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTC
CCTGAAGATCGCAGCCTTCAACATCAGGACATTTGGGAGGACCAAGATGTCCAATG
CCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCC
AGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAAT
CAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAAGAG
CTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAG
CTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGC
CATTCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCC
CCTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCT
ACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTC
AATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACA
AGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACG
CACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTT
CCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGTGACCAACTG
GC C C A AGC CAT C AGT G AC C AC T ATC C AGT GG AGGT GAT GC T G A AGT G A
SEQ ID NO: 19 - a polynucleotide encoding human DNAse I mutant precursor of SEQ ID NO: 2 (secretory signal sequence underlined):
ATGAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGG
GGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCAGGACATTTGGGAGGACCAAGA
TGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCG
CCCTGGTCCAGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGAC
AACCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGG
ACGGAAGAGCTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTG
CGGTGGACAGCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTC
AACCGAGAGCCATTCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTT
GCCATTGTTCCCCTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTC
TATGACGTCTACCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATG
GGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGC
CTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCT
ACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGG
CGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGT
GACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGTG A
SEQ ID NO: 20 - a polynucleotide encoding the secretory signal sequence (SEQ ID NO: 6) of human DNase I:
ATGAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGG
GGCCGTGTCC
SEQ ID NO: 21 - a polynucleotide encoding the mature human DNAse I mutant (without secretory signal sequence) of SEQ ID NO: 5:
CTGAAGATCGCAGCCTTCAACATCAGGACATTTGGGAGGACCAAGATGTCCAATGC
CACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCA
GGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATC
AGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAAGAGC
TATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGC
TACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCC
ATTCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCC
CTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTA
CCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCA
ATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAA
GCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGC
ACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTC
CCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGTGACCAACTGG
CC C A AGC CAT C AGT GACC ACT AT C C AGT GGAGGT GAT GC T GA AGT GA
SEQ ID NO: 22 - a polynucleotide encoding human DNase I, wild-type (WT), precursor of SEQ ID NO: 1:
ATGAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGG
GGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGA
TGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCG
CCCTGGTCCAGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGAC
AACCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGG
ACGGAACAGCTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGC
GGTGGACAGCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCA
ACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTG
CCATTGTTCCCCTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCT
AT GAC GT C T AC CTGGAT GT C C A AG AG A A AT GGGGCTTGG AGGAC GT CAT GTT GAT G
GGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGC
CTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCT
ACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGG
CGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGT
GACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAG
SEQ ID NO: 23 - a polynucleotide encoding the mature wild-type (WT) human DNase I of SEQ ID NO: 4:
CTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGATGTCCAATGC
CACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCA GGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATC
AGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAACAGC
TATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGC
TACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCC
AGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCC
CTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTA
CCTGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCA
ATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAA
GCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGC
ACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTC
CCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGTGACCAACTGG
CC C A AGC CAT C AGT GACC ACT AT C C AGT GGAGGT GAT GC T GA AG
SEQ ID NO: 24 - Mus musculus wild type DNase I, precursor; Genbank Accession No.
NP 034191.3; the secretory signal sequence is underlined:
MRYTGLMGTLLTLVNLLOLAGTLRIAAFNIRTFGETKMSNATLSVYFVKILSRYDIAVIO EVRD SHL VAV GKLLDELNRDKPDTYRYVV SEPLGRKS YKEQ YLF VYRPDQ V SILD S Y Q YDDGCEPCGNDTFSREPAIVKFFSPYTEVQEFAIVPLHAAPTEAVSEIDALYDVYLDVWQ KW GLEDIMFMGDFNAGC SYVT S SQW S SIRLRT SPIF Q WLIPD S ADTT VT STHC AYDRIVV AGALLQ AAVVPN S AVPFDF Q AEY GLSNQL AEAISDHYPVEVTLRKI
SEQ ID NO: 25 - secretory signal sequence of Mus musculus wild type DNase I
MRYT GLMGTLLTL VNLLOL AGT
SEQ ID NO: 26 - mature wild-type (WT) Mus musculus wild type DNase I
LRIAAFNIRTF GETKMSNATL S VYF VKIL SRYDIAVIQEVRD SHL VA V GKLLDELNRDKP DT YRYVV SEPLGRKS YKEQ YLF VYRPDQ V SILD S Y Q YDDGCEPCGNDTF SREP AIVKFF S P YTEV QEF AIVPLHAAPTE AV SEID AL YD VYLD VW QKW GLEDIMFMGDFNAGC S YVTS SQW S SIRLRT SPIF QWLIPD S ADTT VT STHC AYDRIVVAGALLQ AAVVPN S AVPFDF Q AE Y GL SN QL AE AISDH YP VE VTLRKI
SEQ ID NO: 27 - a polynucleotide encoding the secretory signal sequence of Mus musculus wild type DNase I
ATGCGGTACACAGGGCTAATGGGAACACTGCTCACCTTGGTCAACCTGCTGCAGCTG
GCTGGGACT
SEQ ID NO: 28 - a polynucleotide encoding the mature wild-type (WT) Mus musculus wild type DNase I
CTGAGAATTGCAGCCTTCAACATTCGGACTTTTGGGGAGACTAAGATGTCCAATGCT
ACCCTCTCTGTATACTTTGTGAAAATCCTGAGTCGCTATGACATCGCTGTTATCCAAG
AGGTCAGAGACTCCCACCTGGTTGCTGTTGGGAAGCTCCTGGATGAACTCAATCGGG
ACAAACCTGACACCTACCGCTATGTAGTCAGTGAGCCGCTGGGCCGCAAAAGCTAC
AAGGAACAGTACCTTTTTGTGTACAGGCCTGACCAGGTGTCTATTCTGGACAGCTAT
CAATATGATGATGGCTGTGAACCCTGTGGAAATGACACCTTCAGCAGAGAGCCAGC
CATTGTTAAGTTCTTTTCCCCATACACTGAGGTCCAAGAATTTGCGATCGTGCCCTTG
CATGCAGCCCCAACAGAAGCTGTGAGTGAGATCGACGCCCTCTACGATGTTTACCTA GATGTCTGGCAAAAGTGGGGCCTGGAGGACATCATGTTCATGGGAGACTTCAATGC
TGGCTGCAGCTACGTCACTTCCTCCCAGTGGTCCTCCATTCGCCTTCGGACAAGCCC
CATCTTCCAGTGGCTGATCCCTGACAGTGCGGACACCACAGTCACATCAACACACTG
TGCTTATGACAGGATTGTGGTTGCTGGAGCTCTGCTCCAGGCTGCTGTTGTTCCCAAC
TCGGCTGTTCCTTTTGATTTCCAAGCAGAATACGGACTTTCCAACCAGCTGGCTGAA
GCCATCAGTGACCATTACCCAGTGGAGGTGACACTCAGAAAAATCTGA
SEQ ID NO: 29 - a polynucleotide encoding the Mus musculus wild type DNase I, precursor
ATGCGGTACACAGGGCTAATGGGAACACTGCTCACCTTGGTCAACCTGCTGCAGCTG
GCTGGGACTCTGAGAATTGCAGCCTTCAACATTCGGACTTTTGGGGAGACTAAGATG
TCCAATGCTACCCTCTCTGTATACTTTGTGAAAATCCTGAGTCGCTATGACATCGCTG
TTATCCAAGAGGTCAGAGACTCCCACCTGGTTGCTGTTGGGAAGCTCCTGGATGAAC
TCAATCGGGACAAACCTGACACCTACCGCTATGTAGTCAGTGAGCCGCTGGGCCGC
AAAAGCTACAAGGAACAGTACCTTTTTGTGTACAGGCCTGACCAGGTGTCTATTCTG
GAC AGC T AT C A AT AT GAT GAT GGC T GT GA AC CC T GT GGA A AT G AC AC CTTC AGC AG
AGAGCCAGCCATTGTTAAGTTCTTTTCCCCATACACTGAGGTCCAAGAATTTGCGAT
CGTGCCCTTGCATGCAGCCCCAACAGAAGCTGTGAGTGAGATCGACGCCCTCTACG
ATGTTTACCTAGATGTCTGGCAAAAGTGGGGCCTGGAGGACATCATGTTCATGGGAG
ACTTCAATGCTGGCTGCAGCTACGTCACTTCCTCCCAGTGGTCCTCCATTCGCCTTCG
GACAAGCCCCATCTTCCAGTGGCTGATCCCTGACAGTGCGGACACCACAGTCACATC
AACACACTGTGCTTATGACAGGATTGTGGTTGCTGGAGCTCTGCTCCAGGCTGCTGT
TGTTCCCAACTCGGCTGTTCCTTTTGATTTCCAAGCAGAATACGGACTTTCCAACCAG
CTGGCTGAAGCCATCAGTGACCATTACCCAGTGGAGGTGACACTCAGAAAAATCTG
A
SEQ ID NO: 30 - Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader- WPRE Xinact (VR-18013AD)
AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAG
TTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTT
CAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACAC
ACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGG
GCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAG
AGGTT GTCCTGGCGT GGTTT AGGT AGTGT GAGAGGGATCTT GCT ACC AGT GGAAC AG
CCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAG
ACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAG
CCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAA
AGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCT
GTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCC
GTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCA
GCTTCAGGCACCACCACTGACCTGGGACAGTGAATGCCGCCACCATGAGGGGCATG
AAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCCCT
GAAGATCGCAGCCTTCAACATCAGGACATTTGGGAGGACCAAGATGTCCAATGCCA
CCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGG
AGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATCAG
GATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAAGAGCTA TAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTA
CTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCCAT
TCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCT
GCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACC
TGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAAT
GCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGC
CCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCAC
TGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCC
GACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGTGACCAACTGGCC
C A AGC CAT C AGT GAC C AC T ATCC AGT GGAGGT GAT GC T G A AGT GA AGT GGC GGC CG
CTCGAGCTAGCGGCCGCTCTAGAAGATAATCAACCTCTGGATTACAAAATTTGTGAA
AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTT
TAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTAT
AAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC
GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACC
TGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCA
TCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATT
CCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCAC
CTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGA
CCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGC
CCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGACTAG
[00194] (APOE HCR enhancer: bases 1-320; human alpha- 1 -antitrypsin promoter: bases 321-717; Kozak sequence: bases 718-726; human DNasel hyperactive variant with natural full correct leader sequence: bases 727-1575; WPRE X protein inactivated: bases 1576-2212)
SEQ ID NO: 31 - Complete sequence of ApoEHCR enhancer-hAAT promoter-hDNasel wild type-WPRE Xinact (VR-18014AD)
AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAG
TTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTT
CAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACAC
ACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGG
GCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAG
AGGTT GTCCTGGCGT GGTTT AGGT AGTGT GAGAGGGATCTT GCT ACC AGT GGAAC AG
CCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAG
ACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAG
CCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAA
AGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCT
GTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCC
GTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCA
GCTTCAGGCACCACCACTGACCTGGGACAGTGAATGCCGCCACCATGAGGGGCATG
AAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGGGGCCGTGTCCCT
GAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGATGTCCAATGCCA
CCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCGCCCTGGTCCAGG
AGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGACAACCTCAATCAG
GATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGGACGGAACAGCTA TAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGCGGTGGACAGCTA
CTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCAACCGAGAGCCAG
CCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTGCCATTGTTCCCCT
GCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCTATGACGTCTACC
TGGATGTCCAAGAGAAATGGGGCTTGGAGGACGTCATGTTGATGGGCGACTTCAAT
GCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGCCTGTGGACAAGC
CCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCTACACCCACGCAC
TGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGGCGCCGTTGTTCCC
GACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGTGACCAACTGGCC
C A AGC CAT C AGT GAC C AC T ATCC AGT GGAGGT GAT GC T G A AGT GA AGT GGC GGC CG
CTCGAGCTAGCGGCCGCTCTAGAAGATAATCAACCTCTGGATTACAAAATTTGTGAA
AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTT
TAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTAT
AAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC
GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACC
TGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCA
TCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATT
CCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCAC
CTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGA
CCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGC
CCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGACTAG
[00195] (APOE HCR enhancer: bases 1-320; human alpha- 1 -antitrypsin promoter: bases 321-717; Kozak sequence: bases 718-726; human DNasel wild type with natural full correct leader sequence: bases 727-1575; WPRE X protein inactivated: bases 1576-2212)
SEQ ID NO: 32 - a polynucleotide encoding human DNase I, wild-type (WT), precursor of SEQ ID NO: 1 with stop codon:
ATGAGGGGCATGAAGCTGCTGGGGGCGCTGCTGGCACTGGCGGCCCTACTGCAGGG
GGCCGTGTCCCTGAAGATCGCAGCCTTCAACATCCAGACATTTGGGGAGACCAAGA
TGTCCAATGCCACCCTCGTCAGCTACATTGTGCAGATCCTGAGCCGCTATGACATCG
CCCTGGTCCAGGAGGTCAGAGACAGCCACCTGACTGCCGTGGGGAAGCTGCTGGAC
AACCTCAATCAGGATGCACCAGACACCTATCACTACGTGGTCAGTGAGCCACTGGG
ACGGAACAGCTATAAGGAGCGCTACCTGTTCGTGTACAGGCCTGACCAGGTGTCTGC
GGTGGACAGCTACTACTACGATGATGGCTGCGAGCCCTGCGGGAACGACACCTTCA
ACCGAGAGCCAGCCATTGTCAGGTTCTTCTCCCGGTTCACAGAGGTCAGGGAGTTTG
CCATTGTTCCCCTGCATGCGGCCCCGGGGGACGCAGTAGCCGAGATCGACGCTCTCT
AT GAC GT C T AC CTGGAT GT C C A AG AG A A AT GGGGCTTGG AGGAC GT CAT GTT GAT G
GGCGACTTCAATGCGGGCTGCAGCTATGTGAGACCCTCCCAGTGGTCATCCATCCGC
CTGTGGACAAGCCCCACCTTCCAGTGGCTGATCCCCGACAGCGCTGACACCACAGCT
ACACCCACGCACTGTGCCTATGACAGGATCGTGGTTGCAGGGATGCTGCTCCGAGG
CGCCGTTGTTCCCGACTCGGCTCTTCCCTTTAACTTCCAGGCTGCCTATGGCCTGAGT
GACCAACTGGCCCAAGCCATCAGTGACCACTATCCAGTGGAGGTGATGCTGAAGTG
A
SEQ ID NO: 33 - Kozak sequence 5'-GCCGCC ACC-3'
SEQ ID NO: 34 - Anc80L65 VP1 capsid protein
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPF NGLDKGEPVNAADAAALEHDKAYDQQLK AGDNP YLRYNHAD AEF QERLQEDTSF GGN LGRAVFQ AKKRVLEPLGLVEEGAKT APGKKRP VEQ SPQEPD S S SGIGKKGQQP ARKRLN FGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWH CD S T WLGDRVITT S TRTW ALPT YNNHL YKQIS S Q S GGS TNDNT YF GY S TP W GYFDFNRF HCHF SPRDW QRLINNNW GFRPKKLNFKLFNIQ VKE VTTNDGTTTIANNLT S T VQ VF TD S E Y QLP YVLGS AHQ GCLPPFP AD VFMIPQ Y GYLTLNN GS Q A V GRS SF Y CLEYFP S QMLRT GNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQ AGP S SMANQ AKNWLPGPC YRQQRV SKTTNQNNN SNF AWTGATK YHLNGRD SLVNPGP AMATHKDDEDKFFPMS GVLIF GKQGAGN SNVDLDNVMITNEEEIKTTNP VATEEY GT V ATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGG FGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEI Q YT SNYNK S TNVDF A VDTN GV Y SEPRPIGTRYLTRNL
SEQ ID NO: 35 - variant Anc80L65 VP1 capsid protein
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYYLGPFN GLDKGEP VNAAD AAALEHDK AYDQQLK AGDNP YLRYNHAD AEF QERLQEDT SF GGNL GRA VF Q AKKRVLEPLGLVEEGAKT APGKKRP VEQ SPQEPD S S SGIGKKGQQP ARKRLNF GQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPADNNEGADGVGNASGNWHCD S T WLGDRVITT S TRT W ALPT YNNFtL YKQIS S Q S GGS TNDNT YF GY S TP W GYFDFNRFHC HF SPRDW QRLINNNW GFRPKKLNFKLFNIQ VKE VTTNDGTTTIANNLTSTV Q VFTDSEY QLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN NFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGP S SANQ AKNWLPGPC YRQQRV SKTTNQNNN SNF AWT GATK YHLNGRD SLVNPGP AM A THKDDEDKFFPMSGVLIF GKQGAGN SNVDLDNVITNEEEIKTTNP VATEEY GT VATNLQ SANT AP AT GTVNSQGALPGVW QDRD VYLQGPIWAKIPHTDGHFHP SPLMGGF GLKHPP PQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEELQKENSKRWNPEIQYTSNYNK STNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO: 36 - human synapsin promoter
AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGA
CCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC
T AT C AGAGAGGGGGAGGGGA A AC AGGAT GC GGCGAGGC GCGT GC GC AC T GC C AGC
TTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCT
CAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGG
CCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGC
GCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCG
CTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG
SEQ ID NO: 37 - mouse villin promoter cgaagtcagcctagtctgcaaagctagttccaggatggcaagggctacacagagaaaccttgtctcataaaaccaaagtagtagtagtagta gtaatgccatagagaaaattggagtccattcaggatggaccatcctataagatgattctcttgacccaggtaagctaatgtcatggggaaagg ggatgggactgtcctagattaaaaagtgctgaggcgatgcctattctcgatttgattccatatgaaaaggctgataaggcccaagagaagtgg aactgggactctggactgaagacgtgacggccttataaacactggcacttataaacacttataaacactggcacaggcgttcaggtttgaag atcactttcaaaccacagaacagaaagtgctcgctcgtcctcagcgtagcgagcactggctgcagaagagtgatatttagtgaaagctacctt cacaatatctttgcacttatcacatacacgtgtcaaatgtgctaactccctagtccacagatggctgttacactcgtttctgctttcccatctggttg acatttgtcagaaccagaaattagaaatgtgggtatttatttgtgtgctgaggacaccatccagggcttttcacatttcaggcacatggtttacta actgggctacttctccaacggtttgaaaccatttgttttatatttacttattttgtgtgcatgaggtaggcatgtatacgtatgtataggagtcatgca tgtggctgctaccctcaaaatcattgcagatccccagcaagtgaagtcaccgagcgttgtaagttgttatgtgggactgggagccaaggctg ggttctctgcaagagcagccagtggccttaaccatgggaccagctctctaggcctaaggtaatctttagttttttaaaaatatatattctcagccg ggtgtggtggcacacgcctttaatcccagcacttgagaggctgaggtgtaggaattatacacacaggccagctggggtgcagagcttggcc ctgttttttttgttttttctttatgtgcactggtgtcttacctgcgtgtatgtccgtgcaagggtgtcagatcccttggagctggagttaaagacagtt gtgatcacgctgccgttacagatgctggaaattgaacccaggtgtccctagagaagcagccagtgctcttaacttctgagccacccctccaa ccctgcttttagagactcttaaccttttgtgtaatgtgggaactgagtggatcttgcacttaccaagtgtgtgctgcgctgtagcatcactgagcc cgtacccacacgactagtggatacagtttaagggcaaacacttaacaatgacaatagttggatagagtttgaatatagtcctgagctattggtt agcgtgacctttgctgtccttagcatgtgctgtgagaagatagaaaaatgaagacttgagtctagtcctggaacccacagaggcaggcgag aacccactcctgaaagttgttctctgagcttcacatacaacttcacataatagttacaatgataataataattagtaaattcttttagaaggtatatgt tgggagggagagatggctcagcttccaggagcacttgctgctcttgcagaggacctagattcagttcccaggactcatatggtggctcacag ccatctgtaaatccagttccagagggttccacaccctcttctggcctccacaggcaccacatacatagtacacagacatacatgcaggcaaa acacccatacacacataaataaataaggaaacttaaaaggtgcatgtgttggtaaacattgtgcttacacatgctggttgaagacatgtacaac gcacacactgaagagggatctggggctggagagatggctcagcggttaagagcactgactgctcttccgaaggaaggtcctgagttcaaa tcctagcaaccacatggtggctcacaaccatccataatgagatctgacaccctcttctggtgcatctgaagacagctgcagagctacagtgta cttagatatactaataaataaatctttttttaaaaaaatgaagagggatctgagacacctcaaaagagattatgagcagtgactcacgggtgatt atctatcctggagtttttcctttccgcttggcttgcaactgggtggacagacgccccttttcattcacaagaacgggtgctacattatttctgaaca aaacagcacctgcagtatgtttactgtccttgctgactatgagcacgcgcacgcgcgcgcgcacacacacacacacacacacacacacac acacacacacacacacacattcagtctccagagctcttgggaaggtcaagaagaggctgccctcaaacacgatcttcatctttccctcctaaa ggagaccacgattccaaggtggcagaagatctacagggggcagaggcagggaggggnaagcaggccatggtttccagagacctacag cagagggcagcaaggcagatccccaggtccagggcagggaggtggaggcccttgttccgaggagaaggcaggcggcagaacagggt tcaaaggcacaggtttatggcagctcataaaagtggaggtcgtggctcactcagaaaggaggaagaagggaaaggcccttgtgcccactg agcgagggtcatgctgagtaggagagatctgcaggggtgccaggagccccacctgtctgtcccaagggaaccccaagtgtgaactctgg ccttgggtgctgagttccagctacaagaccccaggagtcctactccatccccatccagtgccccctcgccccgccacaccccacccccgac tcccgtgccacttctctagggctggagggtggccagccctggtgggggttgcctacctgcaggtagagcccaggtcctagccggaagtgc accccatccctgaagctgcagagccaagggcggggcacacggcagctcaggctgtcaggctgttgctgggctctaggttcccagggacc tgggcacctacttccccacccccccatccattctctctggggccctatcttcccttatatggtgaaggaagttcctggggggggggtggtggt gaggacaaaggtcgttcggtctcctgcagccagcttgccacaacttcctaagatctcccaggtggtggctgcctcttccagacaggtaaggc aattgggtggggacacatggtgaccacaggtggttggaggggacagggtccttgcttctctctggcagcctgtgctttctgtagcaccttggt ataagtttgggggtgaggtaaggtgctctgaaactctgaaagaagcaagaagccagcaggctgtcttgggccttcaatgaaggaagttcac agaccccctttcctgtaagtcaccttcgcttcatctgtgtagattccctgggaccaaggtggctcctgggactcagatttctacaattaaaatca ggacagtcctgagacttggactccgtgcctgtatttactacttctctctggctgctcatttctgtgttcatgtcttacacatctgaaatggtttctttgt gtcaccattcccctgacactcctgggaggtcgtatccttggcacatgtatcctgggatgtaagctgcagccaccaggagagagggggaga gtcaggagctgtgtcctaggccctattaggcctggacatcacccctttcctagaaatggcccctccatttttcggttaccatgatctattttatatc agagtgggcagtgaaagccaaacctgcccagaagtttgggactcactcagaccaaggttatctgctcagaaatccccctgtcacttgaggtt gggagaatctgcctctgggggcttccaggtcttggttagcaggagggtatcctttgtatagggcatgacctagtctatggtgttactacattcct gtccagttaaaagctggaactaaaacccacggcagcgcccaggattctctacagttgtaccccaagaacaacaagacagtagatatgcaag gataggtagctggggagaagaagaacttaaacccccccaaaggcccacaggttccgttccctagttcacaatgccagtatgagtgctagct actatgggctgtgagttggtagctacaagcatgagtgatgttcatgtgtgtagtgtgtataatctgagcacttgggaggctgaagcaggagga ttgctatatgtttgaggccagcctgagctatagagcgagactttgtctttaagaaaaaaatgaaagcccagcagtggtggcacacgcctttaat cccagcacttgggaggcagaagcaggcagatttctgagttcaaggccagcctggtctatagagtgagttccaggacagccagggctacac agagaaaccctgttttgaaaaaccagaaaaacaaaacaaaacaaaacaaaacaaaacccaaacccaaacccaaacctctcatctctcatct ctctaggctgtgtctgtctaggtggtagagtttggggacttcagacttatatataaataggcctttttatcactggtcagagacgagaaaggtttc agtctgggacacagtgggaccctgagaaagtactccttgccagcccaaaaattctgggaaggcttcctggaggaagtgtgtcccgattgag tctggaagaatatgaaggaagtgggaaagccaaggcagaagcagcctgggtgtaaaggcagcagtcagactactgttctagaaggcaga agagagggttggaagaatgttggtggacagacagttggaacagaaggacaggagggggaggcatccaagattctgaacatgtagctgac ttttggttctctgggtgacaagtgtcccccagggatagggctgtagaaaggggaccaggggtgagccaatgagttcaagttgagggacaca tccagcccagggtccttgctggcaagctaaagaatgagagccctctaaccctccctgaagtttaggggagacaggagagctgaggagatc cttctagggtgaaggagaggtatctgctctgaccaacatggctaggagcagaagcagttggaccagttacccctcagaaccagccatcccc tcttggctctaaggaggctgggcccctttctgtttaagaatcttacttttcttcagagagaggcagcaagcctttgtcccctccctgttggtcaata aacacccctgtgtgtaacattagtttattttactgtcagtttgctccaggacagtccatctggtagacctctgctcctaactcaccaaggtatggc ccacattcctcacccagaagagtgcagaagagagccttagagaaagggtaacagtaacaaagatggccagaataaaacaaaaactactat cctttgtacccaaattggttttgctgaaccaggagggggtgtgtgagtgtatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg tgtgtgtcttgggggacttttcatgctaaagaatatctgatattggcgcccatgccaacaggggtattggggagagtcaggcttctgcaaacac agtaagctgcccaagatggattggtggcctgaatcaccaaggggcaggctgatcagagtggacagaacatcacaagataagccaccctgt ggggctcagaagagggagtttacaagaggtaaaggccaagccatttattatccaagacatgactcaaaatcaaagtgcaaggagagattag ctggagagatggggctgtcagtgtgggacacctgaccttgcacttattagtcactaggccaaggagcagtcacagagggtgactgggtcct actcagcttggagcaggcacgtggagaatgggtgacctccatcctgatggagagggctgagcaccaccaggtacaagtgttccctgtgtct catgccaggattcctggccagttttcaaaggactaaggactcatctctggtggaaacaaagtatccaagccctaagccccattttggtctaatt aaatcagaacccctggggatgcaggctctgagcagcaggagctttttaaaaagctcccaggtgattctgatcagcagctggaacaaacaca gctacaggttcaaacagaaagaggcaaagctagggaaagcttgggatggggagccttcttccaggccagtagatggaggctggttagca gtggtggcagcttctctctgcctgtcatatagctatccatccactcatccatccatacacccacccatccatttatgcacccatccttccatccatc catctatccagctacccacccacgcatccatccaaaccttccttttctccttctttctttcttttttccttcactcattcatttatccaacagagaactg gtattgtactaaatgtgggagatttaattaatttttagaagctctgttgattgactgattgtgcatgtatgtggacaggtacataccacagcacacg tgtggcaatcggagaaaggttttgggtgttgttttctcttcccaccgtgtgggttctggggattgaactcaaattatcgggctggtggcaagtgt ctttaccaccgagccattttgctgacacatcattattattagaaagcatcttatgtagtccaggctggcctcaagcttgctatgtcgccacggatg acctttaactcctgctcttccagcctccacccgagtgctaggtttacaggtgttcaacaccacacccaatttaatgctcaggattttgtactgagc aggcagaaatctgggcaacagctgggcatggtggtgaatgcctttaatcccagcactctgtggggggggggggggaggcggatccctga gttggaggccagtttggtctacagagtttcaggatacctggggctatacagggaaaccctatcccaaacaaacaaacaaacaaacaaaaaa tattctgtgcaataatcacagagattagaggatattagtagggtagtagggctggtgagggagagtcatgctttcttttgtattataatagtaaag tactcacaagatgcattatctatctatctatctatctatctatctatctatctatctatctatctatctacctacctacctacctatccatccatccatcta tcgtatagcccaggctgctttgactctgaatgctcctatttctgggtcaactcttcacccctagtgttgggtttaccaacacccagacatttatttta ttttgttttattttattaatctaggagctcagggtgggactcagggtcttgtgcatgctaagcaagctctctgccacagagctgcagctccagtcc ccattttgttcaggtgactctgtgacagttgtcatattcgcagcgctatgtagctctctccacctcccagttccagcactttctggtcatcccagtg ggcgggcaactctgtgctcaccagtgccctgttccctgtcttcagacctacatatttgcctgtctgaacagttcatgtaaatgggatgcgttcct gtgtattcttttatggctggcccctttatcttagcacagtttgtgttgggccatgtgtcactgctatactctatcttatcatcatcttatggcttaatagt gttcctttgtgtggataaaccactttctgtttcatttactgatggaaatttgtggccccacccccaccctttttttttttatttgagacaaggtctttctgt gtaatcttgcaatcttggctgtcctgagctcactctgtagaccaggctgtgaggctgtccttccacttttgacactcctgtgaacagagtagcca tgaacttcaaagacaattttctgttttggtttgttttttacatttgtgtgtgtatgcgtgtatatgtgcatgtttgtgtcttcaggtgctcacatgtgtgta cctgtgtgtgggacagagaacaaaccgatgtgccattcctcagatactacgcatcttgttaatatgtatgtattatgtatgtttatttagtgtgccc aagtatgcaggtattttgttggagttttcaccttcccttgtgggctctccgcattaaactcagctcctcgggctagtgagcaatgccttcactcgat gagccatctcgctgcccctgctgccacctcctccttatttcccagatgggactacgcactgcactggcctaaagctcaccaagtcatccagag tggctagccagggagactcagggatatgctggcctctgcctccacagtgctagaattacaggcatacatcactgctggaagatttttaacctg aatcctgaggatagagcaggcactctaccaatggagggttctttttgtgtttggtttggtttcctctgcataagatcaggcagtctgaaatagtgt agcctgggctacataacatcttgtctcaaaaagcctatagaggtagggaggtcgaggctaaagaagagccttaagccggctgtgatagcac acaggatagcctgcactatatagcaagaccttgtttcaaaaacatggagggaggggtatgttttaagtgctgggctgtgtaacaggcactaag ggagccaatgtagacatttgactaagaaaggatcatcatcaaagccgggtgggcagggtagaggttggactacagtggtcaagaccccca taggaagccagtttcccttcttcctctgggcctcaagcctggctcgacggccactgctctcacatgccttctcctctaggctcgtccaccccc
SEQ ID NO: 38 - CMV promoter
gcggccgctctagagagcttggcccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttg acattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatg gcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattga cgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacg gtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagttt gttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggag gtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctccggt
SEQ ID NO: 39 - F4/80 promoter
cctctgtctgtctgtctgtctctttggtgtgatgtatgtggtgtgtgtgtattctacaaggttgacatgatgacagaatttaattttcttagcagcaag ctcatggatcctggtgataaatgcagcatgactttactgaaaaggctttgtgatcttgaagagtggattgacttcactgtcggcagcacatgca atctcacttgtttggtgtaatgaaagaagagaatgagaggtggaagggggatggtaatgttgaaaaaaagaatggtacagaggaaactgag gttggagagagatggggtagatggtaagagatggagaaagagggaaggaaatggagagaaagacagagagacagagagagacacac agagagacacacagagacagagaggaagggaaagggaaagagaaaggaagaggaagagggggaggggaaggggaaggggaag gggaagggagagggagaaatgtggacactagccagatttaagggagaaattagggggttgccagtctgtccacctctgatggtggcaact cagcagaaagctgctgggctcagtctggctttgttgagcaaccctgactccaccccttttcttccccacaaagcaagcttttaaagggaaggc tttcttcattgaatgactgccacagtacg
SEQ ID NO: 40 - TMEM119 promoter
gggggtgagcgagggctgctgggaccattgcagggaacaatgataatctaggcttggttcctacccagagagcacgcactcatccttcatg cactcccctgttccaaaccctcactggctccgtactgcctccgaccttccgagactttagcctggctcctgtcaacatctctgacccttactaca tgatcctctctttggtccatgctccagcctaatctaattgcggtggcttgtgcgtggtggcattcccagccaccatacctttacccacgctggtcc ttccatgcggaatgcctttccagggcctgctttgcccgcttctgctcatacacaggcatgccctccaggatggcttcctacctctttcccttggg ggattgatctctctgtcttggggttctcggagcccttgacctgacccctttctgtttggcaaaaaagtaatttacctcggtgtccttctccctggta gtctgtgagctccccaaggctgggctgtgcctgattcacctctggaacttgcttagcacagtgcgtggcctgctgcaggtgttcattgagcact tgccgaatgaatgcatgaatgaatgaatgaatgaatgaatgcaaggggctgctaatccacaggactcctcaggtcagccagacgtcccggt tccaaggcctgccactgactcacctcaggaccctgcttgaaccattagaactcaccctgcctcactttccccctctgtgaaatggggctccaa ctcctattcaagctactatcatttgggggcattgtgaggccacagatcccagaacatcagagtcagaggtagcccagaaagcttcccaccca tccctacaaatgggaaactgaggtctggagagggaagggcagagttgggctccctgtctcaggctcggacccaccatcaggcctgtctcta aaacgaatcccagctcccacgctgcaccctgagcctggaagcctgagccacacaaggacggggaattttccttcccacttccagaggcct ctgaacctccctgagcttgtcccctttggagggtattgggcagcagcgtgggcagaaccccagctcactgtctgggggagcgctgcagga cagccttgtctgtctgtctcagcctgccctggggacccgaggtcagggaggaagtgccgcatctggtcttccccagagcgagagtgtgagc aagggtgggattgcgtgtggcccgagagtagcccctcccctccccctgtccccaccccaaaccctcttaatgaaatcaagctggccctgcg gcccagccggggagggaggaaggaggagggacgggaggagggacgggaggagggagggcgggcaggcgccagcccagagca gccccgggcaccagcacggactctctcttccagcccaggtgccccccactctcgctccattcggcgggagcacccagtcctgtacgccaa ggaactggtgagtcctggggtcccctcct
SEQ ID NO: 41 - MEF2 promoter
acatgcaggcactgtggccccgaaaaactcttcagaggtgagcggctttcaggatgagagggccaacaatggctcatctcctaaagacctc gataaacataatcagtagccccagaaaacacaacagctgtcccaagcctatcccctttgcacctatctcagaaaaggcaacatgcagagtg gtcaggagttcaagtcctcagggccctactctgaagctgtgaccctgacaggtcactttagcgctcagcttccctttcctcatctgtaaaatgg gaatagaaactgaatctgcttcatggggccttgtaagtattacataagctgtcatatgcaaaatgtccatcacatagttcaccgccccaggcat cctagaaactattaagtcacgataaggaggttgctactgtcctgggacctggggagctcggctgtccagtccgaaagggccttctcgccctc gagaccccttcaagtccctttctctccgggcccgggtcccagagtccccttgtgcttgctcctgagggtgacctgcccgtgccccggggcg agcacaggtgcggccctggccacaatgcgggcgtggcgagccgacacccggggaggccaagggcgcctcgagggagggagggag gaggctgacccgggcgtcccgaaggacccgcccggccccggactccgggcggcagccggcctcgcgccccgcccccgccccggga gccccgcccagtcccgtcgcgccgcgccgccccaccgcccgggttggctgccctggaggccacgcgcggcgatttgccacgcgccgc gtcaccgggccgctcccggcctgggctcccgggggctggtcagggaggtggcggcggctgagcggcggagcgggggcggcagggc gcgacgccgccgggccgccccgccctgggaggcgcccgggccctcatcaagtgaccagtatcccttccaggggaacacggtccttcag aggaaagcgagctccaacccgcggccccggcgccaagccgccgtcatcttcttcctgtgccaggtgatcgtctcctcacccacccggaaa aacatagtccgcccccacgtccttgtggaatagcgcccgcttccaagcaccgtgactctctttgccctacccttgtctttcccattccaattactc tagaacccaccgagaggataattcagtcctgaaaagaaactgatggggagaagcgaggaagggaacccaggagggaggggaggcag ggctgcggagggacaccgaggcggcggaggtagtgcgcacgcgcagcacagaacgagttccggtctggccgaggcttgtctcctaaaa atagccccggtgtggggatccgtgcgcggatgtcccggcgagtcccgggctgaaagaggcggctccgggcggcgcgaagcgctggtg gcgggcccgggctgcggcgtgtgcgcgcccgccagctgctccggagatacggtgagggccgcgggcagcggggctcagtccgcgac
SEQ ID NO: 42 - FoxP2 promoter
agtgcagcctcaggggttgaaggtgaaatgtgaaatttgctgtaattacttacccaaaatgtctgtattaaaggctattagaagcagttattttaa gtacttctttatacatctttccctcttctgtttagtatttaaatgtgcaacaaaagtagtggcagatgttactcatctgaagaaactaaaacaagata agtcagtggcaggagtggtggccggttaaagatctcttcgaagtgctttgttcagtaactgttgttttgtctcacattaagttgtaggtggggtgc tgttaaagcttgtccactcacgatggtgttcagctgagacctgtgagttcaagttgtcttgcccagagttaaaggccataaaactaagacagtgt cgttcctgtcttgggtgtaactcctaaaaaactagtcagttcttgcaataggaggaaaattattgcaaataaacctttgatttaccagcttcagtaa gttgttcattctgtctctgtagctgtagtttttaccttaacagtttgaggctaccatttctcttcctttcatgcttatggggtccacacaaaccctgcta ggctacctacagtataaaatcttaaaactccatgtggtgttgtgagctggtagcagatggactttcttttgcatctgctgtgtacagacttggcttt gtactcttttcctgggggagagaaagtctttcttctctgctagttgattcctgtttttgtgaaacagctaagaaaacatctgtggcaggcagaaaa attgacaaaggattgacaaacaccattagctgaaaattttccatttcattgcaactatgcattgtttactttactgtaaatatcttaatacatcatcctc tgaatatgctggcagagaagctggagaactgtgatttcaattaaggttagtaattgatggtatctagtgttcaaaggctgaagcttgattaacat ctgcttggacaaattgtcattgtgaagtggttttgatgtgcattgaagattatttctttctggcttatctgatgttgtggctgtgaaatgcttgtcttgg gtttgcttatttttgtaaactacttcctttggctgtaaattgcagagcactggagctttaccaaaagtgcagtgtataatggtaagcttgtcctaata agggaagcaagaagtgtatttatcacagacatgaaagctaaccgaggacttgagagactcaaactggtgcttttgtctctctctctctgtctttct ctctctcacacacacactcacacactcacacacatgcacacacacacatacacacacacaaaaatgaagcacttactttagaaagattatggt aagcatgctggctcagtcttgaacctttgtcacccctcacgttgcacaccaaagacataccctagtgattaaatgctgattttgtgtacgattgtc cacggacgccaaaacaatcacagagctgcttgatttgttttaattaccagcacaaaatgccatcagtctgggacgtgatcgggcagaggtgt
Definitions
[00196] The term“an enzyme which has a deoxyribonuclease (DNase) activity” is used herein to refer to an enzyme capable of hydrolytic cleavage of phosphodiester linkages in the DNA backbone.
[00197] As used herein, the terms“deoxyribonuclease” and“DNase” are used to refer to any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. A wide variety of deoxyribonucleases is known and can be used in the methods of the present invention. Non-limiting examples of DNases useful in the methods of the present invention include, e.g., DNase I (e.g., recombinant human DNase I (rhDNase I) or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, and DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, and acetylcholinesterase. Also encompassed by the present invention are DNase enzymes which have an extended half-life (e.g., albumin and/or Fc fusions, or protected from binding to actin by modification of actin binding-site; see, e.g., 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 of recombinant human DNase I (SEQ ID NO: 4). For example, in human DNase I hyperactive variant comprising the sequence of SEQ ID NO: 5, the Ala-114 residue is mutated. Exemplary 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, A114C, 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 (in the sequence of SEQ ID NO: 4). Also encompassed are mutations in DNase I with increased DNase I activity. Non-limiting examples of such mutations are, e.g., Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and Al 14Fof recombinant human DNase I (SEQ ID NO: 4). For example, a combination of the Q9R, E13R, N74K and A114F mutations is found in the hyperactive DNase I comprising the sequence of SEQ ID NO: 5. DNase I cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminaied polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides. DNase I acts on single-stranded DNA, double-stranded DNA, and chromatin.
[00198] The term“about” means within an acceptable error range for the particular value as determined by one of ordinary skill 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” can mean within an acceptable standard deviation, per the practice in the art. Alternatively,“about” can mean a range of up to ±20%, up to ±10%, up to ±5%, and up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term“about” is implicit and in this context means within an acceptable error range for the particular value.
[00199] The terms “extracellular DNA”, “cell-free DNA” and “cfDNA” are used interchangeably to refer to extracellular DNA (e.g., of eukaryotic, viral, archaeal, prokaryotic intracellular or extracellular parasites origin), including DNA in extracellular vesicles (e.g., exosomes and microvesicles), found in any bodily fluid and tissue. The cfDNA relevant for the present invention can be found in blood, lymph, liver, nervous tissues, cerebrospinal fluid (CSF), and/or intestine, including DNA in extracellular vesicles (e.g., exosomes and microvesicles) found in these boldily fluids and tissues.
[00200] The terms“treat” or“treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
[00201] 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, etc.) and experimental animal models of diseases (e.g., mice, rats).
[00202] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. 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 employed, the mode of administration, and the like.
[00203] As used herein the term“therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered (e.g., a combination of DNase and another compound) the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
[00204] 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 with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent 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 sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A“tissue specific” promoter may be preferentially active in specific types of tissues or cells.
[00205] The term“liver-specific expression” as used herein refers to a predominant or exclusive expression in the liver, i.e., expression to a substantially greater extent than in other tissues and organs.
[00206] The term“liver-specific promoter” is used herein to refer to a promoter which is predominantly or exclusively active in a liver cell (e.g., hepatocyte) and directs/initiates transcription in the liver to a substantially greater extent than in other tissues and organs. In this context, the term“predominantly” means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (such as 100% of said promoter expression) occurs in liver cells. The ratio of liver expression 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 (e.g., hepatocytes, Kupffer cells, endothelial cells, etc.). Some liver-specific promoters useful in the expression cassettes of the invention include at least one, typically several, hepatic nuclear factor binding sites. Liver- specific promoters useful in the expression cassettes of the invention can be constitutive or inducible promoters. Some non-limiting examples of hepatic promoters useful in the expression cassettes of the invention include: albumin promoter (Alb), human alpha-1 anti-trypsin (hAAT) promoter, thyroxine binding globulin (TBG), Apolipoprotein E hepatic control region promoter, Apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3 A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter PI, murine transthyretin (MTTR) promoter, alpha-fetoprotein (AFP) promoter, lecithin-cholesterol acyl transferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, and mouse prealbumin gene promoter.
[00207] Non-limiting examples of liver-specific promoters include, e.g., albumin promoter (Alb), human alpha-1 anti-trypsin (hAAT) promoter, thyroxine binding globulin (TBG) promoter, Apolipoprotein E hepatic control region promoter, Apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINAl) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3A4) promoter, microRNA 122 (miR-122) promoter, Liver- specific IGF-II promoter PI, murine transthyretin (MTTR) promoter, the alpha-fetoprotein (AFP) promoter, a thyroid hormone-binding globulin promoter, an alcohol dehydrogenase promoter, the factor VIII (FVIII) promoter, a HBV basic core promoter (BCP) and PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, an Hepatic Control Region (HCR)- ApoCII hybrid promoter, an AAT promoter combined with the mouse albumin gene enhancer (Ealb) element, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, the transferrin promoter, a transthyretin promoter, an alpha- fibrinogen and beta-fibrinogen promoters, an alpha 1-antichymotrypsin promoter, an alpha 2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, promoters of the complement proteins (e.g., Clq, Clr, C2, C3, C4, C5, C6, C8, C9, complement Factor I, and Factor H), C3 complement activator and the [alpha] 1 -acid glycoprotein promoter. Additional tissue-specific promoters may be found in the Tissue-Specific Promoter Database, TiProD (Nucleic Acids Research, J4:D104-D107 (2006).
[00208] The term“nervous system-specific promoter” is used herein to refer to a promoter which is predominantly or exclusively active in a nervous system cell and directs/initiates transcription in the nervous system (e.g., central nervous system (CNS), including brain, and/or enteric nervous system (ENS)) to a substantially greater extent than in other tissues and organs. In this context, the term“predominantly” means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (such as 100% of said promoter expression) occurs in cells of the nervous system. The ratio of nervous system expression to non- nervous system expression can vary between different nervous system-specific promoters. In some embodiments, a nervous system-specific promoter may preferentially direct/initiate transcription in a particular CNS and/or ENS cell type (e.g., neurons, glial cells [e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, satellite cells], enteric neurons, intrinsic primary afferent neurons, interneurons, motor neurons, etc.). Nervous system- specific promoters useful in the expression cassettes of the invention can be constitutive or inducible promoters. Some non-limiting examples of nervous system promoters useful in the expression cassettes of the invention include microglia-specific promoters (e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, and Ibal promoters), myeloid-specific promoters (e.g., TTR, CD1 lb, and c-fes promoters), neuron-specific promoters (e.g., CMV, NSE, synapsin [Synl, Synll], CamKII , a-CaMKII, and VGLUT1 promoters), and other neural and glial cell (e.g., oligodendrocytes, astrocytes) type-specific promoters (e.g., glial fibrillary acidic protein [GFAP] promoter).
[00209] The term“intestine-specific promoter” is used herein to refer to a promoter which is predominantly or exclusively active in an intestinal cell and directs/initiates transcription in the intestine to a substantially greater extent than in other tissues and organs. In this context, the term “predominantly” means that at least 50% of said promoter-driven expression, more typically at least 90% of said promoter-driven expression (such as 100% of said promoter expression) occurs in intestine cells. The ratio of intestine expression to non- intestine expression can vary between different intestine-specific promoters. In some embodiments, an intestine-specific promoter may preferentially direct/initiate transcription in a particular intestinal cell type (e.g., enterocytes, goblet cells, enteroendocrine cells, etc.). Intestine-specific promoters useful in the expression cassettes of the invention can be constitutive or inducible promoters. Some non-limiting examples of intestine promoters useful in the expression cassettes of the invention include CB/CMV, GFAP, mi CMV, CMV+I, tetO-CMV, b-acti-CMV, MUC2, Villin, and T3b promoters.
[00210] In some embodiments, e.g., when tissue targeting is mediated by a viral capsid protein, a nucleic acid encoding an enzyme which has a DNase activity can be operably linked to a promoter that allows for efficient systemic expression (e.g., CMV promoter, chicken b-actin promoter (CBA), or EFla promoter).
[00211] The term“portal vein cancer” is used herein to refer to a cancer (e.g., carcinoma, sarcoma, or lymphoma) that has originated in, or metastasized to, an organ or tissue that drains to the portal vein. Non-limiting examples of such organs and tissues are shown in Figure 1 and include, e.g., liver, colon, small and large intestine, stomach, spleen, pancreas, and gall bladder.
[00212] The term “neurodegeneration” is used herein to refer to a separate clinical pathological condition with progressive loss of structure and/or function of neurons, including death of neurons. The neurodegeneration can be primary or secondary. Non-limiting examples of diseases involving primary neurodegeneration include, e.g., Alzheimer's disease (AD), Mild Cognitive Impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion-caused diseases, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism- 17 (FTDP- 17), Lewy body dementia, vascular dementias, Amyotrophic Later Sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), agyrophilic grain disease (AGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel-Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, progressive familial myoclonic epilepsy, striatonigral degeneration, torsion dystonia (e.g., torsion spasm; dystonia musculorum deformans), spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration (e.g., Friedreich's ataxia and related disorders), Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot- Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa), and hereditary optic atrophy (Leber's disease). Secondary neurodegeneration is caused primarily by necrosis. Non-limiting examples of conditions, which may result in secondary neurodegeneration, include destruction of neurons by neoplasm, edema, hemorrhage, stroke, trauma, immune attack, hypoxia, poisoning, metabolic defects, and infections.
[00213] 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 produce untoward 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 regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. [00214] The terms“cytostatic and/or cytotoxic chemotherapy” and“chemotherapy” are used interchangeably herein to refer to a therapy involving administering of a cytostatic and/or cytotoxic agent.
[00215] The terms“anti-cancer agent” and“anti-cancer chemotherapeutic agent” are used herein to refer to any chemical compound, which is used to treat cancer. Anti-cancer chemotherapeutic agents are well known in the art (see, e.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12: 1202-1263 (1990)). Specific examples of chemotherapeutic agents are provided throughout the specification.
[00216] The term“side effect of a chemotherapy” as used herein refers to an undesirable and unintended, although not necessarily unexpected, result of a chemotherapy.
[00217] 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 a cancer treatment to damage the DNA of malignant cells, either directly or by creating charged particles within the affected cells that damage the DNA. Commonly used types of radiation therapy encompassed by the present invention include, e.g., external beam radiation therapy (EBRT or XRT), brachytherapy/sealed source radiation therapy, and systemic radioisotope therapy/unsealed source radiotherapy.
[00218] The terms“side effect of a radiotherapy” or“side effect of a radiation therapy” as used herein refer to an undesirable and unintended, although not necessarily unexpected, result of a radiation therapy. Which side effects develop depend on the area of the body being treated, the dose given per day, the total dose given, the patient's general medical condition, and other treatments given at the same time, and may include, e.g., skin irritation or damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, or a second cancer.
[00219] The term“catabolic state” as used herein refers to a condition characterized by a rapid weight loss and loss of fat and skeletal muscle mass, which may occur in a background of chemotherapy or chemoradiation therapy. Associated clinical events include, for example, immunosuppression, muscle weakness, predisposition to pulmonary embolism, thrombophlebitis, and altered stress response.
[00220] As used herein, the terms“viral vector” and“viral construct” refer to a recombinant viral construct that comprises one or more heterologous nucleotide sequences (e.g., a nucleotide sequence encoding an enzyme which has a DNase activity). In some embodiments, the viral vector is replication deficient. In some embodiments, viral structural and non-structural coding sequences are not present in the viral vector and are provided during viral vector production in trans by a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell line. Depending on the virus, a viral vector can be packaged within a capsid (e.g., an AAV vector) and/or a lipid envelope (e.g., a lentiviral vector).
[00221] The term “polyadenylation signal”, as used herein, relates to a nucleic acid sequence that mediates the attachment of a polyadenine stretch to the 3 ' terminus of the mRNA. Non-limiting examples of polyadenylation signals which can be used on the expression constructs of the invention include, e.g., the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 Elb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like.
[00222] As used in this specification and the appended claims, the singular forms“a,”“an,” and“the” include plural references unless the context clearly dictates otherwise.
[00223] In accordance with the present invention there may be employed conventional pharmacology and molecular biology techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein“Sambrook el al, 1989”); DMA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription and Translation (B.D. Hames & S.J. 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.
DNase Enzyme Targeting to Liver
[00224] The inventors have unexpectedly found that targeting expression of an enzyme which has a DNase activity to the liver can bring about an enhanced clearance of disease-associated cfDNA from the entire circulation, and especially from the porto-sinusoidal circulation. As demonstrated in the Examples section, below, cfDNA can accumulate in the liver, particularly in the periendothelial space and the space of Disse. Degradation of such reservoir of cfDNA via localized expression of an enzyme which has DNase activity can lower the overall level of cfDNA in the circulation.
[00225] As discussed throughout the application, an enzyme which has DNase activity can be efficiently expressed in liver cells through the use of viral vectors (e.g., adeno-associated viral (AAV) vectors, retroviral vectors (e.g., lentiviral vectors), or adenoviral vectors), liposomes, nanoparticles carrying a DNase transgene, or naked DNA. An adult human liver receives nearly one fourth of the cardiac output of blood, filters approximately 1 L of blood per minute, and accounts for 10-15% of the blood volume at any given moment. Such active circulation allows for rapid accumulation of high levels of vector particles within the liver. Vector particles can pass through the fenestrated endothelium along hepatic sinusoids directly to the hepatocytes. Such hepatocytes can be transfected by the vector particles and go on to produce DNase. The produced DNase is secreted into the periendothelial space and the space of Disse, where it can efficiently degrade the concentrated pool of cfDNA.
[00226] The inventors have found that transfection of liver cells to express an enzyme which has DNase activity provides a surprisingly greater reduction in cfDNA, along with substantial and surprising improvement in diseases and conditions associated with high levels of cfDNA in the bloodstream.
[00227] In some embodiments, expression of DNase enzyme in the liver is accomplished by use of nucleic acid expression cassettes that are predominantly expressed in the liver. In some embodiments, such expression cassettes can comprise one or more of the following elements: (a) a hepatic locus control element; (b) a hepatic promoter located 3' to the hepatic locus control element; (c) a coding sequence located 3' to the hepatic promoter, said coding sequence encoding a polypeptide, e.g. a deoxyribonuclease; (d) a polyadenylation signal located 3' to the coding sequence; and (e) an intron located 3' to the hepatic promoter and 5' to the polyadenylation signal. The elements (a), (b), (c), (d) and (e) can be operably linked to express the polypeptide encoded by the coding sequence. In some embodiments, the expression cassettes of the invention direct expression of a therapeutic amount of a polypeptide in liver cells for a period of at least 100 days (such as at least 200 days, at least 300 days, at least 400 days, or at least 500 days). In some embodiments, the polypeptide is a DNase. The DNase may be any DNase described herein, e.g., DNase I.
[00228] The polyadenylation signal may be operably linked to the nucleic acid encoding DNase. Non-limiting examples of polyadenylation signals include, e.g., the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 Elb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like.
[00229] In another aspect, the invention provides vectors that comprise a nucleic acid expression cassette that is predominantly expressed in the mammalian liver. Such expression cassette can comprise, e.g. : (a) a hepatic locus control element; (b) an hepatic promoter located 3' to the hepatic locus control element; (c) a coding sequence located 3' to the hepatic promoter, said coding sequence encoding a polypeptide, e.g. a deoxyribonuclease (DNase); (d) a polyadenylation signal located 3' to the coding sequence; and (e) an intron located 3' to the hepatic promoter and 5' to the polyadenylation signal. Elements (a), (b), (c), (d) and (e) are operably linked to express the polypeptide encoded by the coding sequence.
[00230] Some vectors of the invention are episomal vectors, and some vectors of the inventions are integrating vectors such as integrating viral vectors. In some embodiments, the polypeptide is a deoxyribonuclease (DNase). The DNase may be any DNase described herein, e.g., DNase I.
[00231] In another aspect, the present invention provides methods for treating a disease or condition associated with increased levels of cfDNA. The disease or condition may be any disease or condition described herein or in any of U.S. Patent Nos. 7,612,032; 8,388,951; 8,431,123; 8,535,663; 8,710,012; 8,796,004; 8,871,200; 8,916, 151; 9,072,733; 9,248, 166; 9,770,492; U.S. Pat.
Appl. Pub. Nos. US20170056482, US20170100463, US20150110769, and Int. Appl. Pub. No. W02016/190780, all of which are incorporated by reference herein in their entireties. In one embodiment, the method comprises the steps of: (1) introducing into the liver of a subject a vector comprising a nucleic acid expression cassette, said expression cassette comprising: (a) a liver- specific promoter; (b) a coding sequence located 3' to the liver-specific promoter, said coding sequence encoding an enzyme which has a DNase activity (e.g., any DNAs described herein); (c) a polyadenylation signal located 3' to the coding sequence; and optionally (d) an intron located 3' to the hepatic promoter and 5' to 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 invention, a therapeutic amount of the polypeptide is expressed for at least 100 days (such as at least 200 days, at least 300 days, at least 400 days, and at least 500 days).
[00232] The nucleic acid expression vectors and cassettes of the invention which comprise just the liver-specific promoter are predominantly expressed in the liver, e.g., at least 50% of the expression of the encoded polypeptide occurs within the liver. More typically, at least 90% of the expression occurs within the liver. Some of such expression vectors and cassettes of this aspect of the invention are exclusively expressed within the liver. The nucleic acid expression vectors and cassettes of the invention which comprise a liver-specific/nervous system-specific tandem promoter or a liver-specific/intestine-specific tandem promoter or a liver-specific/nervous system- specific/intestine-specific tandem promoter are expressed both (i) in the liver and (ii) in the nervous system and/or intestine.
[00233] Hepatic, nervous system or intestinal locus control elements may be incorporated into the expression cassettes of the invention that are capable of enhancing the expression of nucleic acid molecules in liver cells, nervous system cells or intestinal cells and confer copy number dependent, position independent expression on the expression cassette. Some hepatic locus control regions useful in the expression cassettes of the invention include, e.g., a matrix attachment region and/or a liver-specific enhancer element.
[00234] In addition to a promoter, an expression cassette and/or a vector may contain one or more additional transcription initiation, termination, and/or enhancer sequences; RNA processing signals (such as splicing and polyadenylation (poly A) signals); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence, such as 5'-GCCGCCACC-3' [SEQ ID NO: 33]); sequences that enhance protein stability; and sequences that enhance secretion of the encoded polypeptide. Examples of suitable polyA sequences include, e.g., SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic poly As. Examples of suitable enhancers include, e.g., the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulin promoter/alpha 1- microglobulin/bikunin enhancer), the Serpinl enhancer, amongst others. In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another. For example, an enhancer may include an Alpha mic/bik enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences.
[00235] In still another embodiment, the expression cassette further contains an intron (e.g., the Promega intron, a truncated chimeric intron (T-chimeric intron), introns described in Int. Pat. Appl. Pub. No. WO 2011/126808).
[00236] Optionally, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence (see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605-619).
[00237] The enhancer may be an ApoE enhancer element of approximately 155 bp derived from apolipoprotein E or ApoE. ApoE is an apolipoprotein that mediates binding, internalization and catabolism of lipoprotein particles and is a ligand for the low-density lipoprotein (ApoB/E) receptor and for the ApoE receptor of hepatic tissues. The genetic enhancer associated with the ApoE gene is a eukaryotic control element that can increase transcription of a nucleic acid specifically in the liver.
[00238] The enhancer may include an element from a hepatic control region (e.g., HCR-1 or HCR-2) associated with the ApoE gene. For example, sequence from HCR-1 is found in the sequence of SEQ ID NO: 17.
[00239] In yet another embodiment, the expression cassette and/or vector further contains a post-transcriptional regulatory element (PRE). An exemplary post-transcriptional regulatory element is the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). For example, a sequence from the WPRE is shown in SEQ ID NO: 16. The WPRE may be effective to increase expression of the protein (e.g., DNase). The WPRE may be modified so as not to produce an active form of a truncated X protein that is encoded in the WPRE. One such modified WPRE is provided in at least the sequence of SEQ ID NO: 16 and in the ApoEHCR enhancenhAAT promoter>hDNaseI (hyperactive)correct leader-WPRE Xinact (bases 1576-2212 of SEQ ID NO: 30; see also Figures 10A and 10B).
[00240] In some embodiments, the cassettes of the invention direct the expression of a therapeutic amount of the polypeptide encoded by the coding sequence for an extended period, typically greater than 200 days, and in some instances greater than 500 days. Expression of the polypeptide encoded by the coding sequence can be measured by any art-recognized means, such as, e.g., by antibody-based assays, such as a Western Blot or an ELISA assay. Again, by way of non-limiting example, expression of the polypeptide encoded by the coding sequence within the expression cassette can be measured in a bioassay that detects an enzymatic or biological activity of the polypeptide.
[00241] Upon entry into the liver cells (e.g., hepatocytes), the vectors can remain episomal (i.e., do not integrate into the genome of a host cell), or can integrate into the host cell genome. Non-limiting examples of episomal vectors include adenoviral vectors, and examples of vector that integrate into the host cell genome include retroviral vectors.
[00242] In some embodiments of the invention, the expression vectors comprising a nucleotide sequence encoding an enzyme which has 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- diolcyloxy propyl -3 -trimethyl ammonium bromide) and DOPE
(dioleoylphosphatidylethanolamine). In some embodiments, cationic liposomes containing DOTMA are effective to target expression vectors of the invention to the liver. In some embodiments, lactoferrin and/or poly L-lysine and/or poly ethyl enemine and/or chitosan is conjugated to an expression vector, with the conjugate effectively targeted to the liver after direct introduction into a subject.
[00243] In some embodiments, the liposomes have dimensions smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver.
[00244] In some embodiments, the expression vector is packaged into a nanovector or is encapsulated by a nanovector.
[00245] In some embodiments, the expression vector is packaged into phosphoramidite nanoparticles.
[00246] Various devices have been developed for enhancing the availability of the nucleic acids of the invention encoding enzymes which has DNase activity to the target cells, including, e.g., catheters or implantable materials containing DNA (G. D. Chapman et ah, Circulation Res. 71 :27-33 (1992)) and needle-free, jet injection devices which project a column of liquid directly into the target tissue under high pressure (P. A. Furth et ah, Anal Biochem. 20:365-368 (1992); (H. L. Vahlsing et ah, J Immunol. Meth. 175:11-22 (1994); (F. D. Ledley et ak, Cell Biochem. 18A:226 (1994)).
[00247] Another approach to targeted liver delivery of the nucleic acids of the invention encoding enzymes which has DNase activity is the use of molecular conjugates, which consist of protein or synthetic ligands to which a nucleic acid-binding agent has been attached for the specific targeting of nucleic acids to cells (R. J. Cristiano et al., Proc. Natl. Acad. Sci. USA 90: 11548-52 (1993); B. A. 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 targeted delivery to many cell types through the use of different ligands (R. J. Cristiano et al., Proc. Natl. Acad. Sci. USA 90: 11548-52 (1993)). For example, lipofection and malaria circumsporozoite protein have been used for the liver-specific delivery of genes (Z. Ding et al., J. Biol. Chem. 270:3667-76 (1995)).
[00248] Preferred routes of administration for the liver delivery of the expression vectors of the invention are intrahepatic, intravenous, and intra-arterial administration. However, other routes of administration allowing liver targeting can be also employed such as, e.g., enteral (e.g., oral), intramuscular, intraperitoneal, etc.
[00249] The expression vectors of the invention can be introduced directly into the liver by any art-recognized means, such as by direct injection into the liver, portal vein injection, or tail vein injection.
[00250] The expression vectors of the invention may be delivered by (1) creating an access site in an artery of a mammal, (2) introducing a guidewire into the artery, (3) introducing a sheath over the guidewire into the artery, (4) advancing a catheter through the sheath into the liver, (5) infusing the expression vector, e.g., rAAV virions, through the catheter into the liver, (6) removing the catheter and the sheath from the artery, and (7) repairing the access site. The catheter may be introduced into the target organ through an appropriate vein or artery. For example, the liver may be accessed via either the portal vein or the hepatic artery. An appropriate vein or artery may be accessed using techniques known in the art, such as the Seldinger technique. See, e.g., Conahan et al., (1977) JAMA 237:446-447, herein incorporated by reference. Other methods of accessing veins and arteries are also known. See, e.g., U.S. Pat. No. 5,944,695 herein incorporated by reference.
[00251] In certain other embodiments, occluding one or more of the blood vessels delivering blood to, or emptying blood from, the liver is conducted. The occluding device can be, for example, a balloon attached to the tip of a catheter. All of the arteries and veins entering or leaving the liver may be occluded so that the expression vector is delivered by way of asanguinous hepatic perfusion, whereby the liver is selectively perfused after excluding normal blood flow from the liver. This may be accomplished by clamping the hepatic artery, portal vein, suprahepatic vena cava, right suprarenal vein and infrahepatic vena cava to achieve the total vascular exclusion of the liver. The portal vein is then cannulated with a catheter of appropriate gauge, while an incision is made in the anterior wall of the infrahepatic vena cava, with a suction cannula placed into the vena cava to collect outflow. Liver perfusion, preferably single pass perfusion, is then performed using a pump. The flow rate may range 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 caval vein incisions are sutured and the liver is revascularized. Cardoso, et al., Human Gene Therapy 4:411-418 at 412 (1993).
[00252] In other embodiments, expression vectors are delivered into the liver by injection into the hepatic artery.
[00253] In a liver organ, the parenchymal cells include hepatocytes, Kupffer cells and the epithelial cells that line the biliary tract and bile ductules. The major constituent of the liver parenchyma are polyhedral hepatocytes (also known as hepatic cells) that presents at least one side to a hepatic sinusoid and apposed sides to a bile canaliculus. Liver cells that are not parenchymal cells include cells within the blood vessels such as the endothelial cells or fibroblast cells.
[00254] In the liver, the hepatic vein is an efferent blood vessel since it normally carries blood away from the liver into the inferior vena cava. Also in the liver, the portal vein and hepatic arteries are afferent blood vessels in relation to the liver since they normally carry blood towards the liver.
[00255] A liver blood vessel includes the portal venous system which transports blood from the gastrointestinal tract and other internal organs (e.g. spleen, pancreas and gall bladder) to the liver. Another liver blood vessel is the hepatic vein. The hepatic vein may also be reached via the inferior vena cava or another blood vessel that ultimately connects to the liver. A needle or catheter may be used to inject the polynucleotide into the vascular system. The injection can be performed under direct observation following an incision and visualization of the tissues blood vessels. Alternatively, a catheter can be inserted at a distant site and threaded so that it resides in the vascular system that connects with the target tissue. In some embodiments, deep injection into the vena cava is performed. In another embodiment, the injection could be performed by using a needle that traverses the intact skin and enters a vessel that supplies or drains from the target tissue.
[00256] In some embodiments, the delivery is performed as follows. The liver and portal vein are visualized through a ventral midline incision. A vector/liposome/naked DNA in 1 ml of various solutions containing heparin to prevent clotting is injected into the portal vein using a needle over approximately 30 sec.
[00257] The efficiency of liver delivery, expression and secretion of the enzyme which has DNase activity can be determined, e.g., by assaying the level of the enzyme in the blood (e.g., blood obtained from the retro-orbital venous sinus), e.g., using immunological assays (e.g., assays with antibodies recognizing the enzyme such as., e.g., a radioimmune assay (RIA) (HGH-TGES 100T kit from Nichols Institute, San Juan Capistrano, Calif., USA). In experimental animal models, the efficiency of liver delivery, expression and secretion of the enzyme which has DNase activity can be determined, e.g., by sacrificing the animals by cervical dislocation and the livers (average weight of 1.5 g) divided into six sections composed of two pieces of median lobe, two pieces of left lateral lobe, the right lateral lobe, and the caudal lobe plus a small piece of right lateral lobe. Each of the six sections may be placed separately into a homogenizing buffer. The homogenates may be centrifuged and the supernatant analyzed for the foreign gene product (i.e., enzyme which has DNase activity). Liver injections can be directed into the inferior cava which was clamped in two locations: proximal and distal to the entry of the hepatic vein into the inferior vena cava. Specifically, the downstream inferior vena cava clamp can be placed between the diaphragm and the entry point of the hepatic vein. The upstream inferior vena cava clamp can be placed just upstream of the entry point of the renal veins. Since the veins of other organs such as the renal veins enter the inferior vena cava at this location, not all of the injection fluid goes into the liver. In some of the animals that receive retrograde injections in the inferior vena cava, the hepatic artery, mesenteric artery, and portal vein can be clamped (occluded).
DNase Enzyme Targeting to Nervous Svstemand/or Intestine
[00258] In an additional aspect, the present invention provides compositions and methods for achieving DNase expression in (i) the liver and (ii) in the nervous system and/or intestine. In some embodiments, such DNase expression is achieved using vectors that comprise a nucleic acid expression cassette that is expressed (i) in the mammalian liver and also (ii) in the nervous system and/or intestine. Such expression cassette can comprise a liver-specific/nervous system-specific tandem promoter or a liver-specific/intestine-specific tandem promoter or a liver-specific/nervous system-specific/intestine-specific tandem promoter.
[00259] As demonstrated in the Examples section below, DNase expression (using tandem promoters) in the nervous system and/or intestine, in addition to its expression in the liver, can lead to superior therapeutic efficacy of DNase-mediated treatment.
[00260] In some embodiments, a DNase enzyme is targeted for expression in both the liver and the nervous system. Delivery of the DNase enzyme to the nervous system may be accomplished by use of nucleic acid expression cassettes wherein DNase is expressed in the nervous system under the control of a nervous system-specific promoter. In some embodiments, delivery of the DNase enzyme to the nervous system is accomplished by the use of nucleic acid expression cassettes that are expressed in both the liver and nervous system due to the use of a tandem promoter comprising a liver-specific promoter and a nervous system-specific promoter. In some embodiments, the nervous system-specific promoter is a central nervous system (CNS)- specific promoter. In some embodiments, the nervous system-specific promoter is the enteric nervous system (ENS)-specific promoter. In one embodiment, the nervous system-specific promoter is human synapsin promoter. In one embodiment, the human synapsin promoter comprises a sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the sequence SEQ ID NO: 36. In one embodiment, the human synapsin promoter comprises the sequence SEQ ID NO: 36. In one embodiment, the human synapsin promoter consists of the sequence SEQ ID NO: 36. In some embodiments, such expression cassettes comprise a liver-specific/nervous system-specific tandem promoter. In one embodiment, the liver-specific/nervous system-specific tandem promoter comprises a hAAT promoter and a human synapsin promoter.
[00261] In some embodiments, a DNase enzyme is targeted for expression in both the liver and the intestine. Delivery of the DNase enzyme to the intestine may be accomplished by use of nucleic acid expression cassettes wherein DNase is expressed in the intestine under the control of an intestine-specific promoter. In some embodiment, delivery of the DNase enzyme to the intestine is accomplished by use of nucleic acid expression cassettes that are expressed in both the liver and the intestine due to the use of a tandem promoter comprising a liver-specific promoter and an intestine-specific promoter. In one embodiment, the intestine-specific promoter is human villin promoter. In one embodiment, the intestine-specific promoter comprises a sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the sequence SEQ ID NO: 37 or a fragment thereof. In one embodiment, the liver- specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter.
[00262] In some embodiments, a DNase enzyme is targeted for expression in the liver, nervous system and intestine. Delivery of the DNase enzyme to nervous system and the intestine may be accomplished by use of nucleic acid expression cassettes that are expressed in both the nervous system and the intestine. In some embodiments, delivery of the DNase enzyme to the nervous system and the intestine is accomplished by use of nucleic acid expression cassettes that are expressed in the liver, nervous system and intestine. In some embodiments, such expression cassettes comprise a nervous system-specific promoter and an intestine-specific promoter. In some embodiments, such expression cassettes comprise DNase-coding sequence under the control of a liver-specific/nervous system-specific/intestine-specific tandem promoter. The liver- specific/nervous system-specific/intestine-specific tandem promoter may comprise a liver-specific promoter, a nervous system-specific promoter and an intestine-specific promoter. The three promoters may be positioned in any order. In one embodiment, the liver-specific/nervous system- specific/intestine-specific tandem promoter comprises a hAAT promoter, a human synapsin promoter, and a human villin promoter.
Parvovirus Vectors including Adeno-Associated Virus Vectors
[00263] In certain embodiments, the vector used in the invention is a parvovirus vector, such as an adeno-associated viral (AAV) vector. The term “parvovirus” as used herein encompasses the family Parvoviridae, including autonomously-replicating parvoviruses and dependoviruses. The autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, HI parvovirus, muscovy duck parvovirus, B 19 virus, and any other autonomous parvovirus now known or later discovered. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., Bernard N. Fields et ah, Virology, Vol. 2, Chapter 69 (4th ed., Lippi ncott-Raven Publishers).
[00264] In some embodiments, the parvovirus vector is a single-stranded parvovirus vector, such as an AAV vector. AAV (genus Dependovirus) normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), or other warm-blooded animals (e.g., bovine, canine, equine, and ovine AAVs). Further information on parvoviruses and other members of the Parvoviridae is provided, e.g., in Kenneth I. Berns,“Parvoviridae: The Viruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed. 1996).
[00265] AAV vectors disclosed herein may be derived from any AAV serotype, including combinations of serotypes (e.g.,“pseudotyped” AAV) or from various genomes (e.g., single- stranded or self-complementary). A“serotype” is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences). Non-limiting examples of AAV serotypes which can be used to develop the AAV expression vectors of the invention include, e.g., AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO (as disclosed, e.g., in U.S. Patent No. 9,790,472, Int. Pat. Appl. Pub. No. WO2017180857 and W02017/180861), AAVLK03, AAVLK06, AAVLK12 (as disclosed, e.g., in Wang et al., Mol. Ther., 2015, 23(12): 1877-1887), AAV-KPl(as disclosed, e.g., in Int. Pat. Appl. Pub. No. W02019191701A1), AAVhu37 (as disclosed, e.g., in Int. Pat. Appl. Pub. No. WO2017180857), AAVrh64Rl (as disclosed, e.g., in Int. Pat. Appl. Pub. No. WO2017180857), Anc80 (based on a predicted ancestor of serotypes AAV1, AAV2, AAV8 and AAV9; 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 now known or later discovered. See, e.g., Fields et al., Virology, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
[00266] Recently, a number of putative new AAV serotypes and clades have been identified
(see, e.g., Gao et ak, (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375-383). The genomic sequences of the various serotypes of AAV and the autonomous parvoviruses, as well as the sequences of the terminal repeats, Rep proteins, and capsid subunits are known in the art, by way of example, Srivistava et al., (1983) J. Virology 45:555; Chiorini et al., (1998) J. Virology 71 :6823; Chiorini et al, (1999) J. Virology 73 : 1309; Bantel-Schaal et al., (1999) J. Virology 73 :939; Xiao et al., (1999) J. Virology 73 :3994; Muramatsu et al., (1996) Virology 221 : 208; Shade et al., (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al., (2004) Virology 33-: 375-383; GenBank Accession number U89790; GenBank Accession number JO 1901 ; GenBank Accession number AF043303; GenBank Accession number AF085716; GenBank Accession number NC 006152; GenBank Accession number Y18065; GenBank Accession number NC 006260; GenBank Accession number NC 006261; International Patent Publication Nos. WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303, the disclosures of which are incorporated by reference herein for teaching parvovirus and AAV nucleic acid and amino acid sequences.
[00267] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VPl, VP2 and VP3) form the capsid. The terminal 145 nucleotides are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wtAAV infection in mammalian cells the Rep genes (i.e. Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively and both Rep proteins have a function in the replication of the viral genome.
[00268] In some embodiments, recombinant AAV (rAAV) vectors comprise one or more nucleotide sequences of interest that are flanked by at least one parvoviral 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 is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins). The terms“AAV Cap protein” or“AAV capsid protein”, as used herein, refer to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VPl, VP2, VP3). Examples of functional activities of Cap proteins (e.g., VPl, VP2, VP3) include the ability to induce formation of a capsid, facilitate accumulation of single-stranded DNA, facilitate AAV DNA packaging into capsids (i.e., encapsidation), bind to cellular receptors, and facilitate entry of the virion into host cells.
[00269] In some embodiments, the AAV vectors may comprise desired proteins or protein variants. A“variant” as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have“nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both.
[00270] AAV vectors are packaged into AAV viral capsids. The sequence of an AAV viral capsid protein defines numerous features of a particular AAV vector. For example, the capsid protein affects capsid structure and assembly, interactions with AAV nonstructural proteins such as Rep and AAP proteins, interactions with host body fluids and extracellular matrix, clearance of the virus from the blood, vascular permeability, antigenicity, reactivity to neutralizing antibodies, tissue/organ/cell type tropism, efficiency of cell attachment and internalization, intracellular trafficking routes, virion uncoating rates, among others. The sequence of a capsid protein (e.g., VP3) may be altered to enhance delivery to the liver.
[00271] AAV constructs may further comprise a sequence encoding one or more capsid proteins (VP1 and/or VP2, and/or VP3 capsid proteins, preferably just VP3 capsid protein) which package the above-mentioned polynucleotide sequence. The sequences coding for the capsid protein(s) for use in the context of the present invention may be taken from any of the known 42 serotypes, such as, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, Anc80, or newly developed AAV-like particles obtained by, e.g., capsid shuffling techniques and/or use of AAV capsid libraries. For some non-limiting examples of capsid protein sequences, see, e.g., U.S. Patent Nos. 9,790,472; 9,677,089; 7,282,199; Int. Pat. Appl. Publ. Nos. WO 2015/054653, WO2017/180857 (AAV8, AAV9, AAVrhlO, AAVhu37, AAVrh64Rl), W02017/180861 (AAVrhlO), WO2017/180854 (AAV8 mutants), and Wang et ah, Mol. Ther., 2015, 23(12): 1877-1887 (AAV8, AAVrhlO, AAV3B, and AAVLK03). When the sequences encoding the capsid proteins derive from a different AAV serotype as the ITRs, the AAV construct is known as a“hybrid” parvovirus genome (i.e., in which the AAV capsid and the AAV terminal repeat(s) are from different AAV) as described in Int. Pat. Appl. Publ. No. WO 00/28004 and Chao et ah, (2000) Molecular Therapy 2:619.
[00272] In some embodiments, the capsid protein(s) mediates efficient targeting of the AAV vector to the liver. In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector to the liver. Some capsid proteins (e.g., VP3 of Anc80, AAV8 and AAV3B) are naturally liver-specific. The invention also encompasses the use of AAV capsid mutants which enhance liver targeting and/or liver specificity. Non-limiting examples of such point mutations to the AAV8 capsid sequence include, e.g., S279A, S671A, K137R, and T252A, as well as AAV8 capsid mutations disclosed in Int. Pat. Appl. Pub. No. WO2017/180854 (e.g., AAV3G1, AAVT20 or AAVTR1, VP3 mutations in amino acids 263-267 [e.g., 263NGTSG267->SGTH ("NGTSG" disclosed as SEQ ID NO: 43 and "SGTH" disclosed as SEQ ID NO: 44) or 263NGTSG267- >SDTH ("NGTSG" disclosed as SEQ ID NO: 43 and "SDTH" disclosed as SEQ ID NO: 45)] and/or amino acids 457-459 [e.g., 457TAN459->SRP], and/or amino acids 455-459 [e.g., 455GGTAN459 ->DGSGL ("GGTAN" disclosed as SEQ ID NO: 46 and "DGSGL" disclosed as SEQ ID NO: 47)] and/or amino acids 583-597).
[00273] In some embodiments, the capsid protein(s) mediates efficient targeting of the AAV vector to the nervous system. In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector both to the liver and to the nervous system. Some capsid proteins would naturally target both the liver and the nervous system. The invention also encompasses the use of AAV capsid mutants which enhance targeting to the liver and/or the nervous system.
[00274] In some embodiments, the capsid protein(s) mediates efficient targeting of the AAV vector to the intestine. In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector both to the liver and to the intestine. Some capsid proteins would naturally target both the liver and the intestine. The invention also encompasses the use of AAV capsid mutants which enhance targeting to the liver and/or the intestine.
[00275] In some embodiments, the capsid protein(s) mediate preferential targeting of the AAV vector to the liver, nervous system and intestine. Some capsid proteins would naturally target the liver, nervous system and intestine. The invention also encompasses the use of AAV capsid mutants which enhance targeting to the liver and/or the nervous system and/or intestine.
[00276] The AAV vectors disclosed herein include a nucleic acid encoding an enzyme which has a DNase activity, such as, e.g., DNase I. In various embodiments, the nucleic acid also may include one or more regulatory sequences allowing expression and secretion of the encoded enzyme, such as e.g., a promoter, enhancer, polyadenylation signal, an internal ribosome entry site (IRES), a sequence encoding a protein transduction domain (PTD), a secretory signal sequence, and the like. Thus, in some embodiments, the nucleic acid may comprise a promoter region operably linked to the coding sequence to cause or improve expression of the protein of interest in transfected cells. Such a promoter may be ubiquitous, cell- or tissue-specific, strong, weak, regulated, chimeric, etc., for example to allow efficient and stable production of the protein in the liver, nervous system and/or intestine. The promoter may be homologous to the encoded protein, or heterologous, although generally promoters of use in the disclosed methods are functional in human cells. Examples of regulated promoters include, without limitation, Tet on/off element- containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters. Other promoters that may be used include promoters that are tissue specific, e.g., for liver, nervous system and/or intestine. Non-limiting examples of liver-specific promoters include, e.g., the albumin promoter (Alb), human alpha- 1 anti -trypsin (hAAT) promoter, thyroxine binding globulin (TBG), apolipoprotein E hepatic control region promoter, apolipoprotein A-II (APOA2) promoter, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) (hAAT) promoter, cytochrome P450 family 3, subfamily A polypeptide 4 (CYP3 A4) promoter, microRNA 122 (miR-122) promoter, liver-specific IGF-II promoter PI, murine transthyretin (MTTR) promoter, and the alpha-fetoprotein (AFP) promoter. Non-limiting examples of nervous system-specific promoters include, e.g., microglia-specific promoters (e.g., F4/80, CD68, TMEM119, CX3CR1, CMV, Ibal), myeloid-specific promoters (e.g., TTR, CD1 lb, c-fes), neuron specific promoters (e.g., CMV, NSE, synapsin [Synl, Synll], CamKII , a- CaMKII, VGLUT 1 ), and other neural and glial cell (e.g., oligodendrocytes astrocytes) type- specific promoters (e.g., GFAP). Non-limiting examples of intestine-specific promoters include, MUC2, Villin, T3b, CB/CMV, GFAP, miCMV, CMV+I, tetO-CMV, and b-acti-CMV. Non limiting examples of ubiquitous promoters include, e.g., viral promoters such as the CMV promoter, the RSV promoter, the SV40 promoter, etc., and cellular promoters such as the phosphoglycerate kinase (PGK) promoter, EF la promoter, CM VE/C AG promoter system, and the b-actin promoter.
[00277] The AAV constructs of the invention may also contain non-resolvable terminal repeats. The expression“non-resolvable terminal repeat”, as used herein, relates to terminal repeats which are not recognized by and resolved (i.e.,“nicked”) by the AAV Rep proteins, such that resolution of the terminal repeat is substantially reduced (e.g., by at least about 50%, 60%, 70%, 80%. 90%, 95%, 98% or greater as compared with a resolvable terminal repeat) or eliminated. Such non-resolvable terminal repeats may be naturally-occurring terminal repeat sequences (including altered forms thereof) and, for example, can be derived from a parvovirus, including an AAV, or can be from another virus or, as a further alternative, can be partially or completely synthetic. The non-resolvable terminal repeat may be a non-AAV viral sequence that is not recognized by the AAV Rep proteins, or it can be an AAV terminal repeat that has been modified (e.g., by insertion, substitution and/or deletion) so that it is no longer recognized by the AAV Rep proteins. Further, a non-resolvable terminal repeat can be any terminal repeat that is non-resolvable under the conditions used to produce the virus vector. Further, an AAV terminal repeat can be modified so that resolution by the AAV Rep proteins is substantially reduced or eliminated. The non-resolvable terminal repeat can be any inverted repeat sequence that forms a hairpin structure and cannot be nicked by the AAV Rep proteins.
[00278] The inverted terminal repeats (ITR) are typically present in at least two copies in the AAV vector, typically flanking the expression cassette containing the nucleotide sequence(s) encoding an enzyme which has a DNase activity. The ITRs typically will be at the 5' and 3' ends of the nucleotide sequence(s) encoding an enzyme which has a DNase activity but need not be contiguous thereto. The ITRs can be the same or different from each other. The term“terminal repeat” includes any viral terminal repeat and/or partially or completely synthetic sequences that form hairpin structures and function as an inverted terminal repeat, such as the“double-D sequence” as described in U.S. Pat. No. 5,478,745 to Samulski et al. An“AAV terminal repeat” may be 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 now known or later discovered. The AAV terminal repeat need not have a wild-type sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, nicking, virus packaging, integration, and/or provirus rescue, and the like. The vector construct can comprise one or more (e.g., two) AAV terminal repeats, which may be the same or different. Further, the one or more AAV terminal repeats can be from the same AAV serotype as the AAV capsid, or can be different. In particular embodiments, the vector construct comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, and/or Anc80 terminal repeat.
[00279] Parvoviral ITR nucleotide sequences are typically palindromic sequences, comprising mostly complementary, symmetrically arranged sequences also referred to as“A,”“B,” and“C” regions. The ITR functions as an origin of replication, a site having a“cis” role in replication, i.e., being a recognition site for trans acting replication proteins such as e.g. Rep 78 (or Rep68) which recognize the palindrome and specific sequences internal to the palindrome. One exception to the symmetry of the ITR sequence is the“D” region of the ITR. It is unique (not having a complement within one ITR). Nicking of single-stranded DNA occurs at the junction between the A and D regions. It is the region where new DNA synthesis initiates. The D region normally sits to one side of the palindrome and provides directionality to the nucleic acid replication step. A parvovirus replicating in a mammalian cell typically has two ITR sequences. It is, however, possible to engineer an ITR so that binding sites are on both strands of the A regions and D regions are located symmetrically, one on each side of the palindrome. On a double-stranded circular DNA template (e.g., a plasmid), the Rep78- or Rep68-assisted nucleic acid replication then proceeds in both directions and a single ITR suffices for parvoviral replication of a circular vector. Thus, one ITR nucleotide sequence can be used in the context of the present invention. Two or another even number of regular ITRs can be used.
[00280] Functional ITR sequences are necessary for the replication, rescue and packaging of AAV virions. The ITR sequences may be wild type sequences or may have at least 80%, 85%, 90%, 95%, or 100% sequence identity with wild type sequences. The ITR sequences may be altered by for example in insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be transduced or target cell.
[00281] The AAV vector can comprise single stranded or double stranded (self complementary) DNA. The single stranded nucleic acid molecule is either sense or antisense strand, as both polarities are equally capable of gene expression. The AAV vector may further comprise a marker or reporter gene, such as a gene 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.
[00282] AAV expression vectors may comprise a nucleic acid that may include a secretory signal sequence allowing secretion of the encoded enzyme which has a DNase activity from the transduced cell. Non-limiting examples of such secretory signal sequences include, e.g., DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, b- glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence.
[00283] In some embodiments, an AAV8-based or an Anc80-based vector is used as the expression vector. The AAV8 and Anc80 vectors are particularly suited for liver targeting and expression. For example, both AAV8 and Anc80 vectors can transfect liver cells with greater efficiency as compared to an AAV2 vector. Both AAV8 and Anc80 also induce lower amounts of neutralizing antibodies than some of the other AAV vectors.
[00284] An AAV8 vector or an Anc80 vector comprising a nucleotide encoding for an enzyme which has a DNase activity can be administered intrahepatically (e.g., via direct organ injection) or systemically, e.g., by intravenous injection, with the AAV8 or Anc80 vector effective to transfect liver cells and mediate effective production of the encoded enzyme which has a DNase activity and its secretion in the periendothelial space and the space of Disse. In some embodiments, an Anc80 capsid protein (e.g., Anc80 VP1 capsid protein comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 9, or an Anc80L65 VP1 capsid protein comprising the sequence of SEQ ID NO:34, or a variant Anc80L65 VP1 capsid protein comprising the sequence of SEQ ID NO: 35) is encoded by a nucleotide sequence in the expression vector. In some embodiments, an AAV8 capsid protein (e.g., AAV8 VP1 or AAV8 VP3) is encoded by a nucleotide sequence in the expression vector. Peripheral administration of the AAV vectors of the invention may include systemic injections, such as, e.g., intramuscular, intravenous, intraperitoneal, and intra-arterial injections.
[00285] In some embodiments, an AAV3-based, or an AAV-LK03, or an AAV-KP1 vector is used as the expression vector. The AAV3, AAV-LK03 and AAV-KP1 vectors are particularly suited for liver targeting and expression. For example, AAV3, AAV-LK03 and AAV-KP1 vectors can transfect liver cells with greater efficiency as compared to an AAV2 vector. Both AAV-LK03 and AAV-KPl also induce lower amounts of neutralizing antibodies than some of the other AAV vectors.
[00286] The desired doses of the DNase enzyme encoding AAV vectors of the invention may be easily adapted by the skilled artisan, e.g., depending on the disease condition, the subject, the treatment schedule, etc. In some embodiments, from 10e5 to 101e4 recombinant viral particles are administered per dose, for example, from 10e6 to lOel l, from 10e7 to lOel l, or from 108 to 1014. In other embodiments, exemplary doses for achieving therapeutic effects may include titers of at least about 10e5, 10e6, 10e7, 10e8, 10e9, lOe 10 or lOel l recombinant viral particles or more.
[00287] The exogenous targeting sequence(s) may replace or substitute part or all of a maj or capsid subunit (e.g., VP3). As a further alternative, more than one exogenous targeting sequence, e.g., two, three, four, five or more sequences, may be introduced into the virion capsid. In alternative embodiments, insertions and substitutions within the minor capsid subunits (e.g., VPl and VP2) may be undertaken. For AAV capsids, insertions or substitutions in VP2 or VP3 may be undertaken.
[00288] The native virion tropism may be reduced or abolished by insertion or substitution of the amino acid sequence. Alternatively, the insertion or substitution of the exogenous amino acid sequence may target the virion to a particular 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 particular embodiments, the targeting peptide or protein may be naturally occurring or, alternately, completely or partially synthetic. Exemplary peptides and proteins include ligands and other peptides that bind to cell surface receptors present in liver cells include ligands capable of binding the Sr-Bl receptor for apoliprotein E, galactose- and lactose-specific lectins, low density lipoprotein receptor ligands, asialoglycoprotein (galactose-terminal) ligands and the like.
[00289] Alternatively, the exogenous targeting sequence may be an antibody or an antigen recognizing moiety thereof. The term “antibody” as used herein 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 species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. Also encompassed by the term“antibody” are bispecific or“bridging” antibodies as known by 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 the corresponding fragments obtained from antibodies other than IgG. Such fragments may be produced by known techniques. The exogenous amino acid sequence inserted into the virion capsid may be one that facilitates purification or detection of the virion. For example, the exogenous amino acid sequence may include a poly-histidine sequence that is useful for purifying the virion over a nickel column, as is known to those skilled in the art or an antigenic peptide or protein that may be employed to purify the virion by standard immunopurification techniques. Alternatively, the amino acid sequence may encode a receptor ligand or any other peptide or protein that may be used to purify the modified virion by affinity purification or any other techniques known in the art (e.g., purification techniques based on differential size, density, charge, or isoelectric point, ion- exchange chromatography, or peptide chromatography).
[00290] Alternatively, the exogenous targeting sequence may be an antibody or an antigen recognizing moiety thereof. The term “ antibody” as used herein 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 species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. Also encompassed by the term “antibody” are bispecific or “bridging” antibodies as known by 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 the corresponding fragments obtained from antibodies other than IgG. Such fragments may be produced by known techniques.
[00291] The exogenous amino acid sequence inserted into the virion capsid 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 alters the virion of the modified parvovirus. For example, the exogenous amino acid sequence may include a poly-histidine sequence that is useful for purifying the virion over a nickel column, as is known to those skilled in the art or an antigenic peptide or protein that may be employed to purify the virion by standard immunopurification techniques. Alternatively, the amino acid sequence may encode a receptor ligand or any other peptide or protein that may be used to purify the modified virion by affinity purification or any other techniques known in the art (e.g., purification techniques based on differential size, density, charge, or isoelectric point, ion-exchange chromatography, or peptide chromatography) .
Adenovirus Vectors
[00292] Adenovirus genome is a linear, 36-Kb double-stranded DNA containing multiple, heavily spliced transcripts. At either end of the genome are inverted terminal repeats (ITRs). Genes are divided into early (E1-E4) and late (L1-L5) transcripts.
[00293] In some embodiments, recombinant adenovirus vectors have two genes deleted: El and E3 (dEl/E3). Deletion of El makes the viral vector replication-deficient. El can be supplied by the adenovirus packaging cell lines (e.g., T 293 or 911). E3 is involved in evading host immunity and is not essential for virus production. Deletion of El and E3 results in a transgene packaging capacity of >8 Kb. In some embodiments, Constructs contain left and right arms to facilitate homologous recombination of the transgene into the adenoviral plasmid.
[00294] Any of the accepted human adenovirus types may be used, such as, e.g., an adenoviral vector based on Ad5. Ad5-based vectors use the Coxsackie- Adenovirus Receptor (CAR) to enter cells. In some embodiments, human adenovirus Type 5 (dEl/E3) is used as a vector. Most adenovirus vectors are engineered such that a transgene replaces the Ad Ela, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Adenovirus vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, nervous system and intestine. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)). Additional examples of the use of adenovirus 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).
Naked DNA Delivery
[00295] In some embodiments, an expression vector comprising a sequence encoding an enzyme which has a DNase activity is administered to the subject as a naked DNA. For efficient liver delivery, such naked DNA can be delivered, e.g., into a liver blood vessel at distal or proximal points. For efficient nervous system delivery, such naked DNA can be delivered, e.g., intravenously (iv), intramuscularly (im), per os (po), intraspinally, intracranially, or intraventricularly. For efficient intestinal delivery, such naked DNA can be delivered, e.g., intravenously (iv), intramuscularly (im), per os (po), or rectally.
Enveloped Viral Vectors
[00296] In various embodiments of the invention, an enveloped viral particle can be used for liver, nervous system and/or intestinal delivery of a nucleic acid encoding an enzyme which has a DNase activity.
[00297] In some embodiments, the viral particle described herein is derived from a virus of the family Retroviridae. In one specific 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 a variety of cell types, to stably integrate transferred genetic material into the genome of the targeted host cell, and to express the transduced gene at significant levels. Vectors derived from the gamma-retroviruses, for example, the murine leukemia virus (MLV), have been used in clinical gene therapy trials (Ross et al., Hum. Gen Ther. 7: 1781-1790, 1996).
[00298] In one specific embodiment, the at least one viral element associated with a nucleotide of interest is a retroviral element. In another specific embodiment, the at least one viral element associated with a nucleotide of interest is a lentiviral element. In one specific embodiment, the at least one viral element associated with a nucleotide of interest is a Psi (y) packaging signal. [00299] In one specific embodiment, the lentiviral particle does not contain gpl20 surface envelope protein and/or gp41 transmembrane envelope protein. In another specific embodiment, the lentiviral particle contains a mutant gpl20 surface envelope protein and/or a mutant gp41 transmembrane envelope protein.
[00300] In some embodiments, the viral particles described herein are replication deficient and only contain an incomplete genome of the virus from which they are derived. For example, in some embodiments, the lentiviral and retroviral particles do not comprise the genetic information of the gag, env, or pol genes (which may be involved in the assembly of the viral particle), which is a known minimal requirement for successful replication of a lentivirus or retrovirus. In these cases, the minimal set of viral proteins needed to assemble the vector particle are provided in trans by means of a packaging cell line. In one specific embodiment, env, tat, vif, vpu and nef genes are lacking in lentiviral particles derived from HIV-1 and are provided in trans or are made inactive by the use of frame shift mutation(s).
[00301] In some embodiments, the RNA molecule incorporated into the lentiviral or retroviral particles (and encoding an enzyme which has a DNase activity) comprises the psi packaging signal and LTRs. To achieve expression of the nucleotide sequence encoding an enzyme which has a DNase activity in the liver, nervous system and/or intestine, such sequence is usually placed under the control of various promoters discussed above.
[00302] In some embodiments of lentiviral and retroviral particles, the RNA molecule together with the gag and pol encoded proteins, provided in trans by the packaging cell line, are then assembled into the vector particles, which then infect cells, reverse-transcribe the RNA molecule that comprises a nucleotide sequence encoding an enzyme which has a DNase activity under the control of a promoter, and either integrate said genetic information into the genome of the target cells or remain episomal (if one or more of the components required for integration are disrupted). If the genetic information for the gag and pol encoded proteins is not present on the transduced RNA molecule, the vector particles are replication deficient, i.e., no new generation of said vector particles will thus be generated by the transduced cell, thus ensuring safety in clinical applications.
[00303] In some embodiments of any of the above methods, the one or more viral elements encodes 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), Murine Stem Cell Virus (MSCV) component(s), Murine Leukemia Virus (MLV) component(s), Avian leukosis virus (ALV) component(s), Feline leukemia virus (FLV) component(s), Bovine leukemia virus (BLV) component(s), Human T-lymphotropic virus (HTLV) component(s), feline sarcoma virus component(s), avian reticuloendotheliosis virus component(s), caprine arthritis encephalitis virus (CAEV) component s), and/or Visna-Maedi virus (VMV) component(s).
[00304] In conjunction with the viral particles described herein, described herein are methods for detecting and/or isolating cells. In some embodiments, such method uses viral particles comprising a selectable marker (e.g., neomycin resistance) and/or a reporter (e.g., GFP or eGFP) as a nucleotide sequence of interest allowing target cells to be selected using, e.g., selection compound exposure or fluorescent activated cell sorting (FACS).
[00305] In some embodiments, the retroviral vectors described herein are derived from murine leukemia virus (MLV). Retroviral vectors encoding MLV are widely available to those skilled in the art, such as PINCO (Grignani et ah, 1998) or the pBabe vector series (Morgenstern and Land, 1990).
[00306] 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 useful viruses can be readily identified and/or prepared by the skilled person.
[00307] In one specific embodiment, the transfer vector comprises: a cytomegalovirus (CMV) enhancer/promoter sequence; the R and U5 sequences from the HIV 5' LTR; the HIV-1 flap signal; an internal enhancer; an internal promoter; a nucleotide sequence of interest; the woodchuck hepatitis virus responsive element; a tRNA amber suppressor sequence; a U3 element with a deletion of its enhancer sequence; the chicken b-globin insulator; and the R and U5 sequences of the 3' HIV LTR.
[00308] The invention also contemplates enveloped viral vectors comprising a heterologous targeting sequence or molecule inserted or substituted into the native envelope. The heterologous targeting sequence or molecule may confer an altered tropism towards liver, nervous system and/or intestine or increase the efficiency of liver, nervous system and/or intestinal delivery of the vector.
Methods and Regimens for Administering Expression Vectors Encoding DNase Enzyme
[00309] In the methods of the invention, expression vectors comprising a nucleic acid encoding and enzyme which has a DNase activity can be administered to a patient at one time or over a series of treatments; once or several times per day.
[00310] The effective amount of 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 determined readily by one of ordinary skill in the art. Analysis of the blood (serum, plasma) or tissue levels of the vector-encoded enzyme which has a DNase activity and comparison to the initial level prior to administration can determine whether the amount being administered is too low, within the right range or too high. Suitable regimes for initial and subsequent administrations are also variable, but are typified by an initial administration followed by subsequent administrations if necessary. Subsequent administrations may be administered at variable intervals, ranging from daily to weekly to monthly to annually to every several years.
[00311] Doses of an expression vector encoding for an enzyme which has a DNase activity depend on the type of condition to be treated, the severity and course of these side effects, the patient’s clinical history, discretion of the attending physician, and response to a prior treatment such as, e.g., chemotherapy, or radiation therapy, and DNase protein therapy. In some embodiments, the effective amount of expression vector is the amount which results in DNase protein level in hepatic portal vein which is from 0.5 to 100 mg/L or from 1000 to 200000 Kunitz units (KU)/L, from 0.5 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.
[00312] The administration of an expression vector, e.g., AAV- or viral-vector, encoding for DNase enzyme, and/or DNase enzyme, according to the methods of the invention can be performed by any suitable route, including, e.g., intravenous, intra-arterial and intrahepatic administration.
Cancer Treatment
[00313] In one aspect is provided a method for treating or ameliorating cancer in a subject suffering from a cancer, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase Il-alpha, DNase Il-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 comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver- specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/nervous system- specific tandem promoter and/or an enhancer element.
[00314] In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/intestine-specific tandem promoter and/or an enhancer element (e.g., CMV enhancer). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
[00315] The amount and manner of administration of the expression vectors of the invention should be effective to treat a cancer, e.g., by one or more of reducing tumor size, reducing the rate of tumor growth, reducing the rate of metastasis, enhancing the therapeutic effects of chemotherapy and/or radiotherapy, prolonging lifespan, reducing the rate of cancer- or treatment- associated weight loss, or increasing the rate of weight gain.
[00316] In some embodiments, the administration of an expression vector encoding an enzyme which has a DNase activity can be combined with administration of the same enzyme as protein (e.g., intravenously, intra-arterially, intrahepatically, intramuscularly, intraperitoneally, enterally, etc.). The enzyme can be, e.g., any one of DNase enzymes (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase Il-alpha, DNase Il-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase).
[00317] The methods of treatment using the vectors of the invention can be used in subjects suffering from a broad range of cancers. The present invention is particularly useful for treating “portal vein cancers”, which include any carcinoma, sarcoma, melanoma, or lymphoma originating in or having metastasized to organs, tissues and structures that drain to the portal vein (see Figure 1). Non-limiting examples of portal vein cancers include, e.g., peritoneal carcinomatosis, lymphomas (e.g., B cell lymphoma, T cell lymphoma, non-Hodgkins lymphomas, Hodgkins lymphoma), stomach cancers, colon cancers, intestinal cancers, colorectal cancers, pancreatic cancers, liver cancers, splenic cancers, cancers of the bile duct, cancers of the gall bladder, sarcomas in a tissue draining to the portal vein (e.g., an angiosarcoma, endotheliosarcoma, hemangiosarcoma, leiomyosarcoma, other sarcomas, and epithelial carcinoma in such tissue), and liver metastatic diseases of any origin.
[00318] Non-limiting examples of other cancers which can be treated by the methods of the invention include, e.g., breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g., non-small cell lung cancer (NSCLC)), renal cancer, thyroid cancer, leukemia (e.g., 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 cancer, bone cancer (including both primary bone cancers (e.g., osteosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, malignant fibrous histiocytoma, adamantinoma, giant cell tumor, and chordoma) and secondary (metastatic) bone cancers), soft tissue sarcoma, basal cell carcinoma, angiosarcoma, hemangiosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstroom’s macroglobulinemia, papillary adenocarcinomas, cystadenocarcinoma, bronchogenic carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, medullary carcinoma, thymoma, sarcoma, etc.
[00319] For combination cancer treatments, an expression vector, e.g., AAV vector or lentiviral vector or adenoviral vector encoding an enzyme which has a DNase activity can be administered before, with, or after a chemotherapeutic agent (or a radiation therapy).
[00320] In some embodiments, the methods of the invention are used to prevent a cancer in a patient, e.g. a patient with a high risk of predisposition to cancer.
[00321] In some embodiments, the methods of the invention are used to prevent a cancer following a positive result from a blood-based DNA screening test (e.g., liquid biopsy) for the detection of predisposition to a cancer.
[00322] In some embodiments, the methods of the invention are used to prevent a cancer following the evaluation of a tumor susceptibility gene. Examples of tumor susceptibility genes include, but are not limited to, ABL1 (ABL), ABL2(ABLL, ARG), AKAP13 (HT31, LBC. BRX), ARAFl, ARHGEF5 (TIM), ATF1, AXL, BCL2, BRAF (BRAFl, RAFB l), BRCA1, BRCA2(FANCD1), BRIP1, CBL (CBL2), CSF1R (CSF-1, FMS, MCSF), DAPKl (DAPK), DEK (D6S23 IE), DUSP6(MKP3,PYST1), EGF, EGFR (ERBB, ERBBl), ERBB3 (HER3), ERG, ETS1, ETS2, EWSR1 (EWS, ES, PNE), FES (FPS), FGF4 (HSTF1,KFGF), FGFR1, FGFR10P (FOP), FLCN, FOS (c-fos), FRAPl, 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 genes, MMD, MOS (MSV), MRAS (RRAS3), MSH type genes, MYB (AMV), MYC, MYCL1 (LMYC), MYCN, NCOA4 (ELE1, ARA70, PTC3), NF1 type genes, 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, RAFl (CRAF), RBI (RB), RET, RRAS2 (TC21), ROS1 (ROS, MCF3), SMAD type genes, SMARCB1(SNF5, INI1), SMURF1, SRC (AVS), STAT1, STAT3, STAT5, TDGF1 (CRGF), TGFBR2, THRA (ERBA, EAR7 etc ), TFG (TRKT3), TIFl (TRIM24, TIF1A), TNC (TN, HXB), TRK, TUSC3, USP6 (TRE2), WNT1 (INTI), WT1, and VHL.
[00323] The susceptibility gene can be evaluated, e.g., by one or more of next-generation sequencing (NGS), whole genome sequencing (WGS), exome sequencing, an ELISA-based method, and PCR amplification. Other methods can be used to evaluate the susceptibility gene as well.
[00324] In another aspect, the invention provides a method for treating or ameliorating a cancer in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and/or nervous system and/or intestine of said subject. In some embodiments, the cancer is accompanied by an increase in the total level or relative content of cell free DNA (cfDNA) of microbial origin and/or is accompanied by an increase in the total level of the cfDNA originating from host cells in blood of said subject. In several embodiments, the method comprises detecting the level of cfDNA of microbial origin or the ratio of the cfDNA of microbial origin to the total level of cfDNA in blood of the subject. Non-limiting examples of methods of detecting the cfDNA of microbial origin include, e.g., PCR, RT-PCR, next-generation sequencing (NGS) and whole genome sequencing (WGS). In some embodiments, cfDNA of microbial origin can be detected, e.g., using PCR or RT-PCR of 16S ribosomal RNA (rRNA), 16S ribosomal DNA (rDNA) or Intergenic Spacer Region. In some embodiments, determining the total level or relative content of cfDNA of microbial origin includes comparing the level of cfDNA of microbial origin or the ratio of the cfDNA of microbial origin to the total level of cfDNA in blood of the subject to a control level or ratio (e.g., a predetermined standard or a corresponding level or ratio determined using age-matched cancer-free subjects).
[00325] In one embodiment of any of the methods of the invention, the subject is human.
Amelioration of Toxicity of Cancer Chemotherapy or Radiation Therapy
[00326] In one aspect, the invention provides a method for preventing or ameliorating a toxicity, or a condition, associated with a cytostatic and/or cytotoxic chemotherapy in a subject suffering from a cancer and receiving or deemed to receive said chemotherapy, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding for an enzyme which has a DNase activity (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., 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 comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system- specific/intestine-specific tandem promoter and/or an enhancer element.
[00327] The amount and manner of administration of the expression vector is effective to ameliorate at least one side effect of said chemotherapy.
[00328] The condition associated with a cytostatic and/or cytotoxic chemotherapy can include, e.g., (i) a catabolic state leading to body weight loss, (ii) bone marrow toxicity and/or catabolic changes in blood biochemistry, (iii) cardiotoxicity (e.g., myocardial necrosis), (iv) gastrointestinal toxicity, (v) suppression of immunity, (vi) neutropenia, and (vii) body weight loss.
[00329] In some embodiments, the cancer is associated with an increased level of cfDNA in blood or cerebrospinal fluid (CSF) or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or CSF or intestine of a healthy age-matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age- matched individuals).
[00330] In some embodiments, the effects of chemotherapy are associated with an increased level of cfDNA in blood or CSF, ascitic fluid, or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of an age- matched individual with a similar cancer profile who does not receive chemotherapy, or an average level of cfDNA in blood or CSF or intestine of several age-matched individuals who have cancer but do not receive chemotherapy).
[00331] In a further aspect, the invention provides a method for increasing the efficacy of a cytostatic and/or cytotoxic chemotherapy in a subject suffering from a cancer and receiving or deemed to receive said chemotherapy, which method comprises administering to the subject a therapeutically effective amount of an expression vector (e.g., Anc80 or AAV8 AAV vector or lentiviral vector or adenoviral vector) encoding an enzyme which has a DNase activity (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS 1L2), DNase II (DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase). The amount and manner of administration of the expression vector is effective to prevent or ameliorate at least one side effect of said chemotherapy, and to prevent or ameliorate toxicity associated with said chemotherapy. In some embodiments, any one of DNase enzyme (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase) is also administered, e.g., intravenously.
[00332] The expression vectors of the invention can be used to ameliorate toxicity and/or increase efficacy of a wide range of different chemotherapeutic agents. Non-limiting examples of such agents include anti-metabolites such as pyrimidine analogs (e.g., 5-fluorouracil [5-FU], floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (e.g., paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (e.g., etoposide, teniposide), DNA damaging agents (e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, nedaplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, aclarubicin, purarubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, nimustine, ranimustine, estramustine, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), pleomycin, peplomycin, mitomycins (e.g., mitomycin C), actinomycins (e.g., actinomycin D), zinostatinstimalamer); enzymes (e.g., L-asparaginase); neocarzinostatin; antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and analogs, imidazol carboxamide, melphalan, chlorambucil, nitrogen mustard-N-oxide hydrochloride, ifosfamide), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa, carboquone, triethylene thiophospharamide), alkyl sulfonates (e.g., busulfan, isoprosulfan tosylate), nitrosoureas (e.g., carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); epoxide type compounds (e.g., mitobronitol); antiproliferative/antimitotic antimetabolites such as folic acid analogs (e.g., methotrexate); platinum coordination complexes (e.g., cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (e.g., estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (e.g., letrozole, anastrozole); antisecretory agents (e.g., breveldin); immunosuppressives (e.g., cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti -angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (e.g., trastuzumab); cell cycle inhibitors and differentiation inducers (e.g., tretinoin); mTOR inhibitors, topoisomerase inhibitors (e.g., doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, irinotecan); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers; chromatin disruptors; sobuzoxane; tretinoin; pentostatin; flutamide; porphimer natrium; fadrozole; procarbazine; aceglatone; radioimmunotherapy (RIT) compounds (e.g., Ibritumomab tiuxetan , Iodine (1311) tositumomab); and targeted radionuclide therapy (TRT) compounds (e.g., samarium- 153 -ED TMP, strontium-89-chloride).
[00333] Non-limiting examples of side effects of cytostatic and/or cytotoxic chemotherapy which can be prevented or ameliorated by administering an expression vector according to the methods of the invention include, for example, bone marrow toxicity, neutropenia, myelopathy (e.g., leukopenia, granulocytopenia, lymphopenia, thrombocytopenia, erythropenia); hematopathy (e.g., plasma fibrinogenopenia); catabolic changes in blood biochemistry; gastrointestinal disorders (e.g., nausea, vomiting, anorexia, body weight loss, heavy feeling of stomach, diarrhea, constipation, stomatitis, esophagitis); pulmonary insufficiency (e.g., chronic pneumonia, lung fibrosis, ARDS, ALS, lung emboli); dermatopathy (e.g., keratinization, pachymenia, chromatosis, epilation, rash, nail alternation, cancer-induced alopecia); nervous system disorders (e.g., paresthesia, depression, deep areflexia, neuroparalysis, auditory disorder, allolalia, disorientation, neurologic manifestation, cerebellar ataxia, somnolence, coma, vertigo, frequency of micturition, frequency of defecation desire); endocrine disorders (e.g., pituitary disorder, adrenal disorder, hyperglycemia, hypoglycemia); genital disorders (e.g., hyposexuality, oligospermia, gynecomastia, menstrual disorder); cardiovascular disorders (e.g., myocardial necrosis, cardiomyopathy, arrhythmia, low blood pressure, tachycardia, cardiac failure); hepatopathy, pancreatic disorder, nephropathy, bladder trouble, hyperuricemia, decrease of immunocompetence, and infection.
[00334] The expression vectors of the invention can be also used to ameliorate toxicity and increase 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/unsealed source radiotherapy. Non-limiting examples of side effects of radiotherapy which can be prevented or ameliorated by administering an expression vector according to the methods of the invention include, for example, skin irritation or damage, fatigue, nausea, vomiting, fibrosis, bowel damage, memory loss, infertility, and a second cancer.
[00335] The expression vectors of the invention can be used to increase efficacy of cancer immunotherapy, including, for example, antibodies (naked monoclonal antibodies, conjugated monoclonal antibodies, chemolabeled antibodies, bispecific monoclonal antibodies), cancer vaccines, oncolytic viruses, Immune checkpoint inhibitors (Drugs that target PD-1 or PD-L1, CTLA-4), Non-specific cancer immunotherapies and adjuvants, CAR-T therapies.
Neurodegeneration Treatment
[00336] In one aspect, the invention provides a method for treating or ameliorating neurodegeneration. The term "neurodegeneration" is used herein to refer to a separate clinical pathological condition with progressive loss of structure and/or function of neurons, including death of neurons. The neurodegeneration can be primary or secondary.
[00337] In some embodiments, the neurodegeneration is associated with an increased level of cfDNA (e.g., microbial cfDNA or cfDNA originating from host cells) in blood or cerebrospinal fluid (CSF) or intestine of a patient, which level is higher than the control level (e.g., the level of cfDNA in blood or CSF or intestine of a healthy age-matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age-matched individuals). In one embodiment, the neurodegeneration is associated with a neurodegenerative disorder. Non-limiting examples of encompassed neurodegenerative disorders include, e.g., Alzheimer's disease (e.g., late-onset Alzheimer's disease), Mild Cognitive Impairment (MCI), Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), and Huntington's disease (HD), prion-caused diseases, progressive supranuclear palsy (PSP), progressive supranuclear palsy (Steel-Richardson- Olszewski), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), agyrophilic grain disease (AGD), Pick disease (PiD), frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism- 17 (FTDP-17), multiple sclerosis, CADASIL Syndrome, ankylosing spondylitis, Dentatorubro-pallido-Luysian atrophy, Kennedy disease, Friedreich’s ataxia, cerebellar ataxia, spinocerebellar ataxia, Lewy body dementia, vascular dementias, familial Danish dementia, familial British dementia, spinal muscular atrophy, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, corticodentatonigral degeneration, Hallervorden-Spatz disease, progressive familial myoclonic epilepsy, striatonigral degeneration, torsion dystonia (e.g., torsion spasm; dystonia musculorum deformans), spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration (e.g., Friedreich's ataxia and related disorders), Shy- Drager syndrome, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot- Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa), and hereditary optic atrophy (Leber's disease), secondary neurodegeneration caused by necrosis (e.g., destruction of neurons by neoplasm, edema, hemorrhage, stroke, trauma, immune attack, hypoxia, poisoning, metabolic defects, and infections), and various taupathies (e.g., Primary age-related tauopathy (PART)/Neurofibrillary tangle-predominant senile dementia, with NFTs similar to AD, but without plaques, CTE, Lytico-Bodig disease,
Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis). In some one embodiments, the neurodegeneration is associated with a nervous system dysfunction such as, e.g., 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, the neurodegenerative disorder is a protein-misfolding associated 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, an amyloidosis (e.g., hereditary cerebral hemorrhage with amyloidosis, primary systemic amyloidosis, secondary systemic amyloidosis, serum amyloidosis, senile systemic amyloidosis, hemodialysis-related amyloidosis, Finnish hereditary systemic amyloidosis, Atrial amyloidosis, Lysozyme systemic amyloidosis, Insulin- related amyloidosis, Fibrinogen a-chain amyloidosis), asthma, or prion disease.
[00338] In some embodiments, the neurodegeneration is caused by, secondary to, or associated with the formation of a misfolded protein due to the presence of DNA (e.g., human, microbial, cell-free, or intracellular). In some embodiments, the neurodegeneration is caused by the formation of a misfolded protein due to the presence of DNA (e.g., human, microbial, cell-free or intracellular).
[00339] In some embodiments, the neurodegeneration is caused by, secondary to, or associated with the formation of a misfolded protein including, but not limited to, b-amyloid, Tau protein, a-synuclein, SOD1, TDP-43, IAPP, ADan, ABri, Fused in sarcoma (FUS) protein, Notch3, Glial fibrillary acidic protein, Seipin, Transthyretin, Serpins, Apolipoproteins, Amyloid b peptide, Lactoferrin, Galectin-7 Comeodesmosin.
[00340] In some embodiments, the methods of the invention can be used to prevent development of a neurodegenerative disease or another disease associated with protein misfolding following the evaluation of the presence of, or activity of, a disease-associated susceptibility gene. Examples of disease-associated susceptibility genes, include, but are not limited to, AD AR1, MDA5 (IFIH1), RNAseH subunits, SamHDl, TREX, TBK1, Optineurin, P62 (sequestosome 1), Progranulin, TDP43, FUS, VCP, CHMP2B, Profilin-1, Amyloid-b, Tau, a-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. The susceptibility gene can be evaluated by one or more of Next-generation sequencing, whole genome sequencing, exome sequencing, an ELISA-based method, and PCR amplification. Other methods can be used to evaluate the susceptibility gene as well.
[00341] In one embodiment of the above methods, prevention or treatment of the misfolded protein aggregate formation in mammalian biological fluids and tissues is achieved by the use of expression vectors disclosed herein, including those comprising the sequence of SEQ ID NO: 30 (ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact) or SEQ ID NO: 31 (ApoEHCR enhancer-hAAT promoter-hDNasel wild type-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 human DNasel hyperactive variant with natural full correct leader sequence, and bases 1576-2212 correspond to the inactivated WPRE X protein. In the sequence of 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 Kozak sequence, bases 727-1575 correspond to human DNasel wild type with natural full correct leader sequence, and bases 1576-2212 correspond to the inactivated WPRE X protein.
[00342] As described in Int. Pat. Appl. Pub. No. WO 2016/190780, cfDNA from the intestine, blood and CSF of patients suffering from neurodegeneration causes neuronal cell death and apoptosis, and treatment with recombinant DNase protein destroying such cfDNA improves the nervous system function in these patients. As shown in the Examples section below, the use of the vectors of the invention for liver-specific, nervous system-specific and/or intestine-specific expression of DNase, provides a further treatment improvement as compared to DNase protein administration.
[00343] The assessment of treatment efficacy for neurodegeneration and neuroinflammation can be performed using any methods known in the art, e.g., according to widely accepted clinical diagnostic criteria of cognitive decline such as MMSE, PANSS, physical function, and/or functional tasks (see, e.g., Holmes et ah, (1999) The British Journal of Psychiatry, 174(1), 45-50; Os et al., (2006) Acta Psychiatrica Scandinavica, 113(2), 91-95; O'Shea et ah, (2002) Physical therapy, 82(9), 888-897; Rochester et al., Arch. Phys. Med. Rehabil. (2004) 85(10), 1578- 1585).
[00344] In some embodiments of the invention, the neurodegeneration is caused by, or aggravated by, increased intestinal permeability and translocation of gut bacteria and DAMPs of microbial origin including microbial cfDNA. Such cfDNA may trigger neuroinflammation and a spectrum of neurodegenerative diseases. cfDNA originating from digestive tract microbiota are subject to“first pass” through porto-sinusoidal circulation in the liver. The NLRP3 inflammasome is considered a key contributor to the development of neuroinflammation. For example, the NLRP3 inflammasome can undergo significant upregulation in the brain, e.g., brain cortical tissue. Aberrant activation of NLRP3 inflammasome signaling has been demonstrated to contribute to pathology in a broad spectrum of neurological diseases (Song, 2017).
[00345] In some embodiments, the invention provides a method for treating neurodegeneration in a subject suffering from neurodegeneration, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS 1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase). The amount and manner of administration of the expression vector (e.g., AAV vector or lentiviral vector or adenoviral vector or liposome or naked DNA) should be effective to treat one or more symptoms or effects from such neurodegeneration.
[00346] Without wishing to be bound by theory, after a nucleic acid encoding an enzyme which has a DNase activity is delivered to the liver, nervous system and/or intestine using the vectors of the invention, the enzyme becomes produced by cells in one or more of these tissues and is secreted into the areas where the enzyme efficiently degrades the concentrated pool of cfDNA present therein (e.g., the periendothelial space, the space of Disse, extracellular nervous tissue space, and intestinal sub-mucus gel layer zone).
[00347] In some embodiments, the use of the expression vectors of the invention is combined with the administration (e.g., intravenous, intra-arterial, intrahepatic, intramuscular, intraperitoneal, or enteral administration) of a DNase protein (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase). Without wishing to be bound by theory, the systemic administration of a DNase protein in conjunction with the above described production of DNase in liver, nervous system and/or intestine using the expression vectors of the invention more quickly degrades cfDNA in both the bloodstream and in any concentrated pool of cfDNA in the liver, nervous system and/or intestine.
[00348] In some embodiments, the expression vectors encoding an enzyme which has a DNase activity can be administered in combination with other treatments useful for treatment of neurodegenerative diseases or other encompassed nervous system dysfunctions (e.g., bipolar disorder, migraine, schizophrenia, epilepsy). Non-limiting examples of such additional treatments include, e.g., 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 (NMD A) receptor inhibitors (e.g., memantine), transcranial magnetic stimulation, transcranial direct current stimulation, etc.
Treatment of Delayed Hypersensitivity Reactions
[00349] In one aspect, the invention provides a method for treating a delayed hypersensitivity reaction (e.g., graft-versus host disease (GVHD)) in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., 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 comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
[00350] Without wishing to be bound by theory, activation of neutrophils can release granule proteins together with double stranded DNA (dsDNA), to form extracellular fibers known as NETs. Complement is activated by NETs. Activation of complement can lead to the symptoms observed in delayed hypersensitivity reactions.
[00351] In some embodiments, the delayed hypersensitivity reaction is associated with an increased level of cfDNA in blood or CSF or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of a healthy age- matched individual or an average level of cfDNA in blood or CSF or intestine of several healthy age-matched individuals). Administration of an expression vector encoding an enzyme which has a DNase activity could inhibit NET formation and lead to less activation of complement. [00352] Without wishing to be bound by theory, the systemic administration of DNase enzyme in conjunction with the above described production of DNase in the liver, nervous system and/or intestine using expression vectors more quickly degrades cfDNA in both the bloodstream and in any concentrated pool of such cfDNA in the liver, nervous system and/or intestine.
[00353] The expression vector encoding DNase enzyme can also be administered in combination with one or more other treatments useful for treating a delayed hypersensitivity reaction such as, e.g., cyclosporine, tacrolimus (FK506), methylprednisolone, prednisone, ATG, sirolimus, mycophenolate mofetil, anti-interleukin-2 (IL-2) receptor, anti-CD5-specific immunotoxin, a pan T-cell ricin A-chain immunotoxin (XomaZyme), the addition of ex vivo cultured mesenchymal cells derived from unrelated donors to conventional steroid therapy, and any combinations thereof.
Treatment of Atherosclerosis
[00354] In one aspect is provided a method for treating atherosclerosis in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., 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 comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
[00355] In some embodiments, atherosclerosis is associated with an increased level of cfDNA in blood or CSF or intestine of the patient, which level is higher than the control level (e.g., the level of cfDNA in blood or cerebrospinal fluid or intestine of a healthy age-matched individual or an average level of cfDNA in blood or cerebrospinal fluid or intestine of several healthy age- matched individuals).
[00356] The expression vector, e.g., Anc80 or another AAV- or lentiviral vector, encoding for DNase enzyme may be administered optionally with additional DNase enzyme. In some embodiments, any one of DNase enzyme (e.g., DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., DNase II-alpha, DNase II-beta), phosphodiesterase I, lactoferrin, or acetylcholinesterase) is also administered, e.g., intravenously. Without wishing to be bound by theory, the systemic administration of DNase enzyme in conjunction with the above described production of DNase in the liver, nervous system and/or intestine more quickly degrades cfDNA in both the bloodstream and in any concentrated pool of such cfDNA in the liver, nervous system and/or intestine.
[00357] The expression vector encoding for DNase enzyme can also be administered in combination with other treatments useful for treating atherosclerosis.
Longevity Improvement
[00358] In one aspect, the invention provides a method for increasing longevity and individual life expectancy and delaying unhealthy manifestations brought by ageing in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of an expression vector encoding an enzyme which has a DNase activity (e.g., DNase I such as, e.g., human recombinant DNase I or bovine pancreatic DNase I), analogues of DNase I (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II (e.g., 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 comprising the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 2 (precursor mutant sequence comprising the secretory signal sequence and also comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 4 (wild-type mature sequence without the secretory signal sequence). In some embodiments, the DNase enzyme comprises the sequence set forth in SEQ ID NO: 5 (mature mutant sequence without the secretory signal sequence and comprising mutations weakening actin-mediated inhibition). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific promoter and/or an enhancer element (e.g., a liver-specific enhancer element). In some embodiments, the DNase enzyme is expressed under the control of a liver-specific/nervous system-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver-specifi c/intestine-specific tandem promoter and/or an enhancer element. In some embodiments, the DNase enzyme is expressed under the control of a liver- specific/nervous system-specific/intestine-specific tandem promoter and/or an enhancer element.
Pharmaceutical Compositions. Formulations and Dosage Forms
[00359] The AAV vectors disclosed herein may be administered in any suitable form, for instance, either as a liquid solution or suspension, as a solid form suitable for solution or suspension in liquid prior to injection. The vectors may be formulated with any appropriate and pharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc. For instance, for injection, a suitable carrier or diluent may be an isotonic solution, a buffer, sterile and pyrogen-free water, or, for instance, a sterile and pyrogen-free phosphate-buffered saline solution. The expression vectors may be formulated in pharmaceutical compositions and dosage forms. Such pharmaceutical compositions and dosage forms can be formulated in any conventional manner using one or more physiologically acceptable carriers and/or excipients. The vector particles may be formulated for administration by, for example, injection. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
[00360] Formulations also include suspensions in liquid or emulsified liquids. The active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.
[00361] The formulations used in the methods of the 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 a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
[00362] Pharmaceutical compositions suitable for parenteral administration may further comprise one or more additional active ingredients (e.g., a DNase protein or another active compound) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the 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 injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
[00363] These compositions can also contain preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may 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 desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
EXAMPLES
[00364] The present invention is also described and demonstrated by way of 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 of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
EXAMPLE 1: Limited ability of DNase enzyme to digest circulating cell free DNA (cfDNA) in tumor patients
[00365] Nine patients with advanced metastatic disease who were admitted to the department of thoracic surgery of Kostushko Hospital (St. Petersburg, Russia) were given a 3 week course of intravenous treatment with bovine pancreatic DNase I protein which has specific activity of 2500 KU/mg (Samson, Russia). The treatment proceeded according following schedule: in week I, 300 mg was administered daily via six 30 minute IV infusions; in week II, 450 mg was administered daily via six 30 minute IV infusions; and in week III, 600 mg was administered daily via six 30 minute IV infusions.
[00366] 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 following last DNase I protein infusion. DNA was isolated from blood plasma using a classic phenol-chloroform method. Electrophoresis of blood cfDNA was performed on 1% agarose gel. DNA was visualized with ethidium bromide. The treatment efficacy was evaluated one week after the last DNase I protein infusion using a spiral CT scan based on RECIST criteria (see, e.g., RECIST 1.1, published in January 2009, www.irrecist.com). The patient summaries and individual blood electrophoretic data are presented in Figures 2A-2IJ. Overall, there was little decrease in the diameter of solid tumors according to the RECIST outcome (Figures 2A-2H). In most patients, there was still cfDNA in circulation after Week III.
[00367] Despite a continuous ascending high dose IV infusion of DNase I enzyme, only one patient with recurrent rectal carcinoma (PVIII) showed complete clearance of circulating cfDNA from plasma as measured by gel electrophoresis. Patient PVIII had a stable disease with no evidence of progression according RECIST criteria. Three other patients who qualified as stable disease patients according to RECIST criteria (PIX, PVII, PI) had little or no increase of circulating cfDNA from plasma as measured by gel electrophoresis.
[00368] Five patients who qualified as progressive disease patients according RECIST criteria (PII, PHI, PIV, PV, and PVI) had a substantial increase of circulating cfDNA from plasma as measured by gel electrophoresis. Also, in 8 out of 9 patients (88%), 3 week continuous intravenous infusions of ascending high doses of DNase enzyme failed to provide a therapeutically effective clearance of circulating cfDNA from plasma.
EXAMPLE 2: Accumulation of cell free DNA (cfDNA) in the liver of tumor bearing animals
[00369] Approximately 12 x 106 of LS174T human colon carcinoma cells were inoculated subcutaneously into the left flank of each of twelve female nu/nu mice (IBCH RAS breed) that were 8 weeks old. The mice received a single IV injection of 99 mTc labeled anti DNA antibodies according the following schedule: three mice received the injection at D5, three mice received the injection at DIO, three mice received the injection at D15 and three mice received the injection at D25. Mice were sacrificed two hours following the injection and tissues and blood were collected for gamma counting.
[00370] AC-30-10 DNA binding antibodies (Genetex) were labeled with 99mTc as follows:
0.024mg/ml solution of antibody in 0.3M citrate buffer (pH4.2) was mixed with lmCi of 99mTc- pertechnetate in 0.25 ml saline and mixed 10-mg/ml solution of SnC12 dihydrate in 0.1N HC1. After 5 min incubation at room temperature, 1.9 ml of saline and 825 mΐ of IgM solution were added. The single injection doses were prepared as follows: 16.6pCi (36852000CPM approx. 0.047pg of 99mTc-IgM) of 99mTc-IgM solution was mixed with 3.3 pg of unlabeled IgM and diluted with saline up to 100 pL volume. Gamma signal was quantified in each tissue using auto gamma counter. Measurement was done in CMP units. Concentrations of 99mTc IgM was quantified based gamma counting data, tissue weight, specific radioactivity of the IgM preparations administered and 99mTc half-life.
[00371] The tissue distribution of radioactivity is presented in Table 1 below:
Table 1: Accumulation of anti-DNA antibodies in tissues of tumor bearing mice. (% of Injected dose /s of tissue).
Figure imgf000116_0001
[00372] Figure 3 shows a gamma-immunoscintigraphic image of a tumor-bearing mouse obtained at Day 15 one hour following injection of 99mTc labeled anti-DNA antibodies. The liver zone (b) in the middle-right portion of the image produced a radioactive signal that is greater than that of tumor zone (a) in the lower left portion of the image. Thus, the liver showed the most accumulation of circulating cfDNA.
EXAMPLE 3: Sourcing of tumor originating cfDNA from liver to blood
[00373] Six 6-week-old female nude mice (IBCH RAS breed) were given subcutaneous injections with 2xl06 of MCF-7 cells into the left flank. At Day 20 following the injection tumor was surgically removed. At Day 25 all mice were given anesthesia and 500 pi blood samples were taken from portal vein, hepatic artery and hepatic vein of each mouse. Plasma was separated from the blood cells by centrifugation at 2000 g for 10 minutes; the clear plasma phase on top was transferred to a new tube, and centrifuged at 14000 g. The supernatant was transferred to a fresh tube for DNA extraction. cfDNA was extracted from blood plasma samples using QIAamp DNA Blood Mini Kit according to manufacturer’s instructions. The human beta globin sequence in cell free DNA was quantified in each sample using RT-PCR (iCycler iQ56 Bio-Rad) with human beta globin primer CAACTTCATCCACGTTCACC (SEQ ID NO: 10). The Ct values of human beta globin sequence in blood plasma sampled from different vessels are presented at the Table 2 below:
Table 2:
Figure imgf000116_0002
[00374] Even though the tumor was surgically removed, only in one mouse was the blood plasma free from circulating cfDNA of human origin. MCF-7 tumors do not produce metastasis when growing subcutaneously. The absence of metastatic nodules in liver or lungs was further confirmed by macroscopic and microscopic examination. Circulating cfDNA originating from the human tumor xenotransplant was detectable in the blood of mice, with the highest quantities detectable in the hepatic vein. These results confirm that liver sinusoids are a depot source of circulating cfDNA.
EXAMPLE 4: Effects of transgenic delivery of DNase and intravenous delivery of
Figure imgf000117_0001
recombinant DNase protein in a pancreatic cancer model
Figure imgf000117_0002
[00375] An AAV vector for liver specific transgenic expression of DNase I (4658_pAAV- AFP-Alb-huDNaseI_mut) was custom manufactured by Sirion Biotech (Martinsried, Germany). A vector map is shown in Figure 5A. The AAV vector is based on AAV serotype 8 which effectively targets liver. The vector comprises the sequence set forth in SEQ ID NO: 2, which codes for a mutant human DNase I enzyme which comprises several mutations which make the enzyme less sensitive to actin inhibition and more catalytically active. The DNase I coding sequence further comprises a secretory signal sequence. The DNase I coding sequence is operably linked at the 5' end to mAFP/Alb enhancer/promoter system (liver-specific mouse alpha fetoprotein enhancer and liver-specific mouse minimal albumin promoter) and at the 3 ' end to hGH polyA signal. The expression cassette is further flanked by AA8 L-ITR and R-ITR.
[00376] The huDNasel mut expression cassette was cloned into an AAV transfer vector and produced in 293T cells via co-transfection of a transfer vector with AAV8 packaging plasmids. Viral particles were purified and concentrated up to 1x1013 GC/ml and kept frozen in PBS.
[00377] A control vector identical to 4658 _pAAV-AFP-Alb-huDNaseI_mut but with non specific for liver CMVE/CAG promoter/enhancer system instead of the liver-specific mAFP/Alb enhancer/promoter system was also produced.
[00378] Human recombinant DNase I protein (rhDNasel) was produced by Catalent (Madison, USA) and kept frozen at lOmg/ml stock solution in ImM CaC12 and 150mM NaCl.
[00379] 56 female B ALB/c nude mice (IBCH RAN) of 6-8 weeks age and 18-22 g weight were used in experiment. The mice were kept in individual ventilation cages at controlled temperature and humidity with four animals in each cage. Each mouse was inoculated subcutaneously at the right flank with MIA PaCa-2 pancreatic tumor cells (1 x 107) in 0.2 ml of PBS supplemented with BD Matrigel (1 : 1) for tumor development. The treatments were started on day 17 after tumor inoculation when the average tumor size reached approximately 170mm3. Each group consisted of eight tumor-bearing mice. The tested vectors and compositions were administered to the mice according to the predetermined regimen as shown in Table 3, below:
Table 3: Experimental Design
Figure imgf000118_0001
[00380] umor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V = 0.5 a x b2 where a and b are the long and short diameters of the tumor, respectively. At the time of routine monitoring, the animals were checked daily for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption by visual inspection only, loss or gain of body weight (measured twice weekly), eye/hair matting, and any other abnormal effects. On day 35, the study was terminated, the mice were humanely sacrificed by CO2, and blood was sampled from the central and hepatic veins. Liver tissues were also sampled and immersed into formalin for fixing.
[00381] Circulating cfDNA was measured in plasma using a 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 diluted first at 1 : 1000 in dimethyl sulphoxide and then at 1 :8 in phosphate- buffered saline. 10 mΐ of plasma samples were applied to 96-well plates. 40 mΐ of diluted
SYBR® Gold was added to each well (final dilution 1 : 10,000) and fluorescence was measured with a 96 well fluorometer at an emission wavelength of 535 nm and an excitation wavelength of 485 nm.
[00382] Serum DNase I activity was measured using ORG590 (Orgentec) according to the manufacturer's protocol. Detection was performed using microplate photometer (Multiscan FC) at 450 nm with a correction wavelength of 620 nm. cfDNA in the liver tissue was quantified by immunohistochemistry (IHC) in liver paraffin slides using AC-30-10 mouse IgG DNA binding antibodies (Sigma) as primary antibodies and Novolink Polymer Detection Systems Novocastra (Leica Biosystems).
[00383] The mean tumor volume over time in female BALB/c nude mice bearing MIA PaCa-2 xenografts dosed with tested vectors and composition (as per Table 3) is shown in Table 4, below:
Table 4: The mean tumor volume over time
Figure imgf000119_0001
[00384] The number of animals with body weight loss at Day 35 in each group of in female
BALB/c nude mice bearing MIA PaCa-2 xenografts and dosed with tested vectors and composition (as per Table 3) is shown in Table 5, below:
Table 5: The number of animals with body weight loss at Day 35
Figure imgf000119_0002
[00385] he results of quantification of circulating cfDNA in blood and DNase I enzymatic activity in central and hepatic vein are summarized in Table 6, below:
Table 6:
Figure imgf000119_0003
Group 7
Figure imgf000120_0001
1.15
Figure imgf000120_0002
1.05
Figure imgf000120_0003
[00386] The IHC microphotographs of liver tissue from different groups are presented in
Figure 4.
[00387] Data from the Tables 4-6, above, and Figure 4 clearly show that liver-specific transgenic expression of DNase I enzyme in combination with chemotherapy not only produced significantly superior results in terms of antitumor efficacy but were also able to completely ameliorate chemotherapy -induced weight loss. Recombinant DNase I enzyme given twice daily in three ascending doses, as well as transgenic expression of DNase I which is not targeted to the liver, demonstrated significantly less antitumor efficacy and amelioration of chemotherapy- induced toxicity than liver-specific transgenic expression of DNase I. Mice treated with liver- specific DNasel mAFP/Alb AAV8 vector had the lowest amount of circulating cfDNA, the lowest amount of cfDNA remaining in the liver, and the highest DNase I enzymatic activity in the hepatic vein.
EXAMPLE 5: Effect of transgenic delivery of DNase on orthotopic and non-orthotopic
Figure imgf000120_0004
growth of pancreatic cancer
[00388] Twelve 8-week old SCID mice (IBCH RAS) divided into 4 experimental groups were used in the experiment. Six mice received 4xl0e6 MIA PaCa-2 pancreatic cancer cells expressing luciferase subcutaneously at the right femur in 100 mΐ of Matrigel (non-orthotopic model). Six mice were implanted in the body -tail of the pancreases with 4x10e6 MIA PaCa-2 pancreatic cancer cells expressing luciferase in 100 mΐ of Matrigel (orthotopic model). The next day, 3 mice with orthotopic MIA PaCa-2 implant received IV injection of mAFP/Alb AAV8 vector 4658_pAAV-AFP-Alb-huDNaseI_mut at l,00xl0el l GC/kg dose and 3 mice with orthotopic MIA PaCa-2 implant received a placebo (PBS) injection. Three mice with non-orthotopic MIA PaCa-2 implant received an IV injection of DNasel mAFP/Alb AAV8 vector at 1.OOxlOel 1 GC/kg dose and three mice with non-orthotopic MIA PaCa-2 implant received a placebo (PBS) injection. Four weeks later at the end of the experiment, tumors were assessed by bioluminescence imaging using an AMI-1000 imaging system. The bioluminescence data are summarized in Table 7, below, with typical images presented in Figure 6.
Table 7:
Figure imgf000120_0005
Figure imgf000121_0002
[00389] Thus, transgenic delivery of DNase I enzyme to the liver completely blocks the development of tumor growth in an orthotopic fashion in an area drained by the portal vein, i.e., the pancreas. Development of same pancreatic cancer xenotransplant in non-orthotopic fashion was also significantly suppressed by injection of DNase I mAFP/Alb AAV8 but not to such an extent as to eliminate tumor growth completely.
EXAMPLE 6: Effects of liver and pulmonary delivery of DNase transgene on growth of
Figure imgf000121_0001
breast carcinoma in a breast cancer model
[00390] ADV-207186 vector was purchased from Vector Biolabs (Malvern, PA). A vector map is shown in Figure 5B. This vector is based on human adenovirus Type 5 (dEl/E3) (which has a natural liver tropism) and contains wild-type human DNase I coding sequence which comprises a secretory signal sequence (SEQ ID NO: l). The DNase I coding sequence is operably linked at the 5' end to liver non-specific CMV promoter and at the 3' end to hGH poly A signal. The expression cassette is further flanked byATT-Ll and ATT-L2.
[00391] MMTV-PyMT female mice IBCH RAS breed were used for the study. When each mouse developed at least three palpable tumors of at least 3 mm c 5 mm, which typically occurred at 8 weeks of age, six mice were injected intravenously with of 0.2xl0u PFU per mice of ADV- 207186 and six mice were injected with 0.2x1011 PFU per mice of ADV-207186 intranasally under anaesthesia. Mice were sacrificed at 14 weeks of age. Six mice were used as control. Tumor size was measured in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V = 0.5 a x b2 where a and b are the long and short diameters of the tumor, respectively. TGI was calculated for each group using the formula: TGI (%) = [l-(Ti-TO)/ (Vi-V0)] c 100; Ti is the average tumor volume of a treatment group on a given day, TO is the average tumor volume of the treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the day of treatment start. Blood was sampled from the central vein at the termination of the study. Circulating cfDNA and serum DNase I activity were measured as described in Example 4. The data are reported in Table 8, below.
Table 8:
Cell Serum
Tumor
TGI free DNase
Treatment Volume
(%) DNA, activity
(mm3)
ng/ml mKuU/ l
Control 355±32 - 137+13 2.15
DNase I Ad Type 5 (dEl/E3) AV IN 209+28 78 110+7 4.95
DNase I Ad Type 5 (dEl/E3) AV IV 54+22 164 34+11 5.2
[00392] The data show that targeting of the liver with an adenoviral vector harboring
DNasel transgene provides superior breast tumor growth inhibition and clearance of blood from circulating cfDNA as compared to the delivery of DNase I transgene to bronchoalveolar system, despite approximately the same level of DNase enzymatic activity in blood.
EXAMPLE 7: Effects of transgenic expression of actin resistant hyperactive DNase in liver
Figure imgf000122_0001
and in muscles on the level of microbial cfDNA and the level of neuroinflammation.
[00393] Increased intestinal permeability and translocation of gastrointestinal microbiota and damage-associated molecular patterns (DAMPs) of microbial origin, including microbial cfDNA, trigger neuroinflammation and spectra of neurodegenerative disease. cfDNA originating from gastrointestinal microbiota are subject to“first pass” through porto-sinusoidal circulation. The effects of muscle and liver delivery of hyperactive actin-resistant DNase I transgene, the effects of injections of wild-type DNase I protein on the level of microbial cfDNA in brain and liver, as well as the level of neuroinflammation in CSF and brain following bacteriophage-induced leaky gut syndrome were further investigated.
[00394] Healthy adult male Wistar rats (n = 24; 12-week-old, 240-280 g) were maintained in individual cages in a P3 room under a 12-h light/dark cycle, at a temperature of 22 to 25 °C and 60 ± 5% atmospheric humidity. All animals had free access to food and water according to the Guide for the Care and Use of Laboratory Animals.
[00395] The DNA molecule coding for the hyperactive actin-resistant DNase I mutant (SEQ ID NO: 5) with DNase I secretory signal sequence replaced by the IL2 secretory signal sequence was synthesized (GeneCust) and cloned into the pLV2 lentiviral vector (Clontech) under the control of the EFla promoter (not 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 lentiviral vector pLV2- IL2ss-DNAseI mut using Lipofectamine 2000 (Life Technology). 5 ml of supernatant was collected at 48 hours after transfection. Lentiviruses were collected by filtering through a 0.45 pm polyethersulfone membrane filter (Millipore), centrifugation during 30 m at 20,000 g. The resulting pellet was resuspended in 1 ml of PBS.
[00396] Leaky gut was induced in 24 rats using a bacteriophage cocktail against Enterobacteriaceae, Staphylococcaceae, and Streptococcaceae families so as to induce microbiota diseases. The cocktail included a Salmonella bacteriophage cocktail 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 Pyobacteriophage phage cocktail 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, 1x106 plaque-forming units/mL of each phage cocktail) were added to drinking water according to the manufacturer’s instruction and administered orally for 10 days. At day 7 of phage treatment, rats were randomized into 4 groups as follows: six rats received intrahepatic injection of 1.0 c 1012 LVP in a volume of 200 pi; 6 rats received injection of 1.0 x 1012 LVP in a volume of 200 pi into a thigh muscle; six rats received twice daily intravenous injections of recombinant DNase I protein at 50mg/kg between day 7 and day 12. The remaining six rats were used as the untreated leaky gut control. An additional six healthy rats were used as a healthy control. At day 12, all rats were sacrificed and blood samples were collected for measurement of the DNase activity in blood. Brain autopsies were collected to perform NLRP3 inflammasome expression analysis and 16S universal bacterial RNA gene quantification.
[00397] The NLRP3 inflammasome is considered a key contributor to the development of neuroinflammation. Aberrant activation of NLRP3 inflammasome signaling has been demonstrated to contribute to pathology in abroad spectrum of neurological diseases (Song, 2017). NLRP3 expression was assessed by collection of 50mg cortical tissue samples. RNA was isolated using the SV total RNA isolation system (Promega). Superscript 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 forward NLRP3 primer and 5'- CACTGTGGGTCCTTCATCTTT-3' (SEQ ID NO: 12) as reverse NLRP3 primer. 16S universal bacterial RNA gene primers (forward: 5'-
TCGTCGGC AGCGT C AGAT GT GT AT A AG AGAC AGCC T AC GGGN GGC W GC AG-3 ' (SEQ ID NO: 13); reverse: 5'-
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 14)) were used to quantify presence of DNA of microbial origin in liver and brain. Gene expression analyses were done using the comparative Ct method.
[00398] Serum DNase I activity was measured using ORG590 (Orgentec) according to the manufacturer's protocol. Detection was performed using microplate photometer (Multiscan FC) at 450 nm with a correction wavelength of 620 nm. Results are summarized at the Table 9, below:
Table 9:
Figure imgf000124_0002
[00399] The data show that induction of a leaky gut led to suppression of endogenous blood
DNase enzyme activity, a significant increase of microbial cfDNA presence in liver and brain cortex and significant upregulation of NLRP3 inflammasome component in brain cortical tissue. Delivery of a hyperactive actin resistant DNase I lentiviral transgene to liver, as compared to the delivery of the same transgene to muscle tissue or to intravenous injections of DNase I protein, almost completely prevented increase of microbial cfDNA in liver and brain and brain inflammasome activation, despite maintaining of comparable DNase enzymatic activity in the central bloodstream.
EXAMPLE 8: Effects of transgenic delivery of hyperactive actin resistant DNase and
Figure imgf000124_0001
intravenous delivery of recombinant DNase protein on GVHD
[00400] The MHC class I and II disparate model, C57BL/6 (H-2b) to BALB/c (H-2d), was used to establish GVHD. All recipients were age-matched females with an age of 2-6 months at the time of bone marrow transplant (BMT). The single cell suspensions of bone marrow cells and splenocytes were prepared in saline for injection. To generate BMT chimeras, recipient BALB/c mice received 1200 rad TBI (137Cs source) split into 2 doses. The mice then received cells from C57BL/6 mice: 5x106 bone marrow cells and 10x 106 splenocytes.
[00401] The experiments were designed to compare the following two treatments: wild-type mouse DNase I protein (50 mg/kg) daily and liver-specific wild-type mouse DNase I mAFP/Alb AAV8 vector as a single intravenous injection. Starting the same day as the BMT, recipients were treated with DNase I via IP route twice a day or by single intravenous delivery of DNase I mAFP/Alb AAV8 at 1.00x1011 GC/kg dose. For histopathology mice were sacrificed on day 16 post-transplant. Tissues were placed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxilin 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 on day 16 post transplantation. Cells recovered from these tissues were stained with CD3, CD4, CD8 and H-2Db antibodies. The numbers of donor-derived T cells (H-2Db+) and CD4+/H-2Db+ and CD8+/H-2Db+ subsets were determined by FACS.
[00402] The survival data are presented in Table 10, below:
Table 10:
Figure imgf000125_0002
[00403] Data on liver histopathology in DNase I-treated
Figure imgf000125_0001
DNase I mAFP/Alb AAV8 treated recipient mice are presented in Figure 7. The left panel shows lymphoplasmacytic infiltration of portal tracts, segmental loss of bile duct epithelial cells, areas of focal necrosis of hepatocytes, and significant amount of nucleiphilic material at the vascular interface in animals treated with DNase I protein only (and not DNasel mAFP/Alb AAV8). The right panel shows that the livers from the animals treated with DNasel mAFP/Alb AAV8 have much lower inflammatory cell density, no necrosis and much less nucleiphilic material at the vascular interface.
[00404] Data on donor-derived T cells in DNase I protein-treated and DNase I mAFP/Alb
AAV8-treated recipient mice are presented in Figure 8. In particular, Figure 8 shows a significant reduction in the total numbers of donor-derived T cells, donor-derived CD4+ and CD8+ T cells in the peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Payer's patches (PP) in DNase I mAFP/Alb AAV8-treated recipients as compared with DNase I protein treated recipients.
[00405] The results show that liver-specific transgenic expression of DNase I enzyme results in doubling of animal survival comparing with DNase I protein treatment. Such survival benefit was accompanied with a significant reduction of total numbers of donor-derived T cells, donor-derived CD4+ and CD8+ T cells in the PLN, MLN and PP in DNase I mAFP/Alb AAV8 treated recipients as compared with DNase I protein treated recipients. In the liver, DNase I mAFP/Alb AAV8 treatment resulted in a significant reduction of lymphoplasmacytic infiltration of portal tracts, prevention of cell loss and clearance of vascular interface from nucleophilic material.
EXAMPLE 9: Cloning of AAV Vector: ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader- WPRE Xinact (VR-18013AD)
[00406] An AAV Promoterless Expression Vector pAAV-MCS-Promoterless was obtained from Cell Bio labs (Cat no. VPK-411; Figure 11). The ITR integrity was checked by performing a Smal restriction enzyme digest followed by gel purification to assess for the presence of the following expected bands: 2681 bp, 666 bp, and 112 bp. The expected 11 bp band would not be seen. If the expected bands were present, the plasmid was amplified by growing in SURE cells (Agilent Technologies) or other similar bacteria. A 5' EcoRI site and a 3' Hindlll site were added to the sequence of SEQ ID NO: 30 (ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader- WPRE Xinact). The polynucleotide of SEQ ID NO: 30 (with 5' EcoRI and 3' Hindlll sites) was synthesized and then digested with EcoRI-HF and Hindlll-HF (available from NEB). The 2.2 kb band was gel-extracted. The pAAV-MCS Promoterless vector was digested with EcoRI-HF and Hindlll-HF, with the resulting 3.5 kb band gel extracted. The digested vector and the polynucleotide of SEQ ID NO: 30 (insert) were ligated for one hour at room temperature in a 3 : 1 insert to vector molar ratio. The ligated construct was transformed into SURE electrocompetent cells and plated on LB-Agar (Amp) plates. Colonies were selected and minipreps were prepared for screening. First, a Smal digest was performed for ITR integrity to determine if the following expected bands were present: 2681 bp, 1650 bp, and 1311 bp. Then, an EcoRI-HF and Hindlll-HF digest was performed to assess if the insert was present. If the insert was present, the following bands would be seen: 3435 bp and 2218 bp. A map of the VR-18013AD vector is shown in Figure 10B.
[00407] Clones were selected if the expected bands were present in each of the Smal digest and the EcoRI-HF and Hindlll-HF digest. For selected clones, a glycerol stock was prepared for long-term storage. The plasmid was also amplified and sequenced. Clones comprising a plasmid with the correct sequence were then cultured and purified by endo-free Gigaprep (ALTA Biotech). The purified plasmid was suitable for packaging by MEE into AAV capsids.
EXAMPLE 10: Cloning of AAV Vector: ApoEHCR enhancer-hAAT promoter-hDNasel [wild tvpeVWPRE Xinact iVR-18014AD)
[00408] An AAV Promoterless Expression Vector pAAV-MCS-Promoterless was obtained from Cell Bio labs (Cat no. VPK-411; Figure 11). The ITR integrity was checked with Smal digest and gel-purified to assess whether the plasmid produced the following expected bands: 2681 bp, 666 bp, and 112 bp. The expected 11 bp band would not be seen. If the expected bands were present, the plasmid was amplified by growing in SURE cells (Agilent Technologies) or other similar bacteria. A 5' EcoRI site and a 3' Hindlll site were added to the sequence of SEQ ID NO: 31 (ApoEHCR enhancer-hAAT promoter-hDNasel (wild type)-WPRE Xinact). The polynucleotide of SEQ ID NO: 31 (with 5' EcoRI and 3' Hindlll sites) was synthesized and then digested with EcoRI-HF and Hindlll-HF (available from NEB). The 2.2 kb band was gel-extracted. The pAAV-MCS Promoterless vector was digested with EcoRI-HF and Hindlll-HF, with the resulting 3.5 kb band gel extracted. The digested vector and the polynucleotide of SEQ ID NO: 31 (insert) were ligated for one hour at room temperature in a 3 : 1 insert to vector molar ratio. The ligated construct was transformed into SURE electrocompetent cells and plated on LB-Agar (Amp) plates. Colonies were selected and minipreps were prepared for screening. First, a Smal digest was performed for ITR integrity to determine if the following expected bands were present: 2681 bp, 1650 bp, and 1311 bp. Then, an EcoRI-HF and Hindlll-HF digest was performed to assess if the insert was present. If the insert was present, the following bands would be seen: 3435 bp and 2218 bp. A map of the VR-18014AD vector is shown in Figure IOC.
[00409] Clones were selected if the expected bands were present for each of the Smal digest and the EcoRI-HF and Hindlll-HF digest. For selected clones, a glycerol stock was prepared for long-term storage. The plasmid was also amplified and sequenced. Clones comprising a plasmid with the correct sequence were then cultured and purified by endo-free Gigaprep (ALTA Biotech). The purified plasmid was suitable for packaging by MEE into AAV capsids.
EXAMPLE 11: Manufacture of ApoEHCR enhancer-hAAT promoter-hDNasel
(hyperactivelcorrect leader- WPRE Xinact (VR-18013AD) and ApoEHCR enhancer-hAAT promoter-hDNasel (wild typeVWPRE Xinact AAV vectors (VR-18014AD)
[00410] The VR-18013AD and VR-18014AD expression plasmids were manufactured by GenScript USA Inc. (Piscataway, NJ 08854, USA). The corresponding hDNAse I (hyperactive) VR-18013AD AAV vector and hDNAsel (wild-type) VR-18014AD AAV vectors were produced using large-scale polyethylenimine transfections of Anc80L65 AAV cis, AAV trans, and adenovirus helper plasmids in near-confluent monolayers of HEK293 cells with further purification on an iodixanol gradient at scale using traditional protocols. The purified ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact (VR-18013AD; transgene cassette of VR-18013AD comprising SEQ ID NO: 30 as shown in Figure 10A; see also the vector map in Figure 10B) and ApoEHCR enhancer-hAAT promoter-hDNasel (wild type)- WPRE Xinact (VR-18014AD; transgene cassette of VR-18014AD comprising SEQ ID NO: 31 as shown in Figure IOC; see also the vector map shown in Figure 10D) vectors were reformulated in PBS supplemented with 35 mM NaCl and 0.001% PF68, and comprising 1.0 xl013 GC/ml (VR- 18014AD) and 5.0 xl012 GC/ml (VR-18013AD).
[00411] The purified VR- 18013 AD AAV vector was analyzed by SDS-PAGE, with the results shown in Figure 12. Three bands of 60, 72, and 90 kDa were observed in a ratio of about 1 : 1 : 10, which corresponds to the VP 1-3 proteins.
[00412] 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-hDNasel (hyperactive)correct leader-WPRE Xinact (VR-18013AD) and ApoEHCR enhancer-hAAT promoter-hDNasel [wild typeVWPRE Xinact (VR-18014AD)
[00413] The transduction efficiency of the following two gene therapy vectors were studied:
ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact and ApoEHCR enhancer-hAAT promoter-hDNasel (wild type)-WPRE Xinact. These gene therapy vector candidates were studied in vitro using HEP G2 human hepatoma derived cell line (ATCC HB-8065) and SK-HEP1 human hepatic adenocarcinoma of endothelial origin (ATCC HTB-52). Cells were seeded to 48 well plates at 5x105 cell/ml. and grown in DMEM Adv, 10% FBS media. 24 h. post seed wells were supplemented with VR-18013AD or VR-18014AD using MOI 104 GC/cell ,105 GC/cell and 106 GC/cell or blank control. Each well was in triplicate.
[00414] The DNase activity was analyzed using fluorescent probe (Terekhov et ah, PNAS 2017; www.pnas.org/content/pnas/114/10/2550.full.pdf) representing hairpin oligonucleotide labeled with fluorescent dye and quencher. The increase in fluorescence was monitored using Variscan Flash plate reader (Thermo Scientific). The culture medium was diluted from 20 to 200 folds by reaction buffer 20 mM Tris-HCl pH 7.5, 1 mM MnC12 and 0.3 mM CaC12. 0.25 mM concentration of florescent probe was used. Complete hydrolysis of florescent probe in reaction conditions resulted in 28 RFU fluorescence under assay conditions. The background probe fluorescence was below 0.3 RFU. Calibration curve for DNase concentration estimation was obtained using serial dilution of pure DNase I Pulmozyme“Domase alfa” (Roche). The DNase concentration in samples was estimated using linear regression. The data are shown in Figures 13 A and 13B.
[00415] The data in Figures 13 A and 13B show that ApoEHCR enhancer-hAAT promoter- hDNasel (hyperactive)correct leader-WPRE Xinact (VR-18013AD) and ApoEHCR enhancer- hAAT promoter-hDNasel (wild type)-WPRE Xinact (VR-18014AD) vectors efficiently transduce cells of hepatocyte origin only and trigger high yield expression and secretion of biologically enhanced DNase I enzyme or wild type DNase I enzyme from transduced G2 cells. DNase concentration estimation was obtained using serial dilution of pure DNase I Pulmozyme“Dornase alfa” (Roche). Transduction appears to be safe for cells since during 72 hours of observation there were no any signs of cell death even at highest VR-18013AD and VR-18014AD doses of 106GC/cell and no morphologic difference between any of transduced cells versus control cells.
EXAMPLE 13: Use of ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact (VR-18013AD) for treatment of Alzheimer’s disease
[00416] The therapeutic effect of single IV injection of VR- 18013 AD (ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact) was studied in 3xTg-AD mice expressing three mutant human transgenes: presenilin (PS)1 (M146V), bARR (Swedish mutation) and tau (P301L). These transgenic mice develop both amyloid and tau pathologies.
[00417] The effect of VR-18013AD on learning and memory deficits was evaluated by performing the following behavioral tests: 1) the Y-maze alternation test, which measures the spatial working memory and is dependent on the integrity of the limbic and non-limbic pathways; and 2) Contextual fear conditioning (CFC), which is highly dependent on hippocampal and amygdala function, but also on the cortex.
[00418] The objective of the study was to quantify the effect of a single VR-18013AD IV injection on amyloid deposition and tau phosphorylation in 3xTg-AD via immunohistochemistry. The impact of VR-18013AD on tau hyperphosphorylation was studied in 3xTg-AD mice using the AT8 mAh antibody specific for pSer202/Thr205 epitopes, which are among the earliest phosphorylated tau epitopes in patients with AD. Total tau was also determined in treated and untreated 3xTg-AD mice. Overactive microglia can induce highly-detrimental neurotoxic effects; hence the attenuation of the microglial response has been proposed as a potential therapeutic approach in AD.
[00419] The design of experiment is summarized in Table 11 below:
Table 11
Figure imgf000130_0001
[00420] At day 0 the following groups o: mice received treatment: (a) 13 six-month old wild type (WT) mice was injected with phosphate buffered saline (PBS); (b) 15 six-month old 3xTg-AD mice were injected with phosphate buffered saline (PBS) only; and (c) 10 six-month- old 3xTg-AD mice injected with VR-18013AD at 1.0x1011 GC/per mouse dose. The data are summarized as follows. In Figure 14, the Y Maze behavioral test shows a slight improvement in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice. In Figure 15, the results of the contextual fear condition test shows a slight improvement in VR- 18013 AD treated 3xTg-AD mice as compared with 3xTg-AD control mice. The data of Figure 16 show that microglia activation was significantly reduced in VR-18013AD treated 3xTg-AD mice as compared with 3xTg-AD control mice, especially in cortical area of brain.
[00421] The data of Figure 17 show that there is a significant reduction of microglia activation in the cortical brain area of VR-18013AD treated 3xTg-AD mice vs 3xTg-AD control mice. The data ofFigure 18 show that treatment with VR-18013AD significantly reduces amyloid deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice. The data of Figure 19 show that treatment with VR-18013AD significantly reduces hyperphosphorylated Tau deposition in VR- 18013 AD treated 3xTg-AD mice vs 3xTg-AD control mice. The photographs in Figure 20 show significant reduction of p-tau in hippocampal region after VR- 18013 AD treated treatment. In summary, a single IV injection of VR-18013AD shows significant therapeutic activity in an Alzheimer’s disease mouse model.
EXAMPLE 14: VR-18014AD as treatment of peritoneal carcinomatosis
[00422] Twelve BALB/c mice (20- 22 g) of the IBCH RAS breed were housed at a controlled temperature of 20-22°C. At day 1, mice were injected intraperitoneally with 200 pi of a cell suspension comprising 5 x 105 CT26 NM!J induced undifferentiated colon carcinoma cells. At day 6 following tumor transplant, mice were randomly assigned to the control group (six mice, single IV injection of PBS) or the treatment group (six mice, single IV injection of VR-18014AD at 1.0 xlO11 GC/per mouse dose). A morphometric assessment and measurement of the number of tumor nodes in each group were performed on day 16 after tumor cell inoculation following mice euthanasia. Mice from the control group had 14-22 tumor nodes per mouse, with part of the nodes forming agglomerates. In contrast VR-18014AD treated mice had just 2-5 nodes of smaller size with no signs of agglomerate formation. Typical morphometric images of control and VR- 18014AD-treated mice are shown in Figure 21.
EXAMPLE 15: cfDNA extracted from mice with induced leaky gut triggers Tau aggregation, and liver-specific DNase gene transfer suppresses the ability of cfDNA to induce Tau aggregation.
[00423] Aggregated tau protein is associated with over 20 neurological disorders, including Alzheimer's disease. Leaky gut was induced in 18 rats as specified in Example 7. The tested vectors and compositions were administered to the mice according to a predetermined regimen as shown in Table 12 below:
Table 12
Figure imgf000132_0001
[00424] cfDNA extraction was performed as follows. Peripheral blood samples (5ml) were collected through cardiopuncture in cell free DNA BCT tubes. Plasma was separated from the cellular fraction with a first centrifugation at 800g at 4°C for 10 min. The plasma was further centrifuged at 13,000g at 4°C for 10 min to pellet and remove any remaining cells, and then stored in 1 ml aliquots at -80°C until DNA extraction.
[00425] For cfDNA extraction, QIAamp Circulating Nucleic Acid (Qiagen) was used following the manufacturer’s protocol.
[00426] An evaluation of cfDNA on Tau aggregation was performed. Full-length human Tau (4R2N) was used. For the assay, a solution comprising 1 mg/ml of Tau monomer in the presence of 2.5 mM heparin in 100 mM HEPES pH 7.4, 100 mM NaCl was incubated in the presence of 25 mΐ of extracted cfDNA at 20°C with cyclic agitation (1 min shaking at 500 rpm followed by 29 min without shaking). The total sample volume was 200 mΐ; the solution also comprised 5 mM Thioflavin T. Aggregation was followed over time by ThT fluorescence (excitation 435nm, emission 485nm).
[00427] The results are summarized in Table 13 below, with both mean and SEM of three replicates indicated:
Table 13
Figure imgf000133_0001
[00428] The data of Table 13 clearly demonstrate that liver-specific transgenic expression of DNase I enzyme significantly suppress the ability of cfDNA extracted from blood of animals with leaky gut to promote Tau aggregation while injections of recombinant DNasel enzyme as well as use of non-liver specific transgenic expression of DNase I enzyme are much less effective.
EXAMPLE 16: Development of AAV vector designed to provide DNase expression in nervous system and /or intestine [00429] In order to develop recombinant adeno-associated virus (rAAV) expression vector VR- 18015 AD containing a liver-specifi c/nervous system-specific tandem promoter, the ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact transgene cassette (SEQ ID NO: 30) was modified by insertion of 485 bp sequence of human synapsin promoter (SEQ ID NO: 36) (P. Colella et al., AAV Gene Transfer with Tandem Promoter Design Prevents Anti-Transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice. Molecular Therapy: Methods & Clinical Development Vol. 12 March 2019) upstream of the hAAT promoter.
[00430] In order to develop recombinant adeno-associated virus (rAAV) expression vector VR-18016AD containing liver-specific/intestine-specific tandem promoter, the ApoEHCR enhancer-hAAT promoter-hDNasel (hyperactive)correct leader-WPRE Xinact transgene cassette was modified by insertion of 685 bp sequence of villin promoter (Madison et al., J. Biol. Chem., 2002, 277(36):33275-33283) upstream of the hAAT promoter.
[00431] The respective ApoEHCR enhancer-hAAT promoter-hSYN promoter-hDNasel (hyperactive)correct leader-WPRE Xinact and ApoEHCR enhancer-hAAT promoter-Vil promoter-hDNasel (hyperactive)correct leader-WPRE Xinact plasmids were manufactured by GenScript USA Inc. (Piscataway, NJ 08854, USA). The corresponding hDNAse I (hyperactive) liver/nervous system tandem VR-18015AD and hDNAse I (hyperactive) liver/intestine tandem VR-18016AD AAV vectors were produced using large-scale polyethylenimine transfections of Anc80L65 AAV cis, AAV trans, and adenovirus helper plasmids in near-confluent monolayers of HEK293 cells with further purification on an iodixanol gradient at scale using traditional protocols. Vectors were reformulated in PBS supplemented with 35 mM NaCl and 0.001% PF68, and comprising 1.0 xl013 GC/ml.
[00432] Comparative effects of VR-18013AD, VR-18015AD and VR-18016AD AAV vectors were tested in 3xTg-AD mice model of Alzheimer’s disease (AD). At day 0 the following groups of mice received treatment: (A) 8 six-month old 3xTg-AD mice was injected IV with VR- 18013AD at 1.0x1011 GC/per mouse dose; (B) 8 six-month old 3xTg-AD mice were injected IV with VR-18015AD at 1.0x1011 GC/per mouse dose; and (C) 8 six-month-old 3xTg-AD mice injected IV with VR-18016AD at 1.0x1011 GC/per mouse dose. Five months later mice were euthanized and brains were sampled. Amyloid 1-42 (Ab1-42) was quantified in brain homogenate using Mouse Amyloid Beta Peptide 1-42 (Ab1-42) enzyme linked immuno-sorbent assay (ELISA) Kit (Cusabio). Phosphorylated tau in brain tissues (p-tau Thrl81) was measured by ELISA with the Cell Like technology kit (D9F4G, Life Technologies), according to the manufacturer instructions.
[00433] The results of Ab1-42 and p-tau Thrl81 ELISAs are summarized in Table 14 below.
Table 14
Figure imgf000135_0002
[00434] The data of Table 14 show that targeted DNase expression in (i) the liver and (ii) nervous system and/or intestine leads to superior therapeutic efficacy in a mouse model of Alzheimer’s disease.
EXAMPLE 17: Effects of transgenic delivery of DNase in a sickle-cell animal model
Figure imgf000135_0001
[00435] An AAV8 vector for liver specific transgenic expression of mouse DNase I was constructed. The DNase I coding sequence further comprised a secretory signal sequence. The DNase I coding sequence was operably linked at the 5' end to m AFP/ Alb enhancer/promoter system (liver-specific mouse alpha fetoprotein enhancer and liver-specific mouse minimal albumin promoter) and at the 3' end to hGH polyA signal. The expression cassette was further flanked by AA8 L-ITR and R-ITR.
[00436] The huDNasel mut expression cassette was cloned into an AAV transfer vector and produced in 293T cells via co-transfection of a transfer vector with AAV8 packaging plasmids. Viral particles were purified and concentrated up to lxlO13 GC/ml and kept frozen in PBS.
A control vector identical to 4658_pAAV-AFP-Alb-huDNaseI_mut but with non-specific for liver CMVE/CAG promoter/enhancer system instead of the liver-specific mAFP/Alb enhancer/promoter system was also produced.
[00437] 30 BERK mice (Tg[Hu-miniLCRalGgAgdbS] Hba2/2 Hbb2/)
(Jackson Laboratories with minor changes) were used. Vaso-occlusive crisis was induced by intraperitoneal injection of TNF-a (0.5 pg). The tested vector was administered to the mice according to the predetermined regimen 3 days before crisis induction as shown in Table 15, below.
Table 15: Experimental Design
Figure imgf000136_0003
[00438] The treatment with AAV vector significantly lowered the number of NETs formec in the lungs of BERK mice (5 animals per group).
Table 16. A number of NETs in the lungs of TNF-a-stimulated BERK mice
Figure imgf000136_0004
[00439] Vector treatment significantly increased the survival rate of TNF-a induced crisis in BERK animals.
Table 17. Survival rate of TNF-a-stimulated BERK mice
Figure imgf000136_0002
of DNase I enzyme significantly decreased NETs formation in BERK mice and prevented death of these animals.
EXAMPLE 18: Effects of transgenic delivery of DNase in a colorectal cancer model
Figure imgf000136_0001
[00441] The plasmid, pAAV-ApoEHCR enhancer-hAAT promoter-hDNase I (hyperactive) -WPRE Xinact (AKA, pCLS-014) was constructed as follows. The cDNA of the transgene expression cassette for AAV-DNase I was synthesized by Genscript (Piscataway, NJ, USA). A 5’ EcoRI site and a 3’ Hindlll site were added to the sequence of CLS-014. The cassette consists of (1) APOE HCR enhancer and human alpha- 1 -antitrypsin (hAAT) promoter, (2) a Kozak sequence, (3) human hyperactive, actin resistant, DNase I-variant containing the natural signal sequence, (4) a woodchuck hepatitis virus posttranscriptional element (WPRE) with the X protein coding region inactivated by mutating the start codon (WPRE-X-inactivated). The CLS-014 cDNA was digested with EcoRI-HF and Hindlll-HF (New England Biolabs Ipswich, MA, USA). An AAV promoterless expression vector, pAAV-MCS-Promoterless (which provides a polyA signal and two flanking AAV2 inverted terminal repeats, ITRs, for the AAV-DNase I cassette), was purchased from Cell Bio labs (Cat no. VPK-411, San Diego, CA, USA). The pAAV-MCS Promoterless vector was digested with EcoRI-HF and Hindlll-HF, and similarly digested pCLS- 014 cassette were ligated for one hour at room temperature in a 3 : 1 insert to vector molar ratio. The ligated construct was transformed into SURE electrocompetent cells (Agilent Technologies, Santa Clara, CA, USA) and plated on LB-Agar-ampicillin plates. Colonies were selected and minipreps were prepared for screening. First, a Smal digest was performed for ITR integrity to determine that the expected bands were present. Bacterial clones were selected when the expected bands were present in each of the Smal digest and the EcoRI-HF and Hindlll-HF digest. The pCLS-014 AAV plasmid was subjected to whole-plasmid sequencing (MGH DNA core) and then plasmid was purified using an endo-free Gigaprep (Qiagen, Carlsbad, CA, USA) performed by Alta Biotech (Aurora, CO, USA). A control AAV plasmid called AAV-null was also engineered. AAV-null contained all components of pCLS-014 AAV plasmid with the exception that it was devoid of the DNase I cDNA.
[00442] An AAV vector for liver specific transgenic expression of DNase I (manufactured by Boston Children Hospital, MA, USA) is based on AAV Anc80 which effectively targets liver. The vector comprises the sequence set forth in SEQ ID NO: 2, which codes for a mutant human DNase I enzyme which comprises several mutations which make the enzyme less sensitive to actin inhibition and more catalytically active.
[00443] The huDNasel mut expression cassette was cloned into an AAV transfer vector and produced in 293T cells via co-transfection of a transfer vector with AAV Anc80 packaging plasmids. Viral particles were purified and concentrated up to Ixl0el2 GC/ml and kept frozen in PBS.
[00444] Male and female wild-type (C57BL/6) mice and male BALB/c nude mice (6-8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, Maine, USA). One million MC38 cells were injected into the portal vein using a 30G needle. After removal of the needle, bleeding was stopped by gently pressing the puncture site with a cotton swab. After injection, the intestine was repositioned and the abdominal wall was closed with non-absorbable sutures. A dose of 1.05x 10el2 GC per mouse vector genomes was systemically administered through the tail vein on day after MC38 injection. Blood was collected before MC38 injection and 21 days after MC38 injection. Livers were harvested at 21 days after MC38 injection.
[00445] AAV-mediated DNase I liver gene transfer reduces the growth of colorectal liver metastases. To determine whether AAV-DNase I injection could inhibit the progression of CRC liver metastasis, a luciferase-expressing colon cancer cell line (MC38) was injected into mice via the portal vein. Four days after MC38 cell injection, mice were given a single IV injection of 1.05x l012 GC/mouse of AAV-DNase I or 1 05 1 Oe l 2 GC/mouse of AAV-null as negative control and monitored for 21 days (Fig. 22A). Bioluminescent imaging shows that metastasis development in AAV-DNase I treated mice was lessened compared to mice in the AAV-null group (p< 0.05) (Figs. 22B and 22C). Single AAV-DNase I administration inhibited the progression of liver metastasis in both male and female mice compared to the AAV-null treated mice (Fig. 22D). AAV- DNase I treatment also significantly decreased the number of metastatic nodules (p< 0 01 , / 0.05) (Fig. 22E). A lower liver weight to body weight ratio in AAV-DNase I treated mice was observed compared to AAV-null treated mice (p< 0.05) (Fig. 22F).
[00446] AAV-mediated DNase I liver gene transfer reduces neutrophil recruitment and NET formation in CRC liver metastasis. In the tumor microenvironment, the presence of immune cells plays a crucial role in cancer development. HE staining revealed the tumor immune cell load following AAV-null and AAV-DNase I treatment at day 21 (Fig. 23 A). The number of immune cell infiltration was significantly higher in AAV-DNase I treated mice than the AAV-null treated group (p< 0.05). In human tumors, neutrophils make up a significant portion of the immune infiltrate in a wide variety of cancer types and increased levels of neutrophils and NETs in tumors is associated with poor prognosis in many patients with advanced cancers (Arelaki et ak, PloS one, 2016, 1 l(5):e0154484; Jin et ak, Annals of Surgical Oncology, 2019, 26(2):635-643). Flow cytometry of tumor tissues revealed that neutrophil infiltration (CD1 lb+/Ly6G+ neutrophils) was significantly reduced in the AAV-DNase I treated group (p< 0.05) (Fig. 23B). Activated neutrophils can release NETs and facilitate capture of circulating cancer cells and support tumor metastasis. The protein level of citrullinated histone H3 (CitH3), a marker of NET formation, was markedly lower in the tumors of AAV-DNase I treated mice compared to AAV-null treated mice (p< 0.01) (Fig. 23C). Similarly, levels of NETs in the circulation as measured by MPO-DNA complexes, were significantly reduced in the AAV-DNase I-treated mice (p< 0.05) (Fig. 23D). Tumor tissues from both AAV-DNase I and AAV-null group were then harvested after 3 weeks of growth and stained for neutrophils (Ly6G), NETs (CitH3), and F-actin. Immunofluorescent images revealed NETs were abundant in the AAV-null treated mice but absent in AAV-DNase I treated mice (Fig. 23E). These findings suggest that AAV mediated DNase I liver gene transfer inhibits neutrophil infiltration and NET formation in tumor tissues.
[00447] AAV-mediated DNase I liver gene transfer recruits CD8+ T cells to CRC liver metastasis. The results above demonstrate that AAV-mediated DNase I liver gene transfer can modulate innate immune responses to the tumor. However, adaptive immune cells in the tumor microenvironment play an equally crucial role in tumor control. CD4+ T cells and CD8+ T cells were analyzed in the metastatic tissue by immunohistochemistry. Compared to the AAV-null treated group, the percentage of CD8+ T cells was greatly increased in the AAV-DNase I treated group (p< 0.05) (Fig. 24A). CD8+ T cells can detect and kill tumors through cytotoxic molecules, such as granzymes and perforin. In contrast, the percentage of CD4+ T cells in the AAV-DNase I- treated mice was not significantly different from those in the AAV-null treated group (Fig. 24B). Similar results of the contrasting percentages of CD4+ and CD8+ T cells in tumor tissues were also detected and confirmed by flow cytometry (Fig. 24C and 24D). The proportion of CD8+ T cells was significantly higher in AAV-DNase I treated mice when compared with AAV-null treated mice (p< 0.05). Though not significant, the proportion of CD4+ T cells had a decreasing trend in AAV-DNase I treated mice. These results suggest that AAV-mediated DNase I liver gene transfer induces anti-tumor immune responses through modulation of both innate and adaptive immune mechanisms.
[00448] The above data show that AAV-mediated DNase I liver gene transfer following a single intravenous injection significantly suppresses the development of liver metastases and rebalances local immune response in tumor tissue through modulation of both innate and adaptive immune mechanisms. Thus, the above data suggest that AAV-mediated DNase I liver gene transfer is a safe and effective modality to inhibit metastasis and represents a novel therapeutic strategy for cancer.
References:
Song, L. et ak,“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.
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Schwarzenbach H. et al.,“Detection and monitoring of cell-free DNA in blood of patients with colorectal cancer” Ann. N.Y. Acad. Sci., 2008, 1 137: 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
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* * *
[00449] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
[00450] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

CLAIMS:
1. A recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/nervous system-specific tandem promoter.
2. The vector of claim 1, wherein the liver-specific/nervous system-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system as compared to other tissues and organs.
3. The vector of claim 1 or claim 2, wherein the liver-specific/nervous system-specific tandem promoter comprises a liver-specific promoter selected from the group consisting of an albumin promoter, an a 1 -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HB V basic core promoter (BCP), a HB V PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a- fibrinogen promoter, a b-fibrinogen promoter, an alpha 1- antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, al- acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINAl) (hAAT) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a-fetoprotein (AFP) promoter.
4. The vector of claim 3, wherein the liver-specific promoter is an anti-trypsin (AAT) promoter.
5. The vector of claim 4, wherein the AAT promoter is a human al -anti -trypsin (hAAT) promoter.
6. The vector of claim 1 or claim 2, wherein the liver-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 15.
7. The vector of claim 6, wherein the liver-specific promoter comprises the sequence SEQ ID NO: 15.
8. The vector of claim 7, wherein the liver-specific promoter consists of the sequence SEQ ID NO: 15.
9. The vector of claim 3, wherein the liver-specific promoter is an albumin promoter.
10. The vector of claim 1 or claim 2, wherein the liver-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 8.
11. The vector of claim 10, wherein the liver-specific promoter comprises the sequence SEQ ID NO: 8.
12. The vector of claim 11, wherein the liver-specific promoter consists of the sequence SEQ ID NO: 8.
13. The vector of any one of claims 1-12, wherein the liver-specific/nervous system-specific tandem promoter comprises a nervous system-specific promoter selected from the group consisting of F4/80 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, CMV promoter, MEF2 promoter, FoxP2 promoter, Ibal promoter, TTR promoter, CD l ib promoter, c-fes promoter, NSE promoter, synapsin promoter, CamKII promoter, cx-CaMKII promoter, VGLUT1 promoter, and glial fibrillary acidic protein (GFAP) promoter.
14. The vector of claim 13, wherein the nervous system-specific promoter is a synapsin promoter.
15. The vector of claim 14, wherein the synapsisn promoter is a human synapsin promoter.
16. The vector of any one of claims 1-12, wherein the liver-specific/nervous system-specific tandem promoter comprises a nervous-specific promoter comprising a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 36.
17. The vector of claim 16, wherein the nervous system-specific promoter comprises the sequence SEQ ID NO: 36.
18. The vector of claim 17, wherein the nervous system-specific promoter consists of the sequence SEQ ID NO: 36.
19. The vector of claim 1 or claim 2, wherein the liver-specific/nervous system-specific tandem promoter comprises a hAAT promoter and a human synapsin promoter.
20. A recombinant adeno-associated virus (rAAV) expression vector comprising (i) a capsid protein and (ii) a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/intestine-specific tandem promoter.
21. The vector of claim 20, wherein the liver-specific/intestine-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs.
22. The vector of claim 20 or claim 21, wherein the liver-specific/intestine-specific tandem promoter comprises a liver-specific promoter selected from the group consisting of an albumin promoter, an a 1 -anti -trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HB V basic core promoter (BCP), a HB V PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a- fibrinogen promoter, a b-fibrinogen promoter, an alpha 1- antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, al- acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINAl) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a-fetoprotein (AFP) promoter.
23. The vector of claim 22, wherein the liver-specific promoter is an anti -trypsin (AAT) promoter.
24. The vector of claim 23, wherein the AAT promoter is a human a 1 -anti -trypsin (hAAT) promoter.
25. The vector of claim 20 or claim 21, wherein the liver-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 15.
26. The vector of claim 25, wherein the liver-specific promoter comprises the sequence SEQ ID NO: 15.
27. The vector of claim 26, wherein the liver-specific promoter consists of the sequence SEQ ID NO: 15.
28. The vector of claim 22, wherein the liver-specific promoter is an albumin promoter.
29. The vector of claim 20 or claim 21, wherein the liver-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 8.
30. The vector of claim 29, wherein the liver-specific promoter comprises the sequence SEQ ID NO: 8.
31. The vector of claim 30, wherein the liver-specific promoter consists of the sequence SEQ ID NO: 8.
32. The vector of any one of claims 20-31, wherein the liver-specific/intestine-specific tandem promoter comprises an intestine-specific promoter selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti-CMV promoter, MUC2 promoter, villin promoter, and T3b promoter.
33. The vector of claim 32, wherein the intestine-specific promoter is a villin promoter.
34. The vector of claim 33, wherein the villin promoter promoter is a human villin promoter.
35. The vector of any one of claims 20-31, wherein the intestine-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 37.
36. The vector of claim 20 or claim 21, wherein the liver-specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter.
37. The vector of any one of claims 1-19, wherein the liver-specific/nervous system-specific tandem promoter is part of the liver-specifi c/nervous system-specific/intestine-specific tandem promoter, which mediates a substantially increased expression of the enzyme in the liver and/or nervous system and/or intestine as compared to other tissues and organs.
38. The vector of claim 37, wherein the liver-specific/nervous system-specific/intestine-specific tandem promoter comprises an intestine-specific promoter selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti-CMV promoter, MUC2 promoter, villin promoter, and T3b promoter.
39. The vector of claim 38, wherein the intestine-specific promoter is a villin promoter.
40. The vector of claim 39, wherein the villin promoter promoter is a human villin promoter.
41. The vector of claim 37, wherein the intestine-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 37.
42. The vector of claim 37, wherein the liver-specific/nervous system-specific/intestine-specific tandem promoter comprises a hAAT promoter, a human synapsin promoter and a human villin promoter.
43. The vector of any one of claims 1-42, wherein the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
44. The vector of any one of claims 1-43, wherein the enzyme which has a DNase activity is DNase I or a mutant or derivative thereof.
45. The vector of claim 44, wherein the DNase I is a human DNase I or a mutant or derivative thereof.
46. The vector of any one of claims 1-45, wherein the nucleic acid comprises the sequence of SEQ ID NO: 30 or SEQ ID NO: 31.
47. The vector of claim 45 or claim 46, wherein the DNase I mutant comprises one or more mutations in an actin binding site.
48. The vector of claim 47, wherein the one or more mutations in the actin-binding site are selected from a mutation at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combinations thereof.
49. The vector of claim 48, wherein one of the mutations in the actin-binding site is a mutation at Ala-114.
50. The vector of any one of claims 44, 45 and 47-49, wherein the DNase I mutant comprises one or more mutations increasing DNase activity.
51. The vector of claim 50, wherein said one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof.
52. The vector of claim 51, wherein said one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, N74K and A114F, and any combinations thereof.
53. The vector of claim 52, wherein one of the mutations increasing DNase activity is Q9R.
54. The vector of claim 52, wherein one of the mutations increasing DNase activity is E13R.
55. The vector of claim 52, wherein one of the mutations increasing DNase activity is N74K.
56. The vector of claim 52, wherein one of the mutations increasing DNase activity is A114F.
57. The vector of claim 44 or claim 45, wherein the DNase I mutant comprises a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 5.
58. The vector of claim 57, wherein the DNase I mutant comprises a sequence having at least 85% sequence identity to the sequence of SEQ ID NO: 5.
59. The vector of claim 58, wherein the DNase I mutant comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 5.
60. The vector of any one of claims 52 and 57-59, wherein the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F.
61. The vector of claim 59, wherein the DNase I mutant comprises the sequence of SEQ ID NO:
5.
62. The vector of claim 61, wherein the DNAse I mutant consists of the sequence of SEQ ID NO:
5.
63. The vector of claim 44 or claim 45, wherein the DNase I mutant comprises a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 2.
64. The vector of claim 63, wherein the DNase I mutant comprises a sequence having at least 85% sequence identity to the sequence of SEQ ID NO: 2.
65. The vector of claim 64, wherein the DNase I mutant comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 2.
66. The vector of any one of claims 63-65, wherein the DNase I mutant comprises the mutations Q9R, E13R, N74K, and A114F.
67. The vector of claim 65, wherein the DNase I mutant comprises the sequence of SEQ ID NO:
2
68. The vector of claim 67, wherein the DNAse I mutant consists of the sequence of SEQ ID NO:
2
69. The vector of claim 44 or claim 45, wherein the DNase I mutant comprises 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 combinations thereof.
70. The vector of claim 44, wherein the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 4.
71. The vector of claim 44, wherein the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 23.
72. The vector of claim 71, wherein the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 23.
73. The vector of claim 72, wherein the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 23.
74. The vector of claim 73, wherein the nucleic acid comprises the nucleotide sequence SEQ ID NO: 23.
75. The vector of claim 44, wherein the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 1.
76. The vector of claim 44, wherein the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 22.
77. The vector of claim 76, wherein the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 22.
78. The vector of claim 77, wherein the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 22.
79. The vector of claim 78, wherein the nucleic acid comprises the nucleotide sequence SEQ ID NO: 22.
80. The vector of claim 44, wherein the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 32.
81. The vector of claim 80, wherein the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 32.
82. The vector of claim 81, wherein the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 32.
83. The vector of claim 82, wherein the nucleic acid comprises the nucleotide sequence SEQ ID NO: 32.
84. The vector of claim 44, wherein the nucleic acid encodes a DNase I comprising the sequence SEQ ID NO: 24.
85. The vector of claim 44, wherein the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 29.
86. The vector of claim 85, wherein the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 29.
87. The vector of claim 86, wherein the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 29.
88. The vector of claim 87, wherein the nucleic acid comprises the nucleotide sequence SEQ ID NO: 29.
89. The vector of claim 44, wherein the nucleic acid encodes a DNase I comprising the sequence of SEQ ID NO: 26.
90. The vector of claim 44, wherein the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 28.
91. The vector of claim 90, wherein the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 28.
92. The vector of claim 91, wherein the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 28.
93. The vector of claim 92, wherein the nucleic acid comprises the nucleotide sequence SEQ ID NO: 28.
94. The vector of claim 44, wherein the nucleic acid encodes a DNase I mutant comprising the sequence SEQ ID NO: 5.
95. The vector of claim 44, wherein the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 21.
96. The vector of claim 95, wherein the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 21.
97. The vector of claim 96, wherein the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 21.
98. The vector of claim 44, wherein the nucleic acid comprises the nucleotide sequence SEQ ID NO: 21.
99. The vector of claim 44, wherein the nucleic acid encodes a DNase I mutant comprising the sequence SEQ ID NO: 2.
100. The vector of claim 44, wherein the nucleic acid comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 19.
101. The vector of claim 100, wherein the nucleic acid comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 19.
102. The vector of claim 101, wherein the nucleic acid comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 19.
103. The vector of claim 102, wherein the nucleic acid comprises the nucleotide sequence SEQ ID NO: 19.
104. The vector of any one of claims 1-103, wherein the enzyme which has a DNase activity is a fusion protein comprising (i) a DNase enzyme or a fragment thereof linked to (ii) an albumin or an Fc or a fragment thereof.
105. The vector of any one of claims 1-104, wherein the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme.
106. The vector of claim 105, wherein the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, the albumin secretory signal sequence, the b-glucuronidase secretory signal sequence, the alkaline protease secretory signal sequence, and the fibronectin secretory signal sequence.
107. The vector of claim 105, wherein the secretory signal sequence comprises the sequence MRGMKLLGALLAL AALLQGAV S (SEQ ID NO: 6) or M YRMQLL S CIAL SL AL VTN S (SEQ ID NO: 7).
108. The vector of claim 107, wherein the secretory signal sequence consists of the sequence MRGMKLLGALLAL AALLQGAV S (SEQ ID NO: 6) or M YRMQLL S CIAL SL AL VTN S (SEQ ID NO: 7).
109. The vector of claim 105, wherein the secretory signal sequence comprises a sequence having at least 80% sequence identity to the sequence of MRGMKLLGALLAL AALLQGAVS (SEQ ID NO: 6) or a sequence having at least 85% sequence identity to the sequence of M YRMQLL S CIAL SL AL VTN S (SEQ ID NO: 7).
110. The vector of claim 105, wherein the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 20.
111. The vector of claim 110, wherein the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 20.
112. The vector of claim 111, wherein the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 20.
113. The vector of claim 112, wherein the sequence encoding the secretory signal sequence comprises the nucleotide sequence of SEQ ID NO: 20.
114. The vector of claim 105, wherein the secretory signal sequence comprises the sequence MRYT GLMGTLLTL VNLLQL AGT (SEQ ID NO: 25).
115. The vector of claim 114, wherein the secretory signal sequence consists of the sequence MRYTGLMGTLLTL VNLLQL AGT (SEQ ID NO: 25).
116. The vector of claim 105, wherein the secretory signal sequence comprises a sequence having at least 80% sequence identity to the sequence of MRYTGLMGTLLTLVNLLQLAGT (SEQ ID NO: 25).
117. The vector of claim 105, wherein the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 85% identical to SEQ ID NO: 27.
118. The vector of claim 117, wherein the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 90% identical to SEQ ID NO: 27.
119. The vector of claim 118, wherein the sequence encoding the secretory signal sequence comprises a nucleotide sequence which is at least 95% identical to SEQ ID NO: 27.
120. The vector of claim 119, wherein the sequence encoding the secretory signal sequence comprises the nucleotide sequence SEQ ID NO: 27.
121. The vector of any one of claims 1-19 and 43-120, wherein the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or nervous system as compared to the corresponding wild-type capsid protein.
122. The vector of any one of claims 20-36 and 43-120, wherein the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or intestine as compared to the corresponding wild-type capsid protein.
123. The vector of any one of claims 37-120, wherein the capsid protein comprises one or more mutations which improve efficiency and/or specificity of the delivery of the vector to the liver and/or nervous system and/or intestine as compared to the corresponding wild-type capsid protein.
124. The vector of any one of claims 121-123, wherein the improved efficiency and/or specificity of the delivery of the vector to the liver results in a substantially increased expression of the enzyme in the liver as compared to other tissues and organs.
125. The vector of claim 121 or claim 123, wherein the improved efficiency and/or specificity of the delivery of the vector to the nervous system results in a substantially increased expression of the enzyme in the nervous system as compared to other tissues and organs.
126. The vector of claim 122 or claim 123, wherein the improved efficiency and/or specificity of the delivery of the vector to the intestine results in a substantially increased expression of the enzyme in the intestine as compared to other tissues and organs.
127. The vector of any one of claims 121-126, wherein the capsid protein comprisesVP3.
128. The vector of any one of claims 121-127, wherein the one or more mutations in the capsid protein are selected from the group consisting of S279A, S671A, K137R, T252A, and any combinations thereof.
129. The vector of claim 128, wherein the one or more mutations in the capsid protein include mutation K137R.
130. The vector of any one of claims 121-123, wherein the capsid protein comprises the sequence SEQ ID NO:34.
131. The vector of claim 130, wherein the capsid protein consists of the sequence SEQ ID NO:34
132. The vector of any one of claims 1-131, wherein the AAV is selected from serotype 1 (AAVl), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrhlO, AAVLK03, AAVLK06, AAVLK12, AAV-KP1, AAV-F, AAVDJ, AAVhu37, AAVrh64Rl, and Anc 80.
133. The vector of claim 132, wherein the AAV is from serotype 8 or Anc 80.
134. The vector of any one of claims 1-133, wherein the nucleic acid further comprises two AAV inverted terminal repeats (ITRs), wherein the ITRs flank the nucleotide sequence encoding the enzyme which has a DNase activity.
135. The vector of any one of claims 1-134, wherein the nucleic acid further comprises one or more enhancers located upstream or downstream of the promoter.
136. The vector of claim 135, wherein the one or more enhancers are selected from the group consisting of an apolipoprotein E (ApoE) enhancer, an alpha fetoprotein enhancer, a TTR enhancer, an LSP enhancer, an al-microglobulin/bikunin enhancer, an albumin gene enhancer (Ealb), a nPE2 enhancer, a Gal4 enhancer, a foxP2 enhancer, aMef2 enhancer, a CMV enhancer, and any combination thereof.
137. The vector of claim 136, wherein the enhancer is an apolipoprotein E (ApoE) enhancer.
138. The vector of claim 136, wherein the enhancer is a hepatic control region (HCR) enhancer.
139. The vector of claim 136, wherein the enhancer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 17.
140. The vector of claim 139, wherein the enhancer comprises the sequence SEQ ID NO: 17.
141. The vector of claim 140, wherein the enhancer consists of the sequence SEQ ID NO: 17.
142. The vector of any one of claims 1-141, wherein the nucleic acid further comprises a polyadenylation signal operably linked to the nucleotide sequence encoding the enzyme which has a DNase activity.
143. The vector of any one of claims 1-142, wherein the nucleic acid further comprises a Kozak sequence.
144. The vector of claim 143, wherein the Kozak sequence comprises the sequence 5'- GCCGCC ACC-3' (SEQ ID NO: 33).
145. The vector of any one of claims 1-144, wherein the nucleic acid further comprises a post- transcriptional regulatory element.
146. The vector of claim 145, wherein the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE).
147. The vector of claim 146, wherein the WPRE does not encode a functional X protein.
148. The vector of claim 145, wherein the post-transcriptional regulatory element comprises a sequence having at least 80% sequence identity to SEQ ID NO: 16.
149. The vector of claim 148, wherein the post-transcriptional regulatory element comprises the sequence SEQ ID NO: 16.
150. The vector of claim 149, wherein the post-transcriptional regulatory element consists of the sequence SEQ ID NO: 16.
151. A pharmaceutical composition comprising the rAAV vector of any one of claims 1-150 and a pharmaceutically acceptable carrier and/or excipient.
152. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and the nervous system in a subject in need thereof, comprising administering to the subject the rAAV vector of any one of claims 1-19, 37-121, and 123-150 or the composition of claim 151.
153. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject the rAAV vector of any one of claims 20-120 and 131-150 or the composition of claim 151.
154. A method for treating and/or preveting a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of the rAAV vector of any one of claims 1-150 or the composition of claim 151.
155. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and nervous system in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific/nervous system- specific tandem promoter.
156. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and nervous system in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver and nervous system cells when administered in vivo.
157. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific/intestine-specific tandem promoter.
158. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver and intestine cells when administered in vivo.
159. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver, nervous system and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the promoter is a liver-specific/nervous system-specific/intestine-specific tandem promoter.
160. A method for delivering an enzyme which has a deoxyribonuclease (DNase) activity to the liver, nervous system and intestine in a subject in need thereof, comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding the enzyme, wherein the vector comprises one or more molecules capable of targeting the nucleic acid to liver, nervous system and intestine cells when administered in vivo.
161. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/nervous system- specific tandem promoter.
162. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver and nervous system cells.
163. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/intestine-specific tandem promoter.
164. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver and intestinal cells.
165. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the promoter is a liver-specific/nervous system- specific/intestine-specific tandem promoter.
166. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid comprising a promoter operably linked to a sequence encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein the vector comprises one or more molecules capable of targeting of the nucleic acid to liver, nervous system and intestinal cells.
167. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and nervous system of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
168. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver and instestine of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
169. A method for treating and/or preventing a neurodegenerative, neurodevelopmental, psychiatric, onclological, or autoimune disease or an infection in a subject in need thereof, said method comprising administering to the subject an expression vector comprising a nucleic acid encoding an enzyme which has a deoxyribonuclease (DNase) activity, wherein said expression vector provides synthesis of the DNase enzyme in the liver, nervous system and intestine of said subject and wherein the microbial cell free DNA (cfDNA) of intestinal origin is detectable in the blood of said patient.
170. The method of any one of claims 154 and 161-169, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, CADASIL Syndrome, Friedreich’s ataxia, cerebellar ataxia, spinocerebellar ataxia, dementia, prion-caused diseases, Lewy body diseases, an amyloidosis, Spinal muscular atrophy, a bipolar disorder, schizophrenia, depressive disorder, autism, autism spectrum disorders, Chronic Fatigue Syndrome, Obsessive-Compulsive Disorder, generalized anxiety disorder (GAD), major depressive disorder (MDD), social anxiety disorder (SAD), attention-deficit/hyperactivity disorder (ADHD), Huntington's disease, Spinal muscular atrophy, Mild Cognitive Impairment (MCI), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), agyrophilic grain disease (AGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel- Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, progressive familial myoclonic epilepsy, striatonigral degeneration, torsion dystonia, spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration, Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot-Marie-Tooth), and hypertrophic interstitial polyneuropathy (Dejerine-Sottas).
171. The method of claim 170, wherein the dementia is selected from fronto-temporal dementia, frontotemporal dementia with parkinsonism- 17 (FTDP-17), familial Danish dementia, and familial British dementia.
172. The method of claim 170, wherein the amyloidosis is selected from hereditary cerebral hemorrhage with amyloidosis, senile systemic amyloidosis, an amyloidosis with a hereditary cerebral hemorrhage, a primary systemic amyloidosis, a secondary systemic amyloidosis, a serum amyloidosis, a senile systemic amyloidosis, a hemodialysis-related amyloidosis, a Finnish hereditary systemic amyloidosis, an Atrial amyloidosis, a Lysozyme systemic amyloidosis, an Insulin-related amyloidosis, and a Fibrinogen a-chain amyloidosis.
173. The method of claim 170, wherein the torsion dystonia is torsion spasm or dystonia musculorum deformans.
174. The method of any one of claims 154 and 161-169, wherein the neurodegenerative disease is secondary to diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, metabolic syndrome, an amyloidosis, asthma, or prion disease.
175. The method of any one of claims 154 and 161-169, wherein the psychitraic disease is selected from anxiety disorders, psychotic disorders, schizophrenia, bipolar disorder, depression, and post-traumatic stress disorder.
176. The method of any one of claims 154 and 161-169, wherein the neurodevelopmental disease is selected from autism, neural tube defects, attention deficit hyperactivity disorder, Dawn syndrome, cerebral palsy, Rett Syndrome, Landau-Kleffner Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Angelman Syndrome, Ataxias and Cerebellar or Spinocerebellar Degeneration, Ataxia Telangiectasia, Attention Deficit- Hyperactivity Disorder, Autism Spectrum Disorders including Asperger Syndrome, Batten Disease, Canavan Disease, Tourette Syndrome, and impairments in vision and/or hearing.
177. The method of any one of claims 154 and 161-169, wherein the onclological disease is selected from astrocytoma, gliomas, glioblastoma, ependymoma, medulloblastoma, central nervous system (CNS) lymphoma, craniopharyngioma, glioblastoma, meningioma, pituitary carcinomas, pituitary adenomas, neurofibromatosis, embryonal tumors, tumors of the pineal region, tumors of meninges, choroid plexus tumors, neuronal and mixed neuronal-glial tumors, germ cell tumors, anaplastic astrocytoma, central neurocytoma, choroid plexus carcinoma, choroid plexus papilloma, dysembryoplastic neuroepithelial tumor, giant-cell glioblastoma, gliosarcoma, hemangiopericytoma, medulloepithelioma, neuroblastoma, neurocytoma, oligoastrocytoma, oligodendroglioma, optic nerve sheath meningioma, pilocytic astrocytoma, pinealoblastoma, pineocytoma, pleomorphic anaplastic neuroblastoma, pleomorphic xanthoastrocytoma, sphenoid wing meningioma, subependymal giant cell astrocytoma, subependymoma, trilateral retinoblastoma, and nervous system metastasis of any origin.
178. The method of any one of claims 154 and 161-169, wherein the autoimune disease is selected from autoimmune encephalitis, autoimmune-related epilepsy, CNS vasculitis, Hashimoto’s encephalopathy, neurosarcoidosis, Neuro-Behcet’s disease, cerebral lupus, diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), gout, asthma, celiac disease, ankylosing spondylitis, vasculitis, pemphigus vulgaris, inflammatory bowel disease (3BD), Crohn's disease, and ulcerative colitis.
179. The method of any one of claims 154 and 161-169, wherein the infection is selected from adenoviral infections, hepatitis B, hepatitis G, poxvirus infections, herpesvirus infections, papillomavirus infections, HIV infections, meningitis (bacterial, fungal, or viral), bacterial persistence in brain, fungal persistence in brain, pancreatitis, and peritonitis.
180. The method of any one of claims 152-179, wherein the vector further comprises one or more molecules capable of targeting of the nucleic acid to liver and/or nervous system and/or intestinal cells.
181. The method of claim 155 or claim 161, wherein the liver-specific/nervous system-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system as compared to other tissues and organs.
182. The method of claim 157 or claim 163, wherein the liver-specific/intestine-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs.
183. The method of claim 159 or claim 165, wherein the liver-specific/nervous system- specific/intestine-specific tandem promoter mediates a substantially increased expression of the enzyme in the liver and/or nervous system and/or intestine as compared to other tissues and organs.
184. The method of any one of claims 155, 157, 159, 161, 163, 165, and 181-183, wherein the liver-specific promoter is selected from the group consisting of an albumin promoter, an al- anti-trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an alpha fetoprotein promoter, an alcohol dehydrogenase promoter, a factor VIII (FVIII) promoter, a HBV basic core promoter (BCP), a HBV PreS2 promoter, a phosphoenol pyruvate carboxykinase (PEPCK) promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an apolipoprotein E (ApoE) promoter, a low density lipoprotein promoter, a pyruvate kinase promoter, a phosphenol pyruvate carboxykinase promoter, a phenylalanine hydroxylase promoter, a lecithin-cholesterol acyl transferase (LCAT) promoter, an apolipoprotein H (ApoH) promoter, an apolipoprotein A-II promoter (APOA2), a transferrin promoter, a transthyretin promoter, an a- fibrinogen promoter, a b-fibrinogen promoter, an alpha 1- antichymotrypsin promoter, an a2-HS glycoprotein promoter, an haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a promoter of a complement protein, al- acid glycoprotein promoter, a LSP1 promoter, a serpin peptidase inhibitor promoter, a clade A member 1 (SERPINAl) (hAAT) promoter, a Cytochrome P450 family 3 subfamily A polypeptide 4 (CYP3A4) promoter, a microRNA 122 (miR-122) promoter, a liver-specific IGF-II promoter PI, a transthyretin (MTTR) promoter, and an a-fetoprotein (AFP) promoter.
185. The method of claim 184, wherein the liver-specific promoter is an anti -trypsin (AAT) promoter.
186. The method of claim 185, wherein the AAT promoter is a human al -anti -trypsin (hAAT) promoter.
187. The method of any one of claims 155, 157, 159, 161, 163, 165, and 181-183, wherein the liver-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 15.
188. The method of claim 187, wherein the liver-specific promoter comprises the sequence SEQ ID NO: 15.
189. The method of claim 188, wherein the liver-specific promoter consists of the sequence SEQ ID NO: 15.
190. The method of claim 189, wherein the liver-specific promoter is an albumin promoter.
191. The method of any one of claims 155, 157, 159, 161, 163, 165, and 181-183, wherein the liver-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 8.
192. The method of claim 191, wherein the liver-specific promoter comprises the sequence SEQ ID NO: 8.
193. The method of claim 192, wherein the liver-specific promoter consists of the sequence SEQ ID NO: 8.
194. The method of any one of claims 155, 159, 161, 165, and 181-193, wherein the nervous system-specific promoter is selected from the group consisting of F4/80 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, CMV promoter, MEF2 promoter, FoxP2 promoter, Ibal promoter, TTR promoter, CDl lb promoter, c-fes promoter, NSE promoter, synapsin promoter, CamKII promoter, a-CaMKII promoter, VGLUT1 promoter, and glial fibrillary acidic protein (GFAP) promoter.
195. The method of claim 194, wherein the nervous system-specific promoter is a synapsin promoter.
196. The vector of claim 195, wherein the synapsisn promoter is a human synapsin promoter.
197. The method of of any one of claims 155, 159, 161, 165, and 181-193, wherein the nervous system-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 36.
198. The method of claim 197, wherein the nervous system-specific promoter comprises the sequence SEQ ID NO: 36.
199. The method of claim 198, wherein the nervous system-specific promoter consists of the sequence SEQ ID NO: 36.
200. The method of any one of claims 155, 159, 161, 165, and 181-199, wherein the liver- specific/nervous system-specific tandem promoter comprises a hAAT promoter and a human synapsin promoter.
201. The method of any one of claims 157, 159, 163, 165, and 181-193, wherein the intestine- specific promoter mediates a substantially increased expression of the enzyme in the liver and/or intestine as compared to other tissues and organs.
202. The method of any one of claims 157, 159, 163, 165, 181-193, and 201, wherein the intestine-specific promoter is selected from the group consisting of CB/CMV promoter, glial fibrillary acidic protein (GFAP) promoter, miCMV promoter, CMV+I promoter, tetO-CMV promoter, b-acti-CMV promoter, MUC2 promoter, villin promoter, and T3b promoter.
203. The method of claim 202, wherein the intestine-specific promoter is a villin promoter.
204. The method of claim 203 wherein the villin promoter promoter is a human villin promoter.
205. The method of any one of claims 157, 159, 163, 165, 181-193, and 201, wherein the intestine-specific promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 37.
206. The method of any one of claims 157, 159, 163, 165, 181-193, and 201, wherein the liver- specific/intestine-specific tandem promoter comprises a hAAT promoter and a human villin promoter.
207. The method of any one of claims 157, 159, 163, 165, 181-193, and 201, wherein the liver- specific/intestine-specific tandem promoter comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 37.
208. The method of any one of claims 155-207, wherein the vector is used as a naked DNA.
209. The method of any one of claims 155-207, wherein the vector is a viral vector.
210. The method of claim 209, wherein the viral vector is an adeno-associated virus vector, an adenovirus vector, a retrovirus vector, or a hepatotropic virus vector.
211. The method of claim 210, wherein the retrovirus vector is a lentivirus vector.
212. The method of claim 210, wherein the hepatotropic virus vector is a hepatitis B virus (HBV) vector.
213. The method of any one of claims 155-212, wherein the enzyme which has a DNase activity is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
214. The method of claim 213, wherein the DNase is DNase I or a mutant or derivative thereof.
215. The method of claim 214, wherein the DNase I is human DNase I or a mutant or derivative thereof.
216. The method of claim 214 or claim 215, wherein the DNase I mutant comprises one or more mutations in an actin binding site.
217. The method of claim 216, wherein the one or more mutations in the actin-binding site are selected from a mutation at Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, Ala-114, and any combinations thereof.
218. The method of claim 217, wherein one of the mutations in the actin-binding site is a mutation at Ala- 114.
219. The method of claim 214 or claim 215, wherein the DNase I mutant comprises one or more mutations increasing DNase activity.
220. The method of claim 219, wherein said one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, A114F, and any combinations thereof.
221. The method of claim 220, wherein said one or more mutations increasing DNase activity are selected from the group consisting of Q9R, E13R, N74K, and A114F.
222. The method of claim 221, wherein one of the mutations increasing DNase activity is Q9R.
223. The method of claim 221, wherein one of the mutations increasing DNase activity is E13R.
224. The method of claim 221, wherein one of the mutations increasing DNase activity is N74K.
225. The method of claim 221, wherein one of the mutations increasing DNase activity is A114F.
226. The method of claim 214 or claim 215, wherein the DNase I mutant comprises 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 combinations thereof.
227. The method of claim 214 or claim 215, wherein the DNase I mutant is a long acting form of DNase.
228. The method of claim 214, wherein the DNase I comprises the sequence SEQ ID NO: 4.
229. The method of claim 214, wherein the DNase I comprises the sequence SEQ ID NO: 1.
230. The method of claim 214, wherein the DNase I mutant comprises the sequence SEQ ID NO: 5.
231. The method of claim 214, wherein the DNase I mutant comprises the sequence SEQ ID NO: 2.
232. The method of any one of claims 155-231, wherein the sequence encoding the enzyme which has a DNase activity comprises a sequence encoding a secretory signal sequence, wherein said secretory signal sequence mediates effective secretion of the enzyme into the hepatic porto-sinusoidal circulation upon administration of the vector to the subject.
233. The method of claim 232, wherein the secretory signal sequence is selected from the group consisting of DNase I secretory signal sequence, IL2 secretory signal sequence, albumin secretory signal sequence, b-glucuronidase secretory signal sequence, alkaline protease secretory signal sequence, and fibronectin secretory signal sequence.
234. The method of claim 232, wherein the secretory signal sequence comprises a sequence having at least 80% sequence identity to MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or a sequence having at least 80% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
235. The method of claim 234, wherein the secretory signal sequence comprises a sequence having at least 95% sequence identity to MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or a sequence having at least 95% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
236. The method of claim 235, wherein the secretory signal sequence comprises the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
237. The method of claim 236, wherein the secretory signal sequence consists of the sequence MRGMKLLGALLALAALLQGAVS (SEQ ID NO: 6) or MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7).
238. The method of any one of claims 155-237, wherein the nucleic acid further comprises one or more hepatic control region (HCR) enhancers located upstream or downstream of the promoter.
239. The method of claim 238, wherein the HCR enhancer comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 17.
240. The method of claim 239, wherein the HCR enhancer comprises the sequence SEQ ID NO:
17.
241. The method of claim 240, wherein the HCR enhancer consists of the sequence SEQ ID NO: 17.
242. The method of any one of claims 155-241, wherein the nucleic acid further comprises a Kozak sequence.
243. The method of claim 242, wherein the Kozak sequence comprises the sequence 5'- GCCGCC ACC-3' (SEQ ID NO: 33).
244. The method of any one of claims 155-243, wherein the nucleic acid further comprises a post-transcriptional regulatory element, wherein the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE) that does not encode a functional X protein.
245. The method of any one of claims 152-244, wherein the nucleic acid comprises a sequence having at least 80% sequence identity to the sequence SEQ ID NO: 19.
246. The method of claim 245, wherein the nucleic acid comprises the sequence SEQ ID NO:
19.
247. The method of claim 246, wherein the nucleic acid consists of the sequence SEQ ID NO:
19.
248. The method of any one of claims 152-247, wherein the administration of the nucleic acid results in the expression of the enzyme and its secretion into the hepatic porto-sinusoidal circulation of the subject.
249. The method of any one of claims 155-248, wherein the nucleic acid is administered in a dose and regimen which is sufficient to decrease the level of the cell free DNA (cfDNA) in the porto-sinusoidal circulation of said subject.
250. The method of any one of claims 152-249, said method further comprising selecting a subject with an elevated level of cell-free DNA (cfDNA) in the bloodstream as compared to a level of cfDNA in the bloodstream of a normal healthy subject.
251. The method of any one of claims 152-250, said method further comprising administering a deoxyribonuclease (DNase) enzyme to the subject.
252. The method of claim 251, wherein the DNase is selected from the group consisting of DNase I, DNase X, DNase g, DNaselLl, DNaselL2, DNase 1L3, DNase II, DNase Ila, DNase IIb, Caspase-activated DNase (CAD), Endonuclease G (ENDOG), Granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and mutants or derivatives thereof.
253. The method of claim 252, wherein the DNase is DNase I or a mutant or derivative thereof.
254. The method of any one of claims 152-253, wherein the subject is human.
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