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WO2025214477A1 - Traitement de cardiomyopathies génétiques avec des vecteurs de thérapie génique aav - Google Patents

Traitement de cardiomyopathies génétiques avec des vecteurs de thérapie génique aav

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Publication number
WO2025214477A1
WO2025214477A1 PCT/CN2025/088527 CN2025088527W WO2025214477A1 WO 2025214477 A1 WO2025214477 A1 WO 2025214477A1 CN 2025088527 W CN2025088527 W CN 2025088527W WO 2025214477 A1 WO2025214477 A1 WO 2025214477A1
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Prior art keywords
aav
seq
vector
vector construct
ctnt
Prior art date
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Pending
Application number
PCT/CN2025/088527
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English (en)
Inventor
Jinzhao Hou
Ting Yu
Yanqun Shu
Carol Xiufeng HUANG
Jiao YUE
Dianyang CHEN
Hui Liu
Marius P. SUMANDEA
Pooja Agarwal
Yen Sin ANG
Wesley Yonemoto
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Skyline Therapeutics Shanghai Co Ltd
Skyline Therapeutics Ltd
Biomarin Pharmaceutical Inc
Original Assignee
Skyline Therapeutics Shanghai Co Ltd
Skyline Therapeutics Ltd
Biomarin Pharmaceutical Inc
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Filing date
Publication date
Application filed by Skyline Therapeutics Shanghai Co Ltd, Skyline Therapeutics Ltd, Biomarin Pharmaceutical Inc filed Critical Skyline Therapeutics Shanghai Co Ltd
Publication of WO2025214477A1 publication Critical patent/WO2025214477A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A01K2217/077—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out heterozygous knock out animals displaying phenotype
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • gene therapy vector constructs such as recombinant adeno-associated virus (rAAV) gene therapy vector constructs and virus particles useful in the treatment and prevention of cardiomyopathy by increasing expression of human cardiac muscle troponin T.
  • rAAV recombinant adeno-associated virus
  • the basic units of muscle are sarcomeres, which are made up of thick and thin protein filaments.
  • Cardiac muscle troponin T (cTnT) encoded by the TNNT2 gene, is one of the three components of the troponin complex which is formed from troponin T, troponin I and troponin C.
  • the troponin complex is present on the actin thin filament of cardiac muscle, and it regulates muscle contraction in response to changes in intracellular calcium ion concentration.
  • cTnT is the largest of the three subunits, and is responsible for interacting with tropomyosin and actin.
  • Hypertrophic cardiomyopathy is characterized by thickening (hypertrophy) of the cardiac muscle. The thickening usually occurs in the interventricular septum between the left and right ventricles. In some people, thickening of the cardiac muscle wall impedes the flow of blood from the heart. In others, there is not a physical obstruction of blood flow, but the left ventricle pumps blood less efficiently. Functionally, it is characterized by left ventricular hyper-contractile state, diastolic dysfunction, ischemia and obstruction.
  • the symptoms are variable; some have no symptoms, while others may have chest pain; shortness of breath, especially with physical exertion, palpitations; lightheadedness; dizziness; and fainting. Individuals, even without symptoms, are at risk of arrhythmias that can be life threatening and lead to sudden death. A small number of affected individuals develop heart failure, which may require heart transplantation.
  • restrictive cardiomyopathy the cardiac walls stiffen, without necessarily becoming hypertrophic (thicker) . Because the cardiac walls are rigid, they are unable to relax and fill with blood even if contractility is normal, because the relaxation is abnormal. When the left ventricle is unable to stretch and fill with blood, pressure builds up causing abnormal heart rhythms and symptoms of heart failure.
  • An echocardiogram for RCM shows biatrial dilatation, normal or mildly reduced left ventricular (LV) and right ventricular ejection fraction, and nondilated ventricles.
  • Doppler imaging shows a restrictive filling pattern with tissue Doppler showing an elevated E/e’ ratio. Many individuals with restrictive cardiomyopathy do not have symptoms.
  • symptoms include those of heart failure, i.e., shortness of breath; fatigue; dizziness; fainting; persistent cough when lying down; swelling of legs, ankles and feet; and palpitations. Even individuals without symptoms, however, are at risk for sudden cardiac arrest due to arrhythmia, which will result in death if not treated urgently.
  • Dilated cardiomyopathy is characterized by enlargement of the left ventricle chamber and contractile dysfunction.
  • the right ventricle may also be dilated and dysfunctional. It is the third most common cause of heart failure and the most frequent reason for heart transplantation.
  • Echocardiographic features of DCM are left ventricular (LV) dilation and systolic dysfunction. Other frequent characteristics are LV dyssynchrony, right ventricular (RV) dysfunction, atrial dilation, functional mitral and tricuspid regurgitation, and secondary pulmonary hypertension. Many individuals have symptoms of heart failure, noted above.
  • Treatment of dilated cardiomyopathy is essentially the same as treatment of chronic heart failure, with angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs) , beta blockers, aldosterone antagonists, cardiac glycosides, diuretics, inotropic agents, neprilysin inhibitor and nitrates.
  • ACE angiotensin-converting enzyme
  • ARBs angiotensin II receptor blockers
  • beta blockers beta blockers
  • aldosterone antagonists aldosterone antagonists
  • cardiac glycosides diuretics
  • inotropic agents inotropic agents
  • neprilysin inhibitor and nitrates may be used to prevent blood clotting.
  • the embodiments described herein relate to a vector construct, a recombinant replication deficient AAV particle, cells, and pharmaceutical compositions for delivering functional human cardiac troponin T protein (cTnT) to a subject in need thereof, particularly a subject with cardiomyopathy.
  • the embodiments described herein also relate to the use of such AAV particles or such vector constructs to deliver a gene encoding or expressing cTNT to the heart (e.g. cardiomyocytes) of patients (human subjects) diagnosed with cardiomyopathy.
  • the disclosure provides a recombinant vector construct comprising a nucleic acid encoding or expressing a functional cardiac troponin T protein (cTnT) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 2 or comprising an amino acid sequence at least 95%identical to any of SEQ ID NOs: 71-81; (b) a heterologous cardiomyocyte-specific promoter; (c) optionally, an intron, for example, an intron comprising a nucleic acid sequence at least 95%identical to any of SEQ ID NOs: 24-31; (d) a polyadenylation signal, (e) optionally, a stuffer sequence, and (f) optionally, one or both of 5’ and 3’ AAV inverted terminal repeat (ITR) sequences.
  • cTnT functional cardiac troponin T protein
  • the disclosure provides a recombinant vector construct comprising a nucleic acid sequence encoding or expressing a cardiac troponin T protein (cTnT) comprising (a) an amino acid sequence of SEQ ID NO: 2.
  • the disclosure also provides a recombinant vector construct comprising a nucleic acid sequence encoding or expressing a cTnT protein comprising an amino acid sequence which excludes exon 5 (SEQ ID NO: 101) .
  • Such vector constructs optionally further comprise one or more of the following: (b) a heterologous cardiomyocyte-specific promoter; (c) an intron, for example, an intron comprising a nucleic acid sequence at least 95%identical to any of SEQ ID NOs: 24-31; (d) a polyadenylation signal, (e) a stuffer sequence, and (f) one or both of 5’ and 3’ AAV inverted terminal repeat (ITR) sequences.
  • the intron is located between exon 2 and exon 3 of the nucleic acid encoding SEQ ID NO: 2 or 71-81.
  • the disclosure provides a recombinant vector construct comprising: (a) a nucleic acid encoding a functional cardiac troponin T protein (cTnT) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 2 or comprising an amino acid sequence at least 95%identical at any of SEQ ID NOs: 71-81; (b) a heterologous cardiomyocyte-specific promoter, (c) an intron, for example, comprising a nucleic acid sequence at least 95%identical to any of SEQ ID NOs: 24-31; (d) a polyadenylation signal, (e) a stuffer sequence; and (f) optionally, one or both 5’ and 3’ AAV inverted terminal repeat (ITR) sequences.
  • cTnT functional cardiac troponin T protein
  • the stuffer sequence has a length of at least 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 750bp, 800bp, 850bp, 900bp. 950bp, 1000bp, 1050bp, 1100bp, 1150bp, 1200bp, 1250bp, 1300bp, 1350bp, 1400bp, 1450bp, 1500bp, 1550bp, 1600bp, 1650bp, 1700bp, 1750bp, 1800bp, 1850bp, 1900bp, 1950bp, or 2000bp.
  • the vector construct is about 4 to about 5kb in size, about 4.5 to about 5 kb in size, or about 4.6 to about 4.8 kb in size.
  • the nucleic acid encoding (and expressing) cTNT comprises a nucleotide sequence at least 97%, 98%or 99%identical to SEQ ID NO: 1 or comprises a nucleotide sequence at least 97%, 98%or 99%identical to any of SEQ ID NOs: 1 or 3-20.
  • the cardiomyocyte-specific promoter comprises a nucleotide sequence at least 80%identical to SEQ ID NO: 21.
  • the intron comprises the nucleotide sequence of any of SEQ ID NOs: 24-31 or fragments thereof.
  • the intron is located after position 41of SEQ ID NO: 1 or the corresponding position 41 in any of SEQ ID NOs: 3-20.
  • the polyadenylation signal is a growth hormone polyadenylation signal, optionally comprising a nucleotide sequence at least 90%identical to SEQ ID NO: 38, or a fragment thereof.
  • the vector construct comprises a nucleotide sequence at least 97%, 98%or 99%identical to any of SEQ ID NO: 39-43 or a nucleotide sequence at least 97%, 98%or 99%identical to any of SEQ ID NO: 49-53.
  • the vector construct is an rAAV vector construct and is about 4 to about 5 kb in size.
  • the rAAV vector construct comprises a stuffer sequence, e.g., in order to increase the size of the insert to the desired size.
  • the vector construct comprises AAV 5'ITR and/or AAV 3'ITR from AAV2.
  • the rAAV particle comprises an AAV capsid with cardiac tropism, for example an AAV6 type or AAV9 type capsid.
  • the disclosure provides a method of producing rAAV particles comprising any of the vector constructs or capsids disclosed herein comprising (a) providing a cell permissive for AAV replication with one or more nucleic acid constructs comprising (i) any of the vector constructs disclosed herein, (ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein (s) in the cell; and (iii) a nucleotide sequence encoding one or more AAV capsid proteins which is operably linked to a promoter that is capable of driving expression of the capsid protein (s) in the cell; (b) culturing the cell under conditions permitting expression of the Rep and the capsid proteins; and optionally (c) recovering the AAV particle.
  • the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In some embodiments, the cell is an insect cell.
  • the method of producing rAAV particles comprises the steps of (a) providing a mammalian cell comprising one or more nucleic acid constructs that comprise (i) a vector construct disclosed herein, (ii) a nucleotide sequence encoding one or more AAV Rep proteins operably linked to a promoter, and (iii) a nucleotide sequence encoding one or more AAV capsid proteins operably linked to a promoter, (b) culturing the mammalian cell under conditions conducive to the expression of the Rep and capsid proteins, and (c) recovering rAAV particles.
  • the disclosure also provides a population of rAAV particles produced by any of the methods disclosed herein, optionally enriched for particles comprising full length or nearly full-length vector genomes by steps that reduce the number of empty capsids.
  • the disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the vector constructs disclosed herein, any of the rAAV particles disclosed herein or the population of rAAV particles produced by any of the methods disclosed herein, in an aqueous suspension with a sterile pharmaceutically acceptable excipient.
  • the disclosure also provides a method of delivering a human cTnT coding sequence, comprising administering to a patient with cardiomyopathy any of the vector constructs disclosed herein, any of the rAAV particles disclosed herein or the population of rAAV particles produced by any of the methods disclosed herein, or any of the pharmaceutical compositions disclosed herein.
  • Such methods include methods of treating cardiomyopathy by administering any of these products to a patient with cardiomyopathy.
  • the cardiomyopathy may be hypertrophic cardiomyopathy, restrictive cardiomyopathy or dilated cardiomyopathy.
  • the patient exhibits a mutation in one or both cTnT alleles.
  • Such methods also include methods of reducing mutated cTnT protein expression in heart tissue of a subject with cardiomyopathy.
  • the method normalizes cardiomyocyte and/or cardiac function, improves cardiac contractility, improves cardiac relaxation, reduces systolic dysfunction, reduces diastolic dysfunction, reduces ventricular dilation, reduces atrial dilation, reduces dilated left ventricular end-diastolic diameter (LVEDD) and/or reduces left ventricular posterior wall thickness (LVPWTs) , prevents or reduces arrhythmia, prevents cardiac arrest, reduces fainting, reduces dizziness, reduces fatigue, reduces shortness of breath, reduces chest pain, reduces leg swelling, reduces symptoms of heart failure, and/or reduces the amount or frequency of concomitant medications administered to the patient to treat heart failure.
  • LVEDD left ventricular end-diastolic diameter
  • LVPWTs left ventricular posterior wall thickness
  • Figure 1 depicts example schematics showing components of AAV vector constructs described herein.
  • Figures 2A-2B depict transgene hTNNT2 mRNA levels and exogenous cTnT/endogenous cTNT protein levels observed after administration of various doses of rAAV particles comprising an intron-containing vector construct (Format A) and intronless vector construct (Format E) to wild type (WT) induced pluripotent stem cells differentiated into cardiomyocytes (iPSC-CM) .
  • rAAV particles comprising an intron-containing vector construct (Format A) and intronless vector construct (Format E) to wild type (WT) induced pluripotent stem cells differentiated into cardiomyocytes (iPSC-CM) .
  • Figures 3A-3B depict transgene hTNNT2 mRNA levels and exogenous cTnT/endogenous cTnT protein levels observed after administration to WT human iPSC-CM of various doses of rAAV particles comprising vector constructs with different intron sequences at different positions (Formats A-D) .
  • Figures 4A-4F depict results observed after administration to WT mice of rAAV particles comprising vector constructs with different intron sequences at different positions (Formats A-E) .
  • Figure 4A depicts vector copy numbers
  • Figure 4B depicts transgene hTNNT2 mRNA levels
  • Figures 4C-4D depict exogenous cTnT protein levels
  • Figures 4E-4F depict exogenous cTnT/endogenous cTnT protein levels.
  • Figures 5A-5F depict results observed after administration to WT and TNNT2 R141W/R141W homozygous mice of rAAV particles comprising vector constructs with different intron sequences at different positions (Formats A and B) .
  • Figure 5A depicts vector copy numbers
  • Figure 5B depicts transgene hTNNT2 mRNA levels
  • Figures 5C-5D depict exogenous cTnT protein levels
  • Figures 5E-5F depict exogenous cTnT/endogenous cTnT protein levels.
  • Figures 6A-6C depict echocardiography results (ejection fraction, dilated left ventricular end-diastolic diameter (LVEDD) and left ventricular posterior wall thickness (LVPWTs) observed after administration to TNNT2 R141W/R141W homozygous mice of rAAV particles comprising vector constructs with different intron sequences at different positions (Formats A and B) .
  • LVEDD left ventricular end-diastolic diameter
  • LVPWTs left ventricular posterior wall thickness
  • Figures 7A-7F depict results observed after administration to WT iPSC-CMs of rAAV particles comprising vector constructs with wild type or different codon optimized sequences #1-8 and 10-16.
  • Figure 7A -7B depicts transgene hTNNT2 mRNA levels
  • Figures 7C-7F depict exogenous cTnT protein levels.
  • Figures 8A-8D depict results observed after administration to WT mice of rAAV particles comprising vector constructs of Format A, with wild type sequence or three different codon optimized sequences #8, 11 and 13.
  • Figure 8A depicts vector copy numbers
  • Figure 8B depicts transgene hTNNT2 mRNA levels
  • Figures 8C depicts exogenous cTnT protein level
  • Figure 8D depicts exogenous cTnT/endogenous cTnT protein levels.
  • Figures 9A and 9B depict results observed from WT mice and after administration to TNNT2 R141W/R141W homozygous (homo) mice of rAAV particles comprising vector constructs with or without intron sequences (Formats B and E) .
  • Figure 9A depicts the echocardiography showing ejection fraction in the treated mice.
  • Figure 9B depicts the ratio of heart weight (HW) to body weight (BW) at necropsy.
  • WT C57BL/6 mice injected with DPBS
  • Homo TNNT2 R141W/R141W mice injected with DPBS.
  • Figures 10A-10C depict results observed from WT mice and after administration to TNNT2 R141W/R141W homozygous (homo) mice of rAAV particles comprising vector construct (Format B) of 4 different doses.
  • Figure 10A depicts the echocardiography results showing ejection fraction.
  • Figure 10B depicts the ratio of heart weight (HW) to body weight (BW) at necropsy.
  • Figure 10C depicts the survival curves.
  • Figure 11 depicts long-term echocardiography results showing ejection fraction in WT mice and after administration to TNNT2 R141W/R141W homozygous (homo) mice of rAAV particles comprising vector construct (Format B) .
  • Figures 12A and 12B depict the expression of luciferase in WT mouse tissues.
  • Figure 12A depicts the luciferase activities in heart tissues (DPBS, mice injected with DPBS) .
  • Figure 12B depicts the relative luciferase activities of each promoter in different tissues with the activity in heart set as 1.
  • Figures 13A and 13B depict the expression of luciferase in WT mouse tissues.
  • Figure 13A depicts the luciferase activities in heart tissues (DPBS, mice injected with DPBS) .
  • Figure 13B depicts the relative luciferase activities of each promoter in different tissues with the activity in heart set as 1.
  • nucleic acids or vector constructs encoding functionally active therapeutic cTnT protein, AAV vector genomes and replication deficient rAAV particles comprising such vector constructs, and pharmaceutical compositions comprising such vector constructs, vector genomes and AAV particles.
  • the vector constructs comprise hTNNT2 gene (which encodes cTnT protein) and one or more expression control elements.
  • the compositions and methods of the invention may provide improved AAV virus production yield and/or enhanced or specific expression of cTnT protein in the heart, particularly in cells of the myocardium (cardiomyocytes) .
  • methods of making the vector constructs, AAV vector genomes and replication deficient rAAV particles comprising such vector constructs are also provided herein. Further provided herein are methods of treating a deficiency in functional wild-type cTnT, and methods of treating cardiomyopathy, including HCM, RCM or DCM.
  • AAV adeno-associated virus
  • the methods comprise the steps of culturing a cell that has been transfected with any of the AAV vector constructs provided herein (in association with various AAV cap and rep genes) and recovering recombinant therapeutic AAV particles from the transfected cell or supernatant of the transfected cell culture.
  • the cells useful for recombinant AAV production include mammalian cells such as HEK293, HeLa, CHO, NSO, SP2/0, PER. C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5.
  • Cells useful for recombinant AAV production also include any cell type susceptible to baculovirus infection, including insect cells such as High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38.
  • a medicament for the treatment of a subject suffering from cardiomyopathy including HCM, RCM or DCM, or deficiency in functional wild-type cTnT protein.
  • the subject suffering from cardiomyopathy is a human.
  • the medicament is administered by intravenous (IV) administration or subcutaneously.
  • the medicament is administered intramyocardially or by retrograde coronary sinus infusion.
  • administration of the medicament results in increased levels of functional cTnT in cardiac muscle cells.
  • administration of the medicament slows progression of the cardiomyopathy or reverses progression of the cardiomyopathy.
  • administration of the medicament alleviates symptoms of cardiomyopathy, including improving contractile function, reducing dilation of chambers, reducing rigidity of heart muscle, improving relaxation of heart muscle, altering plasma biomarkers (e.g., NT-proBNP, Troponin) , improving ability to perform physical activities, and/or reducing the incidence or severity of end-stage heart failure.
  • the medicament is also for co-administration with a prophylactic and/or therapeutic corticosteroid for the prevention and/or treatment of any toxicity associated with administration of the AAV particle.
  • cardiomyopathy therapy provided herein optionally further includes administration, e.g. concurrent administration, of other therapies that are used to treat cardiomyopathy.
  • vector or “gene delivery vector” may refer to a particle that functions as a gene delivery vehicle, and which comprises nucleic acid (i.e., the vector genome comprising any of the vector constructs described herein) packaged within, for example, an envelope or capsid.
  • a gene delivery vector may be a viral gene delivery vector or a non-viral gene delivery vector.
  • the term “vector” may be used to refer only to the vector genome or vector construct.
  • Viral vectors suitable for use herein may be a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus.
  • the parvovirus may be an adenovirus-associated virus (AAV) .
  • AAV adenovirus-associated virus
  • AAV is a standard abbreviation for adeno-associated virus.
  • Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus.
  • serotypes of AAV There are numerous serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, Handbook of Parvoviruses, Vol. 1, pp. 169-228 (1989) ; and Berns, Virology, pp. 1743-64, Raven Press, (New York) (1990) ; Gao et al., Meth. Mol. Biol. 807: 93-118 (2011) ; Ojala et al., Mol. Ther.
  • an “AAV vector construct” refers to nucleic acids, either single-stranded or double-stranded, having at least one of (i) an AAV 5’ inverted terminal repeat (ITR) sequence and (ii) an AAV 3’ ITR, flanking a protein-coding sequence (in one embodiment, a functional therapeutic protein-encoding sequence, e.g.
  • a single-stranded AAV vector refers to nucleic acids that are present in the genome of an AAV virus particle, and can be either the sense strand or the anti-sense strand of the nucleic acid sequences disclosed herein. The size of such single-stranded nucleic acids is provided in bases.
  • a double-stranded AAV vector refers to nucleic acids that are present in the DNA of plasmids, e.g., pUC19, or genome of a double-stranded virus, e.g., baculovirus, used to express or transfer the AAV vector nucleic acids.
  • the size of such double-stranded nucleic acids in provided in bases or base pairs (bp) .
  • the AAV vector constructs provided herein in single strand form may be less than about 5.5 kb in length, or less than about 5.4 kb in length, or less than about 5.3 kb in length, or are less about 5.2 kb in length.
  • the AAV vector constructs in single strand form may also be at least about 3.0 kb in length.
  • the AAV vector constructs provided herein in single strand form are less than about 7.0 kb in length, or are less than 6.5 kb in length, or are less than 6.4 kb in length, or are less than 6.3 kb in length, or are less than 6.2 kb in length, or are less than 6.0 kb in length, or are less than 5.8 kb in length, or are less than 5.6 kb in length, or are less than 5.5 kb in length, or are less than 5.4 kb in length, or are less than 5.3 kb in length, or are less than 5.2 kb in length.
  • the AAV vector constructs in single strand form may also be at least about 4.0 kb in length.
  • the AAV vector constructs are about 4.5 kb to about 5 kb in length.
  • Oversized AAV vectors are randomly truncated at the 5’ ends and lack a 5’ AAV ITR. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in oversized AAV vectors lacks the 5’ AAV ITR and possibly portions of the 5’ end of the target protein-coding gene, and the antisense strand in oversized AAV vectors lacks the 3’ ITR and possibly portions of the 3’ end of the target protein-coding gene.
  • a functional transgene is produced in oversized AAV vector infected cells by annealing of the sense and antisense truncated genomes within the target cell.
  • the AAV hTNNT2 vectors and/or viral particles comprise at least one ITR.
  • inverted terminal repeat refers to the art-recognized regions found at the 5’ and 3’ termini of the AAV genome which function in cis as origins of DNA replication and as packaging signals for the viral genome.
  • AAV ITRs together with the AAV rep coding region, provide for efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a host cell genome. Sequences of certain AAV-associated ITRs are disclosed by Yan et al., J. Virol. 79: 364-79 (2005) which is herein incorporated by reference in its entirety.
  • ITR sequences that find use herein may be full length, wild-type AAV ITRs or fragments thereof that retain functional capability, or may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as origins of replication.
  • AAV ITRs useful in the recombinant AAV cTnT vectors of the embodiments provided herein may be derived from any known AAV serotype and, in certain embodiments, derived from the AAV2 serotype.
  • control sequences or “expression control sequences” or “regulatory elements” or “regulatory region” refers to DNA sequences that control the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • transcription regulatory element refers to nucleotide sequences of a gene involved in regulation of genetic transcription including a promoter, plus response elements, activator and enhancer sequences for binding of transcription factors to aid RNA polymerase binding and promote expression, and operator or silencer sequences to which repressor proteins bind to block RNA polymerase attachment and prevent expression.
  • cardiomyocyte-specific transcription regulatory element or “cardiomyocyte-specific expression control element” refers to a regulatory element or region that produces preferred gene expression specifically in cardiomyocytes, e.g., a promoter whose activity in cardiac cells is at least 2-fold or at least 5-fold higher than in any other non-cardiac cell type.
  • the cardiomyocyte-specific promoter provides expression in cardiomyocytes at least 5-fold higher than in skeletal muscle cells.
  • the cardiac-specific or cardiomyocyte-specific promoter is operably linked to the nucleic acid sequence encoding the cTnT protein which means that the promoter is combined with the coding nucleic acid so as to enable the expression of said coding nucleic acid under the control of the promoter in cardiomyocytes when integrated into the genome of the cell or present as an extragenomic nucleic acid construct in the cell.
  • a mutated native promoter when linked to its corresponding gene may still be considered heterologous because the sequence does not occur naturally.
  • Regulatory elements include, for example, a promoter, an enhancer element, intron, transcription termination sequences, polyadenylation sequence, or post-transcriptional regulatory elements for increasing the expression level of the desired protein.
  • SV40 early gene enhancer and the enhancer of the long terminal repeat (LTR) of Rous Sarcoma Virus can, for example, be derived from SV40 (Sambrook et al (1989) , Molecular Cloning: A Laboratory Manual) .
  • hGH human growth hormone
  • any other element which is known in the art to support efficiency or specificity of expression may be added to the expression vector, such as the Woodchuck hepatitis post-transcriptional regulatory element (wPRE) and stuffer sequences to balance the payload to approximately 4700 bp.
  • wPRE Woodchuck hepatitis post-transcriptional regulatory element
  • stuffer sequences to balance the payload to approximately 4700 bp.
  • other elements can be introduced to inactivate the expression of genes in other tissues, such as sequences encoding miRNAs.
  • an “intron” is broadly defined as a sequence of nucleotides that is removable by RNA splicing. “RNA splicing” means the excision of introns from a pre-mRNA to form a mature mRNA. Introns may be upstream, downstream, or within the coding region of a gene. Insertion of an intron into a nucleotide sequence can be accomplished by any method known in the art.
  • Heterologous refers to a nucleotide or polypeptide sequence that is non-native or native to an AAV or a host cell, but is not present in its native location or position within the viral genome or host cell genome. For example, artificial manipulation of isolated segments can result in a novel combination of sequences that are not normally linked together.
  • operably linked is used to describe the connection between regulatory elements and a gene or its coding region.
  • gene expression is placed under the control of one or more regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers.
  • a gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.
  • a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.
  • the recombinant AAV vector construct comprises (a) a nucleic acid comprising an AAV2 5’ inverted terminal repeat (ITR) (which may or may not be modified as known in the art) , (b) a cardiomyocyte-specific transcription regulatory region, (c) a functional cTnT protein coding region, (d) optionally, one or more introns, I a polyadenylation sequence, and (f) an AAV2 3’ ITR (which may or may not be modified as known in the art) .
  • ITR inverted terminal repeat
  • the vector construct comprises a nucleic acid encoding a functionally active cTnT protein, for example, a hTNNT2 gene.
  • the cTnT encoding sequence may be wild-type, codon optimized, or a variant.
  • other optional elements can be introduced as part of the cTnT encoding sequence, such as epitope-tag sequences (myc, FLAG, HA, His, and the like) , or fluorochromes such as GFP, YFP, RFP.
  • wild-type hTNNT2 gene encoding cTnT protein is the isoform that has the nucleotide sequence of SEQ ID NO: 1
  • wild-type cTnT protein is the isoform has the amino acid sequence of SEQ ID NO: 2.
  • isolated when used in relation to a nucleic acid molecule of the present disclosure typically refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid may be present in a form or setting that is different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • variant refers to a polynucleotide (or polypeptide) having a sequence substantially similar to a reference polynucleotide (or polypeptide) .
  • Procedures for the introduction of nucleotide and amino acid changes in a polynucleotide, protein or polypeptide are known to the skilled artisan (see, e.g., Sambrook et al. (1989) ) .
  • a variant can have deletions, substitutions, additions of one or more nucleotides at the 5’ end, 3’ end, and/or one or more internal sites in comparison to the reference polynucleotide.
  • variants and/or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques.
  • variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis.
  • a variant of a polynucleotide including, but not limited to, a DNA, can have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%or more sequence identity to the reference polynucleotide as determined by sequence alignment programs known by skilled artisans.
  • a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide.
  • a variant of a polypeptide can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%or more sequence identity to the reference polypeptide as determined by sequence alignment programs known by skilled artisans.
  • the amino acid substitutions can be conservative or non-conservative. It is preferred that the substitutions are conservative substitutions, i.e. a substitution of an amino acid residue by an amino acid of similar polarity, which acts as a functional equivalent.
  • the amino acid residue used as a substitute is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue can be substituted with another hydrophobic residue, or a polar residue can be substituted with another polar residue having the same charge.
  • Functionally homologous amino acids which may be used for a conservative substitution comprise, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.
  • non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.
  • uncharged polar amino acids comprise serine, threonine, glutamine, asparagine, tyrosine and cysteine.
  • Examples of charged polar (basic) amino acids comprise histidine, arginine and lysine.
  • charged polar (acidic) amino acids comprise aspartic acid and glutamic acid.
  • cTnT protein also considered as functional protein, e.g. functional cTnT protein, are variants which differ from their naturally occurring counterparts by addition, substitution or deletion of one or more (e.g. 2, 3, 4, 5, 10, or 15 or more) amino acids. Additional amino acids may be present within the amino acid sequence of the original cTnT protein (i.e. as an insertion) , or they may be added to one or both termini of the protein. Such insertions, substitutions or deletions can take place at any position provided they do not impair the capability of the polypeptide to fulfill the function of the naturally occurring cTnT protein and/or rescue the disease in the treated subject.
  • insertions, substitutions or deletions can take place at any position provided they do not impair the capability of the polypeptide to fulfill the function of the naturally occurring cTnT protein and/or rescue the disease in the treated subject.
  • variants of cTnT protein also comprise proteins in which, compared to the original polypeptide, one or more amino acids are lacking. Such deletions may affect any amino acid position provided that it does not impair the ability to fulfill the normal function of the cTnT protein and/or rescue the disease.
  • variants of the cTnT protein also refer to proteins which differ from the naturally occurring protein by structural modifications, such as modified amino acids.
  • Modified amino acids are amino acids which have been modified either by natural processes, such as processing or post-translational modifications, or by chemical modification processes known in the art.
  • Typical amino acid modifications comprise phosphorylation, glycosylation, acetylation, O-Linked N-acetylglucosamination, glutathionylation, acylation, branching, ADP ribosylation, crosslinking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization and/or covalent or non-covalent bonding to hosphatidylinositol, flavine derivatives, lipoteichonic acids, fatty acids or lipids.
  • modifications have been extensively described in the literature, e.g., in Proteins: Structure and Molecular Properties, T. Creighton, 2 nd edition, W. H. Freeman and Company, New York (1993) .
  • identity means that two or more referenced entities are the same, when they are “aligned” sequences.
  • two polypeptide sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion.
  • two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion.
  • the identity can be over a defined area (region or domain) of the sequence.
  • An “area” or “region” of identity refers to a portion of two or more referenced entities that are the same.
  • two polypeptide or nucleic acid sequences are identical over one or more sequence areas or regions they share identity within that region.
  • aligned sequence refers to multiple polynucleotide or polypeptide (amino acid) sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence.
  • Substantial homology means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e.g., a biological function or activity) of the reference molecule, or relevant/corresponding region or portion of the reference molecule to which it shares homology.
  • Percent (%) nucleic acid sequence identity or homology is defined as the percentage of nucleotides in a candidate sequence that are identical with a reference sequence after aligning the respective sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Unless otherwise specified, “percent (%) identity” is calculated as the number of identical nucleotides between the candidate sequence and reference sequences divided by the number of nucleotides of the candidate sequence (i.e. the full length candidate sequence) . In some cases (when specified) , “percent (%) identity” is calculated as the number of identical nucleotides between the candidate sequence and reference sequences divided by the number of nucleotides of the reference sequence (i.e. the full length reference sequence) .
  • Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Percent (%) amino acid sequence identity or homology e.g., with respect to the cTnT amino acid sequences identified herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a cTnT polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Unless otherwise specified, “percent (%) identity” is calculated as the number of identical amino acids between the candidate sequence and reference sequences divided by the number of animo acids of the candidate sequence (i.e. the full length candidate sequence) .
  • percent (%) identity is calculated as the number of identical amino acids between the candidate sequence and reference sequences divided by the number of amino acids of the reference sequence (i.e. the full length reference sequence) . Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Codon optimization or “codon optimized” refers to changes made in the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the non-codon optimized sequence. It does not change the amino acid for which each codon encodes.
  • An “AAV virion” or “AAV viral particle” or “AAV vector particle” or “AAV virus” refers to a viral particle composed of at least one AAV capsid protein and an encapsulated AAV vector construct as described herein. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell) , it is typically referred to as a “recombinant AAV vector particle” or simply an “AAV vector” .
  • Production of AAV vector particles necessarily includes production of AAV vector genome, as such a vector genome is contained within an AAV vector particle. It is understood that reference to the polynucleotide AAV vector construct encapsulated within the vector particle, and replication thereof, refers to the AAV vector genome.
  • AAV virus refers to an AAV virion, AAV viral particle, AAV vector particle, or AAV virus that comprises a heterologous polynucleotide that encodes a therapeutic protein such as the cTnT described herein.
  • An “AAV vector construct” or “AAV vector genome” as used herein refers to a vector construct comprising one or more gene of interest, e.g. a polynucleotide encoding a protein of interest (also called transgenes) , that are flanked by at least one AAV terminal repeat sequences (ITRs) and operably linked to one or more expression control elements.
  • ITRs AAV terminal repeat sequences
  • therapeutic protein refers to a polypeptide that has a biological activity that replaces or compensates for the loss or reduction of activity of an endogenous protein.
  • a functional cTnT protein is a therapeutic protein for cardiomyopathy.
  • Cardiomyopathy refers to an inherited disease caused by mutations in troponin T characterized, for example, by symptoms of palpitations, chest pain, shortness of breath, fatigue and dizziness, fainting, arrhythmias, sudden cardiac death, heart failure, and/or swelling in feet, ankles, legs or belly.
  • Genesenor disease or disorder refers to an inherited disease or disorder caused by a mutation in a subject’s endogenous gene of interest (a “mutated endogenous gene of interest” ) which in turn produces a defective endogenous gene therapy product (protein or RNA) that is non-functional or has reduced or aberrant activity.
  • a mutant endogenous gene of interest a defective endogenous gene therapy product
  • RNA defective endogenous gene therapy product
  • Deficiency in gene therapy product refers to an inherited condition caused by a reduced level of endogenous functional gene therapy product, due to absence of the endogenous gene therapy product, reduced production of the endogenous gene therapy product, or production of a mutated defective endogenous gene therapy product that is non-functional or has reduced or aberrant activity.
  • a deficiency in gene therapy product includes genetic diseases and/or disorders.
  • “Therapeutically effective” or “gene therapy” as used herein refers to any therapeutic intervention of a subject having a disease or disorder that benefits from administration of a gene therapy product, e.g., wherein the therapeutic invention ameliorates symptoms of the disease or disorder.
  • the therapy ameliorates a deficiency in an endogenous functional gene therapy product, increases level of functional gene therapy product in the subject, e.g., in heart tissue and/or cardiomyocytes, and/or ameliorates symptoms of the disease or disorder, including reduces the frequency, duration or severity of symptoms of the disease or disorder.
  • Cardiac Tropinin T (cTnT) protein mutation or a “mutation in functional wild-type cTnT protein” as used herein refers to an inherited condition caused by altered DNA sequences encoding for cTnT protein.
  • the mutation in the amino acid sequence in the cTnT protein may result in a gain of function (GOF) effect or have a dominant negative effect on properly functioning wild type cTnT protein.
  • GAF gain of function
  • These effects can manifest as cardiomyopathy, for example, hypertrophic cardiomyopathy, restrictive cardiomyopathy or dilated cardiomyopathy.
  • “Therapeutically effective for cardiomyopathy” or “cardiomyopathy therapy” as used herein refers to any therapeutic intervention of a subject having cardiomyopathy that ameliorates the characteristic alterations to functional wild-type cTnT, reduction in mutant protein levels and replacement with wildtype cTnT protein within the cell, e.g. in myocardium, ameliorates symptoms, or reduces the frequency, duration or severity of symptoms.
  • Cardiomyopathy gene therapy refers to any therapeutic intervention of a subject having cardiomyopathy that involves the replacement or restoration or increase of cTnT through the delivery of one or more nucleic acid molecules to the cells of the subject that encode or express functional cTnT.
  • cTnT gene therapy refers to gene therapy involving an adeno associated viral (AAV) particle comprising a vector construct that encodes human cTnT.
  • AAV adeno associated viral
  • the gene therapy involves transfecting a plasmid that encodes human cTnT.
  • Treat” or “treatment” as used herein refers to preventive or therapeutic treatment which refers to a treatment administered to a subject who exhibits signs or symptoms of pathology, i.e., cardiomyopathy, for the purpose of diminishing or eliminating those signs or symptoms or ameliorating their progression, severity or duration.
  • the signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective.
  • “Ameliorate” as used herein refers to the action of lessening the severity of symptoms, progression, or duration of a disease.
  • stably treating refers to using a therapeutic vector construct, AAV particle or cell administered to a subject where the subject stably expresses a therapeutic protein encoded or expressed by the vector construct, AAV particle or cell.
  • Stably encoded or expressed therapeutic protein means that the protein is encoded or expressed for a clinically significant length of time.
  • “Clinically significant length of time” as used herein means expression at therapeutically effective levels for a length of time that has a meaningful impact on the quality of life of the subject, e.g., demonstrated by reduced signs or symptoms of disease.
  • significant length of time is expression for at least six months, for at least eight months, for at least one year, for at least two years, for at least three years, for at least four years, for at least five years, for at least six years, for at least seven years, for at least eight years, for at least nine years, for at least ten years, or for the life of the subject.
  • the term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • a “subject” refers to an animal that is the object of treatment, observation or experiment.
  • “Mammal, ” as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals.
  • Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes, and, in particular, humans.
  • the mammal is a human, including an infant, child or juvenile human.
  • a “pharmaceutically acceptable carrier” is one that is not toxic or unduly detrimental to cells and is preferably sterile.
  • exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, saline or phosphate buffered saline.
  • Pharmaceutically acceptable carriers include physiologically acceptable carriers.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the recombinant vector construct of the disclosure may be used itself as gene therapy, or may be used to produce rAAV particles by methods described herein, comprising providing to a suitable host cell the recombinant vector construct, together with Rep and Cap genes.
  • the vector constructs described herein comprise a nucleic acid sequence that encodes a functional cTnT protein.
  • the recombinant vector construct may comprise a nucleic acid encoding functional human cTnT operably linked to a heterologous expression control element, e.g. a promoter and/or enhancer; optionally an intron; and optionally a polyadenylation (polyA) signal.
  • the heterologous expression control element may be a heterologous cardiomyocyte-specific promoter as described herein.
  • the recombinant vector construct may comprise (a) one or both of (i) an AAV 5’ inverted terminal repeat (ITR) sequence and (ii) an AAV 3’ ITR, (b) a heterologous cardiomyocyte-specific promoter, and (c) a nucleic acid encoding a functional human cTnT, optionally wherein the AAV ITRs are AAV2 ITRs.
  • the nucleic acid encoding the functional cTnT is operably linked to the cardiomyocyte-specific promoter.
  • the vector construct may include one or more additional expression control elements, for example: an enhancer; an intron (optionally linked to an exon or fragment thereof) ; and/or a polyadenylation (polyA) signal.
  • additional expression control elements for example: an enhancer; an intron (optionally linked to an exon or fragment thereof) ; and/or a polyadenylation (polyA) signal.
  • the recombinant AAV vector construct comprises a nucleic acid comprising (a) an AAV2 5’ inverted terminal repeat (ITR) (which may or may not be modified as known in the art) , (b) a cardiomyocyte-specific transcription regulatory region, a functional cTnT protein coding region, (c) an intron, preferably between exons 2 and 3 of the nucleic acid encoding functional cTnT, (d) optionally an exon or fragment thereof, (e) a polyadenylation sequence, (f) optionally a stuffer sequence, and (g) an
  • the rAAV particles also comprise an AAV capsid with cardiac tropism, optionally an AAV6 or AAV9 type capsid.
  • AAV capsids with cardiac tropism include AAV1, 6, 7 and 9.
  • constructs encoding a functional cTnT polypeptide, wherein the constructs comprise one or more of the individual elements of the above described constructs and combinations thereof, in one or more different arrangements or orientation (s) .
  • Another embodiment provided herein is directed to the above-described constructs in an opposite orientation.
  • the AAV vector constructs provided herein in single strand form range from about 3.0kb to about 5.5kb in size.
  • the vector construct is an AAV vector genome about 4 kb to about 5.5kb in size, or about 4.5 kb to about 5 kb in size.
  • AAV vectors When AAV vectors are produced from recombinant vector constructs, they may lack a portion of the 5’ or 3’ ends of the recombinant vector construct. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in AAV vectors lacks the 5’ AAV ITR and possibly portions of the 5’ end of the target protein-coding gene, and the antisense strand in AAV vectors lacks the 3’ ITR and possibly portions of the 3’ end of the target protein-coding gene. A functional transgene is produced from these truncated strands in AAV-infected cells by annealing of the sense and antisense truncated strands within the infected cell.
  • the rAAV particles of the invention may comprise recombinant vector constructs that comprise at least one ITR, and a substantial portion of a nucleotide sequence encoding a functional cTnT, such as a fragment of any of SEQ ID NOs: 1, or 3-20, most preferably SEQ ID NO: 1, or alternative codon optimized versions SEQ ID NOs: 3-17, that is greater than 50%, 60%, 70%, 80%, or 90%of the length of the nucleotide sequence.
  • the recombinant vector construct may comprise at least one ITR, a cardiomyocyte-specific promoter, and a substantial portion of a nucleotide sequence encoding a functional cTnT.
  • Generation of the vector constructs can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989) ) .
  • AAV vector constructs can be replicated and packaged into infectious AAV particles, preferably replication deficient AAV particles, when present in a host cell that has been transfected with polynucleotide (s) encoding and expressing rep and cap gene product.
  • polynucleotide (s) encoding and expressing rep and cap gene product.
  • a “protein of interest” is any functional cTnT protein, including naturally-occurring and non-naturally occurring variants thereof.
  • a polynucleotide encoding one or more cTnT proteins of interest can be inserted into the viral vectors disclosed herein, wherein the polynucleotide is operably linked with the promoter.
  • the promoter can drive the expression of the protein (s) of interest in a host cell (e.g., human myocardium) .
  • the vector construct of the disclosure comprising SEQ ID NO: 1 encodes a functional cTnT protein of SEQ ID NO: 2.
  • the encoded functional cTnT comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%or 100%identical (over its length) to SEQ ID NO: 2.
  • the encoded functional cTnT comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%to 100%identical to SEQ ID NOs: 71, 76 or 77.
  • the functional cTnT comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%to 100%identical to any of SEQ ID NOs: 72-75 and 78-81.
  • the encoded functional cTnT excludes the amino acid sequence encoded by exon 5 (SEQ ID NO: 58) .
  • the present disclosure also provides an isolated nucleic acid molecule which encodes such functional wild-type cTnT protein, such as SEQ ID NO: 1.
  • the nucleic acid molecule encodes a functional cTnT and comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 1, or at least 100, 200, 300, 400 or 500 consecutive nucleotides of SEQ ID NO: 1.
  • the nucleic acid molecule encoding a functional cTnT is a codon optimized variant, for example, comprises the nucleotide sequence of any of SEQ ID NOs: 3-17.
  • the nucleic acid molecule encoding or expressing a functional cTnT is a variant, for example, comprising the nucleotide sequence of any of SEQ ID NOs: 18-20.
  • the nucleic acid molecule encodes a functional cTnT and comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to any of SEQ ID NOs: 3-20, or at least 100, 200, 300, 400 or 500 consecutive nucleotides of SEQ ID NO: 3-20.
  • the nucleic acid molecule comprises a nucleotide sequence at least 80%, 85%or 90%identical to SEQ ID NO: 1 or comprises a nucleotide sequence at least 80%, 85%or 90%identical to the nucleotide sequence of any of SEQ ID NOs: 3-20, and encodes or expresses a functional cTnT comprising an amino acid sequence at least 90%identical to SEQ ID NO: 2 or comprises an amino acid sequence at least 80%, 85%or 90%identical to SEQ ID NOs: 71-81 (preferably SEQ ID NOs: 71, 76 or 77) .
  • the nucleic acid molecule comprises a nucleotide sequence at least 80%, 85%or 90%identical to SEQ ID NO: 1 or comprises a nucleotide sequence at least 80%, 85%or 90%identical to the nucleotide sequence of any of SEQ ID NOs: 3-20, and encodes or expresses a functional cTnT comprising an amino acid sequence at least 95%identical to any of SEQ ID NOs: 2 or 71-81.
  • the nucleic acid molecule comprises a nucleotide sequence at least 80%, 85%or 90%identical to SEQ ID NO: 1 or comprises a nucleotide sequence at least 80%, 85%or 90%identical to the nucleotide sequence of any of SEQ ID NOs: 3-20, and encodes or expresses a functional cTnT comprising an amino acid sequence at least 98%identical to any of SEQ ID NOs: 2 or 71-81.
  • the expressed functional cTnT excludes the amino acid sequence encoded by exon 5 (SEQ ID NO: 58) .
  • the nucleotide sequence of the gene of interest is codon optimized, preferably codon optimized for more efficient expression in humans, or for more efficient expression in a target organ, target tissue and/or target cells of humans.
  • Target organs, tissues or cells include heart tissue and/or cardiomyocytes.
  • the adaptiveness of a nucleotide sequence encoding a gene therapy product to the codon usage of human cells may be expressed as codon adaptation index (CAI) .
  • a codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed human genes.
  • the relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid.
  • CAI is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Kim et al., Gene. 1997, 199: 293-301; zur Megede et al., Journal of Virology, 2000, 74: 2628-2635) . In certain embodiments, a gene of interest has a CAI of at least 0.75, 0.80, 0.85, 0.90, 0.95, or 0.99.
  • Codon optimization can be performed, for example, using the DNA2.0 codon optimization algorithm, see Villalobos et al., “Gene Designer: a synthetic biology tool for constructing artificial DNA segments, ” BMC Bioinformatics, vol. 7, article no: 285 (2006) or Operon/Eurofins Genomics codon optimization software or other codon optimization tools, e.g. Grote et al., “Jcat: a novel tool to adapt codon usage of a target gene to its potential expression host, ” Nucleic Acids Res. 33: W526-31 (2005) .
  • the nucleotide sequence of the gene of interest can be adjusted to reduce CpG di-nucleotide content and optionally remove any extra ORF in the sense and anti-sense direction.
  • CpG di-nucleotide content has been shown to activate TLR9 in dendritic cells leading to potential immune activation and CTL responses. Reducing CpG content may reduce liver inflammation and ALT.
  • the nucleotide sequence of the gene of interest has a CpG di-nucleotide content of less than 25, less than 20, less than 15, or less than 10.
  • the nucleotide sequence of the gene of interest has a GC content of less than 65%, less than 60%, or less than 55%.
  • codon optimization or CpG reduction does not change the amino acid for which each codon encodes. It simply changes the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the non-optimized sequence.
  • the nucleotide sequence encoding the cTnT protein can be modified to improve expression efficiency of the protein.
  • the methods that can be used to improve the transcription and/or translation of a gene herein are not particularly limited.
  • the nucleotide sequence can be modified to better reflect host codon usage to increase gene expression (e.g., protein production) in the host (e.g., a mammal) .
  • one or more of the splice donors and/or splice acceptors in the nucleotide sequence of the protein of interest is modified to reduce the potential for extraneous splicing.
  • one or more introns can be inserted within or adjacent to the nucleotide sequence of the protein of interest to optimize AAV vector packaging and enhance expression.
  • the nucleic acid molecule when expressed in a suitable system (e.g. a host cell) , produces a functional cTnT protein and at a relatively high level. Since the cTnT that is produced is functional, it will have a conformation which is the same as at least a portion of the wild type cTnT. In certain embodiments, a functional cTnT protein produced as described herein effectively treats a subject suffering from deficiency in wild-type cTnT protein and/or cardiomyopathy.
  • a suitable system e.g. a host cell
  • nucleic acid molecule provided herein. This could be done, for example, using chemical synthesis of a given sequence. Further, suitable methods would be apparent to those skilled in the art for determining whether a nucleic acid described herein encodes or expresses a functional protein. For example, one suitable in vitro method involves inserting the nucleic acid into a vector construct, transducing host cells with the vector, and assaying for exogenous cTnT expression. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into mice with a cardiomyopathy model of disease and assaying for reduced cardiomyopathy symptoms. Regulatory Elements
  • the nucleic acid sequence encoding cTnT is operably linked to one or more heterologous expression control elements.
  • the expression control element is a cardiomyocyte-specific promoter, for example, a human cardiac troponin T (hTNNT2) promoter, cTnT413 promoter, mouse alpha-myosin heavy chain ( ⁇ MHC) promoter, human ⁇ MHC promoter, human beta-myosin heavy chain ( ⁇ MHC) or fragments, rat cardiac myosin light chain 2 (MLC-2) promoter, or fragments or variants thereof.
  • hTNNT2 human cardiac troponin T
  • cTnT413 promoter
  • mouse alpha-myosin heavy chain ( ⁇ MHC) promoter human ⁇ MHC promoter
  • human beta-myosin heavy chain ( ⁇ MHC) or fragments rat cardiac myosin light chain 2 (MLC-2) promoter, or fragments or variants thereof.
  • fragments or variants of hTNNT2 promoter include a cardiomyocyte-specific promoter sequence comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 21 (over the length of the SEQ ID NO) .
  • fragments or variants of cTnT413 promoter include a cardiomyocyte-specific promoter sequence comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 102 (over the length of the SEQ ID NO) .
  • fragments or variants of mouse ⁇ (alpha) MHC promoter include a cardiomyocyte-specific promoter sequence comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 103 (over the length of the SEQ ID NO) .
  • fragments or variants of human ⁇ MHC promoter include a cardiomyocyte-specific promoter sequence comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 104 (over the length of the SEQ ID NO) .
  • fragments or variants of human ⁇ (beta) MHC promoter include a cardiomyocyte-specific promoter sequence comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 105 (over the length of the SEQ ID NO) .
  • fragments or variants of rat ⁇ MHC promoter include a cardiomyocyte-specific promoter sequence comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 106 (over the length of the SEQ ID NO) .
  • fragments or variants of rat MLC-2 promoter include a cardiomyocyte-specific promoter sequence comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 107 (over the length of the SEQ ID NO) .
  • the vector construct also comprises an intron, optionally adjacent to an exon or fragment thereof.
  • the location and size of the intron in the vector can vary.
  • the intron is located between the promoter and the sequence encoding the protein of interest.
  • the intron is located downstream of the sequence encoding the protein of interest.
  • the intron is located within the promoter.
  • the intron is located within the sequence encoding the protein of interest, preferably between exons of the sequence encoding the protein of interest.
  • the intron may comprise all or a portion of a naturally occurring intron within the sequence encoding the protein of interest.
  • the intron is a heterologous or hybrid intron.
  • the intron may be 5’ to the cTnT coding sequence or within the cTnT coding sequence, for example between exon 2 and exon 3.
  • the intron may be located after position 41 of SEQ ID NO: 1 or the corresponding position 41 in any of SEQ ID NOs: 3-20.
  • the intron enhances expression of the cTnT protein.
  • Examples of intron sequences include CBA hybrid intron, hTNNT2 intron sequences 1 or 2, or fragments or variants thereof.
  • the intron may comprise a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to any of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, or any combination thereof (over the length of the SEQ ID NO) .
  • the vector construct further comprises a stuffer sequence that does not encode functional protein.
  • stuffer sequences include a reversed sequence of human albumin 5’ UTR noncoding region comprising the nucleotide sequence of any of SEQ ID NO: 23-37, or a fragment thereof.
  • the stuffer sequence is used to increase the length of the insert between the two ITRs, such that the AAV vector construct is about 3.5 kb to about 5.5 kb in length, or about 4 kb to about 5 kb in length, or about 4.5 kb to about 5 kb in length.
  • the vector construct further comprises a transcription termination region such as a polyadenylation signal sequence.
  • a transcription termination region such as a polyadenylation signal sequence.
  • polyadenylation signal sequences include, but are not limited to, human growth hormone (hGH) poly (A) , bovine growth hormone (bGH) poly (A) , rabbit ⁇ -globin (RGB) poly (A) , or functional fragments or variants thereof.
  • the transcriptional termination region is an hGH polyA comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 38 (over the length of the SEQ ID NO) .
  • the transcriptional termination region is an RGB polyA comprising a nucleotide sequence at least, or more than, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or 99%identical to SEQ ID NO: 108 (over the length of the SEQ ID NO) .
  • regulatory elements can be used in the vector constructs, for example enhancers to further increase expression level of the protein of interest in a host cell, a ribosome binding sequence, and/or a consensus splice acceptor or splice donor site.
  • the regulatory element can facilitate maintenance of the recombinant DNA molecule extrachromosomally in a host cell and/or improve vector potency (e.g. scaffold/matrix attachment regions (S/MARs) ) .
  • S/MARs scaffold/matrix attachment regions
  • Example embodiments include vector constructs of Formats A, B, C, D and E, which each comprise a 5’ and 3’ AAV ITR.
  • Format A comprises (a) a hTNNT2 promoter, optionally with a 5’ UTR fragment of the hTNNT2 gene, (b) a CBA hybrid intron adjacent the promoter and upstream (5’ ) of the transgene, (c) nucleic acid encoding functional cTnT protein, (d) human growth hormone polyA signal, and (e) a stuffer sequence (non-functional, non-coding nucleotide sequence added to increase the size of the AAV vector genome) .
  • Construct A1 SEQ ID NO: 39
  • Construct A2 SEQ ID NO: 44
  • Construct A3 SEQ ID NO: 49
  • Format B comprises (a) a hTNNT2 promoter, optionally with a 5’ UTR fragment of the hTNNT2 gene (b) a hTNNT2 intron 2 located within the coding region of the transgene, e.g. between exons 2 and 3, (c) nucleic acid encoding functional cTnT protein, (d) human growth hormone polyA signal, and (e) a stuffer sequence.
  • Construct B1 SEQ ID NO: 40
  • Construct B2 SEQ ID NO: 45
  • Construct B3 SEQ ID NO: 50
  • Format C comprises (a) a hTNNT2 promoter, optionally with a 5’ UTR fragment of the hTNNT2 gene (b) a chimeric intron2D+CBA hybrid intron located within the coding region of the transgene, e.g. between exons 2 and 3, (c) nucleic acid encoding functional cTnT protein, (d) human growth hormone polyA signal, and (e) a stuffer sequence. See, e.g. Construct C1 (SEQ ID NO: 41) , Construct C2 (SEQ ID NO: 46) or Construct C3 (SEQ ID NO: 51) .
  • Format D comprises (a) a hTNNT2 promoter, optionally with a 5’ UTR fragment of the hTNNT2 gene (b) a chimeric intron comprising intron2D+intron 1 partial+intron 2A located within the coding region of the transgene, e.g. between exons 2 and 3, (c) nucleic acid encoding functional cTnT protein, and (d) human growth hormone polyA signal. See, e.g. Construct D1 (SEQ ID NO: 42) , Construct D2 (SEQ ID NO: 47) or Construct D3 (SEQ ID NO: 52) .
  • Format E comprises (a) a hTNNT2 promoter, (b) nucleic acid encoding functional cTnT protein, (c) human growth hormone polyA signal, and (d) a stuffer sequence. See, e.g. Construct E1 (SEQ ID NO: 43) , Construct E2 (SEQ ID NO: 48) or Construct D3 (SEQ ID NO: 53) .
  • the above-described AAV vector construct comprises an insert between the two ITRs that comprises a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%or 99%identical to any of SEQ ID NOs: 39-43.
  • the vector construct comprises a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%or 99%identical to a nucleotide sequence that is complementary to or is a negative (-) strand of any of SEQ ID NOs: 39-43.
  • the vector constructs include a nucleic acid encoding a Flag-tag linked to the nucleic acid encoding cTNT, e.g. any of SEQ ID NOs: 30-34 which correspond to SEQ ID NOs. 39-45, respectively.
  • the above-described AAV vector construct comprises an insert between the two ITRs that comprises a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%or 99%identical to any of SEQ ID NOs: 49-53 and further comprises one or more stuffer sequence (s) or additional intron sequences (e.g., partial intron 1) sufficient in length such that the AAV vector construct is a length of about 3.5 kb to about 5.5 kb in length, or about 4 kb to about 5 kb in length, or about 4.5 kb to about 5 kb in length.
  • stuffer sequence s
  • additional intron sequences e.g., partial intron 1
  • the vector construct comprises a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%or 99%identical to a nucleotide sequence that is complementary to or is a negative (-) strand of any of SEQ ID NOs: 49-53 and further comprises one or more stuffer sequence (s) or additional intron sequences (e.g., partial intron 1) sufficient in length such that the AAV vector construct is a length of about 3.5 kb to about 5.5 kb in length, or about 4 kb to about 5 kb in length, or about 4.5 kb to about 5 kb in length.
  • stuffer sequence s
  • additional intron sequences e.g., partial intron 1
  • the AAV vector construct may comprise any known 5’ ITR and 3’ ITR sequences such as AAV2-ITR sequences.
  • Example ITR sequences include but are not limited to SEQ ID NOs: 99-100 including any complementary sequences and/or combinations thereof.
  • Polynucleotides and polypeptides including modified forms can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques known to those of skill in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition) . Methods of Gene Delivery.
  • a gene delivery vector may be a viral gene delivery vector, such as a viral particle, or a non-viral gene delivery vector, such as a vector construct or nucleic acid encoding the protein of interest.
  • Viral vectors include parvovirus, an adenovirus, a retrovirus, a gamma-retrovirus, a lentivirus, a herpes simplex virus, a vaccinia virus, a measles virus, a vesicular stomatitis virus, a polio virus or a reovirus.
  • the parvovirus may be an adenovirus-associated virus (AAV) .
  • non-viral systems may be used, including using naked DNA (with or without chromatin attachment regions) or conjugated DNA that is introduced into cells by various transfection methods.
  • Example methods include electroporation, sonoporation, biolistic, or the use of a "gene gun” , which shoots DNA coated gold particles into the cell using, for example, high pressure gas or an inverted . 22 calibre gun ( Gene Gun System (BIO-RAD) ) , microinjection, lasers, elevated temperature, ultrasound, hydrodynamic gene transfer, magnetotransfection, chemical transfection (e.g.
  • DNA may be integrated from a donor construct that comprises the gene of interest and intron as described herein, flanked by homology arms.
  • DNA from a donor construct may also be integrated into a genome via targeted gene editing methods with nucleases such as ZFNs, TALENs, meganucleases, or CRISPR-Cas9.
  • the targeted nuclease cleaves a target site in a gene, and the donor construct, which comprises homology arms complementary to the regions flanking the target site, facilitate homology directed repair which causes integration of the sequence between the homology arms.
  • the donor construct, or donor template may be for example a single-stranded donor oligonucleotides (ssODN) , double-stranded DNA (e.g. PCR product) , minicircle or virus (rAAV or lentivirus) ) .
  • ssODN single-stranded donor oligonucleotides
  • rAAV or lentivirus minicircle or virus
  • a suitable viral gene delivery vector such as a viral particle may be used to deliver a nucleic acid.
  • viral gene delivery vectors suitable for use herein may be a parvovirus, an adenovirus, a retrovirus, a gamma-retrovirus, a lentivirus, a herpes simplex virus, a vaccinia virus, a measles virus, a vesicular stomatitis virus, a polio virus or a reovirus.
  • the parvovirus may be an adenovirus-associated virus (AAV) .
  • AAV adenovirus-associated virus
  • the present disclosure provides viral particles for use as gene delivery vectors (comprising an AAV vector construct provided herein) based on animal parvoviruses, in particular dependoviruses such as infectious human or simian AAV, and the components thereof (e.g., an animal parvovirus genome) for introduction and/or expression of a cTnT protein in a mammalian cell.
  • dependoviruses such as infectious human or simian AAV
  • the components thereof e.g., an animal parvovirus genome
  • the term "parvoviral” as used herein thus encompasses dependoviruses such as any type of AAV.
  • Dependoviruses are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture.
  • the genus Dependovirus includes AAV, which normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) , primates (e.g., serotypes 1 and 4) , and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, mice, rats, and ovine adeno-associated viruses) in addition to birds and reptiles. Further information on parvoviruses and other members of the Parvoviridae is described in Kenneth I.
  • AAV rep and cap genes are genes encoding replication and encapsidation proteins, respectively.
  • AAV rep and cap genes have been found in all AAV serotypes examined to date, and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (i.e., they are “coupled” together as adjoining or overlapping transcriptional units) , and they are generally conserved among AAV serotypes.
  • AAV rep and cap genes are also individually and collectively referred to as "AAV packaging genes.
  • the AAV cap genes for use herein encode Cap proteins which are capable of packaging AAV vectors in the presence of rep and adeno helper function and are capable of binding target cellular receptors.
  • the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.
  • the AAV sequences employed for the production of AAV can be derived from the genome of any AAV serotype.
  • the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide a similar set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms.
  • genomic sequence of AAV serotypes and a discussion of the genomic similarities. (See, e.g., GenBank Accession number U89790; GenBank Accession number J01901; GenBank Accession number AF043303; GenBank Accession number AF085716; Chiorini et al., J. Virol.
  • 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 form the capsid.
  • the assembly-activating protein (AAP) rapidly chaperones capsid assembly and prevents degradation of free capsid proteins (Grosse et al., J. Virol. 91 (20) : e01198-17 (2017) .
  • the terminal 145 nt 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 encode the Rep proteins, Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the p19 promoter.
  • the cap genes encode the VP proteins, VP1, VP2, and VP3. The cap genes are transcribed from the p40 promoter.
  • the ITRs employed in the vectors of the present embodiment may correspond to the same serotype as the associated cap genes, or may differ. In one embodiment, the ITRs employed herein correspond to an AAV2 serotype and the cap genes correspond to an AAV6 or AAV9 serotype.
  • the AAV VP proteins are known to determine the cellular tropicity of the AAV virion.
  • the VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes.
  • the ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped AAV particles comprising the capsid proteins of a serotype (e.g., AAV1, 6, 7 or 9) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2) .
  • pseudotyped rAAV particles are a part of the present disclosure.
  • the AAV ITR sequences for use in the context of the present disclosure are derived from AAV1, AAV2, AAV4, AAV6 and/or AAV9.
  • the Rep (e.g., Rep78 and Rep52) coding sequences are in one embodiment derived from AAV1, AAV2, AAV4, AAV6 and/or AAV9.
  • sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the context of the present disclosure may however be taken from any serotype, such as from AAV1, AAV2, AAV3 or 3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or from simian AAVs or mixed serotypes (see, e.g., US Patent No. 8,318,480 for its disclosure of non-natural mixed serotypes) , or mutated, chimeric or shuffled proteins obtained by e.g.
  • capsid shuffling techniques or with chimeric swapped variable regions and/or variant glycan binding sequences and/or variant GH loop.
  • the capsid sequences comprise an amino acid sequence at least 85%, 90%, 95%or 98%identical to any of the VP1, VP2 or VP3 capsid sequences of any of SEQ ID NOs: 82-98.
  • amino acid sequences of various capsids are published. See, e.g., AAVRh. 1 /hu. 14 /AAV9 AAS99264.1 (SEQ ID NO: 82) AAVRh. 8 SEQ97 of U.S. Pat. Pub. 2013/0045186 (SEQ ID NO: 83) AAVRh. 10 SEQ81 of U.S. Pat. Pub. 2013/0045186 (SEQ ID NO: 84) AAVRh. 74 SEQ 1 of Int’l. Pat. Pub.
  • Modified "AAV" sequences also can be used in the context of the present disclosure, e.g. for the production of AAV gene therapy vectors.
  • Such modified sequences e.g. sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity (e.g., a sequence having about 75-99%nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP, can be used in place of wild-type AAV ITR, Rep, or VP sequences.
  • the present disclosure provides materials and methods for producing recombinant AAV particles in insect or mammalian cells that comprise any of the vector constructs described herein.
  • the helper functions for producing AAV are provided by one or more helper plasmids or helper viruses comprising adenoviral or baculoviral helper genes.
  • adenoviral or baculoviral helper genes include, but are not limited to, E1A, E1B, E2A, E4 and VA, which can provide helper functions to AAV packaging.
  • Helper viruses of AAV are known in the art and include, for example, viruses from the family Adenoviridae and the family Herpes viridae.
  • helper viruses of AAV include, but are not limited to, SAdV-13 helper virus and SAdV-13-like helper virus described in US Publication No. 20110201088 (the disclosure of which is incorporated herein by reference) , and helper vectors pHELP (Applied Viromics) .
  • SAdV-13 helper virus and SAdV-13-like helper virus described in US Publication No. 20110201088 the disclosure of which is incorporated herein by reference
  • helper vectors pHELP Applied Viromics
  • the insect or mammalian cell can be transfected with the helper plasmid or helper virus, the vector construct and the plasmid encoding the AAV cap genes; and the recombinant AAV virus can be collected at various time points after co-transfection.
  • the recombinant AAV virus can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or a time between any of these two time points after the co-transfection.
  • Recombinant AAV particles can also be produced using any conventional methods known in the art suitable for producing infectious recombinant AAV.
  • a recombinant AAV can be produced by using an insect or mammalian cell that stably expresses some of the necessary components for AAV particle production.
  • a plasmid (or multiple plasmids) comprising AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell.
  • the insect or mammalian cell can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the viral vector construct comprising the 5'a nd 3'A AV ITR (and the nucleotide sequence encoding the heterologous protein, if desired) .
  • a helper virus e.g., adenovirus or baculovirus providing the helper functions
  • the viral vector construct comprising the 5'a nd 3'A AV ITR (and the nucleotide sequence encoding the heterologous protein, if desired) .
  • the advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV particle.
  • adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into packaging cells.
  • both the viral vector construct containing the 5'a nd 3'A AV ITRs and the rep-cap genes can be stably integrated into the DNA of producer cells, and the helper function can be provided by a wild-type adenovirus to produce the recombinant AAV.
  • an AAV particle useful as a gene delivery vector
  • the method comprising the steps of: (a) providing a cell permissive for AAV replication (e.g. an insect cell or a mammalian cell) with one or more nucleic acid constructs comprising: (i) a nucleic acid molecule (e.g.
  • recombinant vector construct provided herein that is flanked by at least one or both AAV inverted terminal repeat nucleotide sequence; (ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein (s) in the cell; (iii) a nucleotide sequence encoding one or more AAV capsid proteins which is operably linked to a promoter that is capable of driving expression of the capsid protein (s) in the cell; (iv) and optionally AAP and MAAP contained in the VP2/3 mRNA (b) culturing the cell defined in (a) under conditions conducive to the expression of the Rep and the capsid proteins; and, optionally, (c) recovering the AAV gene delivery vector, and optionally, (d) purifying the AAV particle.
  • the recombinant vector construct of (i) comprises (1) at least one AAV ITR, (2) a heterologous cardiomyocyte-specific transcription regulatory region as described herein, and (3) a nucleic acid encoding a functional cTnT.
  • the recombinant vector construct of (i) comprises both a 5'a nd 3'A AV ITR.
  • a method provided herein for producing a AAV gene delivery vector comprises: providing to a cell permissive for AAV replication (a) a nucleotide sequence encoding a template for producing vector genome, e.g. vector construct of the present disclosure (as described in detail herein) ; (b) nucleotide sequences sufficient for replication of the template to produce a vector genome (the first expression cassette defined above) ; (c) nucleotide sequences sufficient to package the vector genome into an AAV capsid (the second expression cassette defined above) , under conditions sufficient for replication and packaging of the vector genome into the AAV capsid, whereby AAV particles comprising the vector genome encapsulated within the AAV capsid are produced in the cell.
  • the viral particles comprising the vector constructs described herein may be produced using any cell type such as mammalian and invertebrate cell types which allows for production of AAV or biologic products and which can be maintained in culture.
  • AAV viral particles There are a number of methods for generating AAV viral particles: for example, but not limited to, transfection using vector and AAV helper sequences in conjunction with coinfection with one of the AAV helper viruses (e.g., adenovirus, herpesvirus, or vaccinia virus) or transfection with a recombinant AAV vector, an AAV helper vector, and an accessory function vector.
  • AAV helper viruses e.g., adenovirus, herpesvirus, or vaccinia virus
  • WO1996039530 WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353, WO2001023597, WO2015191508, WO2019217513, WO2018022608, WO2019222136, WO2020232044, WO2019222132; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995) ; O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994) ; Samulski et al., J. Vir. 63: 3822-8 (1989) ; Kajigaya et al., Proc. Nat'l.
  • AAV viral particles 6,001,650, herein incorporated by reference in its entirety
  • This method does not require the use of an infectious helper virus, enabling AAV viral particles to be produced without any detectable helper virus present.
  • This is accomplished by use of three vectors for AAV viral particle production, namely an AAV helper function vector, an accessory function vector, and an AAV viral particle expression vector.
  • the nucleic acid sequences encoded by these vectors can be provided on two or more vectors in various combinations.
  • the host cell can be transfected with the helper plasmid or helper virus, the viral construct and the plasmid encoding the AAV cap genes; and the AAV viral particles can be collected at various time points after co-transfection.
  • wild-type AAV and helper viruses may be used to provide the necessary replicative functions for producing AAV viral particles (see, e.g., U.S. Pat. No. 5,139,941, herein incorporated by reference in its entirety) .
  • a plasmid, containing helper function genes, in combination with infection by one of the well-known helper viruses can be used as the source of replicative functions (see e.g., U.S. Pat. No. 5,622,856 and U.S. Pat. No. 5,139,941, both herein incorporated by reference in their entireties) .
  • a plasmid, containing accessory function genes can be used in combination with infection by wild-type AAV, to provide the necessary replicative functions.
  • Other approaches, described herein and/or well known in the art can also be employed by the skilled artisan to produce AAV viral particles.
  • any one of the AAV vectors disclosed in the present application can be used in the method as the viral construct to produce the rAAV virions.
  • AAV helper refer to AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
  • AAV helper functions include both of the major AAV open reading frames (ORFs) , rep and cap.
  • the Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters.
  • the capsid (Cap) expression products supply necessary packaging functions.
  • AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vector genomes.
  • nucleotide sequences encoding VP proteins can be operably linked to a suitable expression control sequence.
  • nucleotide sequences encoding Rep proteins can be operably linked to a suitable expression control sequence such as eukaryotic promoters.
  • the nucleotide sequences can be operably linked to eukaryotic promoters such as the SV40 promoter, CMV promoter, RSV promoter, UBC promoter, EF1A promoter, PGK promoter, dihydrofolate reductase promoter, the b-actin promoter, TRE (Tet, Tet-On, Tet-Off) promoter, Cumate controlled systems (CuR/CuO) (See US2004/0205834) , the temperature-induced HSP70 promoter, p5 promoter, p10 promoter, p19 promoter, and the p40 promoter.
  • eukaryotic promoters such as the SV40 promoter, CMV promoter, RSV promoter, UBC promoter, EF1A promoter, PGK promoter, dihydrofolate reductase promoter, the b-actin promoter, TRE (Tet, Tet-On, Tet-Off) promoter, Cumate controlled systems (Cu
  • nucleotide sequences can be operably linked to baculoviral promoters such as the polyhedrin (Polh) promoter, ⁇ IE1 promoter, p5 promoter, p10 promoter, p19 promoter, the p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.
  • baculoviral promoters such as the polyhedrin (Polh) promoter, ⁇ IE1 promoter, p5 promoter, p10 promoter, p19 promoter, the p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.
  • Rep proteins For production, cells with AAV helper functions produce Rep proteins to promote production of rAAV. It has been found that infectious particles can be produced when at least one large Rep protein (Rep78 or Rep68) and at least one small Rep protein (Rep52 and Rep40) are expressed in cells. In a specific embodiment all four of Rep 78, Rep68, Rep52 and Rep 40 are expressed. Alternately, Rep78 and Rep52, Rep78 and Rep40, Rep 68 and Rep52, or Rep68 and Rep40 are expressed. Examples below demonstrate the use of the Rep78/Rep52 combination. Rep proteins can be derived from AAV-2 or other serotypes. In one or more embodiments, nucleotide sequences encoding Rep proteins can be operably linked to a suitable expression control sequence.
  • nucleotide sequences encoding Rep proteins can be operably linked to a suitable expression control sequence such as eukaryotic promoters.
  • a suitable expression control sequence such as eukaryotic promoters.
  • the nucleotide sequences can be operably linked to eukaryotic promoters such as the SV40 promoter, CMV promoter, RSV promoter, UBC promoter, EF1A promoter, PGK promoter, dihydrofolate reductase promoter, the b-actin promoter, TRE (Tet, Tet-On, Tet-Off) promoter, Cumate controlled systems (CuR/CuO) (See US2004/0205834) , and the temperature-induced HSP70 promoter, p5 promoter, p10 promoter, p19 promoter, and the p40 promoter.
  • nucleotide sequences can be operably linked to baculoviral promoters such as the polyhedrin (Polh) promoter, ⁇ IE1 promoter, p5 promoter, p10 promoter, p19 promoter, the p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.
  • baculoviral promoters such as the polyhedrin (Polh) promoter, ⁇ IE1 promoter, p5 promoter, p10 promoter, p19 promoter, the p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.
  • the AAV cap genes are present in a plasmid or bacmid.
  • the plasmid can further include an AAV rep gene which may or may not correspond to the same serotype as the cap genes.
  • nucleotide sequences encoding AAP can be operably linked to a suitable expression control sequence.
  • the nucleotide sequences can be operably linked to eukaryotic promoters.
  • the nucleotide sequences can be operably linked to baculoviral promoters such as the polyhedrin (Polh) promoter, ⁇ IE1 promoter, p5 promoter, p10 promoter p19 promoter, the p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.
  • non-AAV helper function refers to non-AAV derived viral and/or cellular functions upon which AAV is dependent for its replication.
  • captures proteins and RNAs that are required in AAV replication including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.
  • non-AAV helper function vector refers generally to a nucleic acid molecule that includes nucleotide sequences providing accessory functions.
  • An accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virion production in the host cell.
  • infectious viral particles as they exist in nature, such as adenovirus, herpesvirus or vaccinia virus particles.
  • accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid. In particular, it has been demonstrated that the full-complement of adenovirus genes are not required for accessory helper functions.
  • adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., (1970) J. Gen. Virol. 9: 243; Ishibashi et al, (1971) Virology 45:317. Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions. Carter et al., (1983) Virology 126: 505. However, adenoviruses defective in the E1 region, or having a deleted E4 region, are unable to support AAV replication. Thus, E1A and E4 regions are likely required for AAV replication, either directly or indirectly.
  • Ad mutants include: E1B (Laughlin et al. (1982) , supra; Janik et al. (1981) , supra; Ostrove et al., (1980) Virology 104: 502) ; E2A (Handa et al., (1975) J. Gen. Virol. 29: 239; Strauss et al., (1976) J. Virol.
  • Particularly preferred accessory function vectors comprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A 72 kD coding region, an adenovirus E1A coding region, and an adenovirus E1B region lacking an intact E1B55k coding region.
  • Such vectors are described in International Publication No. WO 01/83797.
  • mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2/0, PER. C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells.
  • nucleic acids such as vectors, e.g., insect-cell compatible vectors
  • methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors into such cells and methods of maintaining such cells in culture.
  • nucleic acids such as vectors, e.g., insect-cell compatible vectors
  • the nucleic acid construct encoding AAV in insect cells is an insect cell-compatible vector.
  • “Expression vector” refers to a vector including a recombinant polynucleotide including expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) , artificial chromosomes, and viruses that incorporate the recombinant polynucleotide.
  • An "insect cell-compatible vector” or “vector” as used herein refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell.
  • Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell-compatible. The vector may integrate into the insect cells genome but the presence of the vector in the insect cell need not be permanent and transient episomal vectors are also included.
  • the vectors can be introduced by any means known, for example by chemical treatment of the cells, electroporation, or infection.
  • the vector is a baculovirus, a viral vector, or a plasmid.
  • the vector is a baculovirus, i.e. the construct is a baculoviral vector. Baculoviral vectors and methods for their use are described in the above cited references on molecular engineering of insect cells.
  • the insect cell line used can be from Spodoptera frugiperda, such as SF9, SF21, SF900+, drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyx mori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines.
  • Spodoptera frugiperda such as SF9, SF21, SF900+, drosophila cell lines
  • mosquito cell lines e.g., Aedes albopictus derived cell lines
  • domestic silkworm cell lines e.g. Bombyx mori cell lines
  • Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines.
  • insect cells are cells from the insect species which are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38.
  • Baculoviruses are enveloped DNA viruses of arthropods, two members of which are well known expression vectors for producing recombinant proteins in cell cultures. Baculoviruses have circular double-stranded genomes (80-200 kbp) which can be engineered to allow the delivery of large genomic content to specific cells.
  • the viruses used as a vector are generally Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV) (Kato et al., Appl. Microbiol. Biotechnol. 85 (3) : 459-70 (2010) .
  • Baculoviruses are commonly used for the infection of insect cells for the expression of recombinant proteins.
  • expression of heterologous genes in insects can be accomplished as described in for instance U.S. Pat. No. 4,745,051; EP 127,839; EP 155,476; Vlak et al., J. Gen. Virol. 68: 765-76 (1988) ; Miller et al., Ann. Rev. Microbiol. 42: 177-9 (1988) ; Carbonell et al., Gene, 73 (2) : 409-18 (1998) ; Maeda et al., Nature, 315: 592-4 (1985) ; Lebacq-Veheyden et al., Molec. Cell.
  • the baculovirus shuttle vector or bacmids are used for generating baculoviruses.
  • Bacmids propagate in bacteria such as Escherichia coli as a large plasmid. When transfected into insect cells, the bacmids generate baculovirus.
  • the methods provided herein are carried out with any mammalian cell type which allows for replication of AAV or production of biologic products, and which can be maintained in culture.
  • mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2/0, PER. C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells.
  • rAAV particles can also be produced using methods disclosed in one or more embodiments.
  • rAAV particles can be produced by using an insect or mammalian cell that stably expresses some of the necessary components for rAAV particle production.
  • a plasmid or multiple plasmids including AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell.
  • a plasmid (or multiple plasmids) including a selectable marker, such as a neomycin resistance gene can be integrated into the genome of the cell.
  • the insect, fungal, or mammalian cell can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the viral vector including the 5'a nd 3'A AV ITR (and the nucleotide sequence encoding the heterologous protein, if desired) .
  • a helper virus e.g., adenovirus or baculovirus providing the helper functions
  • the viral vector including the 5'a nd 3'A AV ITR (and the nucleotide sequence encoding the heterologous protein, if desired) e.g., adenovirus or baculovirus providing the helper functions
  • the viral vector including the 5'a nd 3'A AV ITR (and the nucleotide sequence encoding the heterologous protein, if desired) .
  • the advantages of this method are that the cells are selectable and are suitable for large-scale production of the rAAV
  • a suspension of transfected cells is purified through a multi-step process to remove process impurities, including recombinant baculoviruses and host cells, and enrich for the virions comprising the recombinant parvoviral (rAAV) vector construct.
  • method provided herein may comprise the step of affinity-purification of the rAAV vector construct using an anti-AAV antibody, in one embodiment an immobilized antibody.
  • the anti-AAV antibody is a monoclonal antibody.
  • One antibody for use herein is a single chain camelid antibody or a fragment thereof as e.g.
  • the antibody for affinity-purification of rAAV is an antibody that specifically binds an epitope on an AAV capsid protein, whereby in one embodiment the epitope is an epitope that is present on capsid protein of more than one AAV serotype.
  • the antibody may be raised or selected on the basis of specific binding to AAV6 capsid or AAV9 capsid but at the same time also it may also bind other AAV capsids.
  • the methods provided herein for producing rAAV particles produce a population of rAAV particles.
  • the population is enriched for particles comprising full length or nearly full-length vector genomes by steps that reduce the number of empty capsids, e.g. centrifugation steps, or chromatography steps such as ion-exchange or metal affinity.
  • the population of rAAV particles produced by the methods provided herein are used, for example, for administration in any of the treatment methods described herein.
  • Host Organism and/or Cells are used, for example, for administration in any of the treatment methods described herein.
  • a host cell comprising the vector construct described above.
  • the vector construct is capable of being replicated, or capable of expressing the nucleic acid molecule provided herein in the host cell.
  • cardiomyopathy therapeutics that are host cells comprising a vector construct comprising a nucleic acid encoding cTnT, for use in cardiomyopathy cell therapy.
  • the cells may be autologous or allogeneic to the subject.
  • the term "host” refers to organisms and/or cells which harbor a nucleic acid molecule or a vector construct of the present disclosure, as well as organisms and/or cells that are suitable for use in expressing a recombinant gene or protein.
  • a host cell may be in the form of a single cell, or a population of similar or different cells.
  • a host cell may be, for example, a bacterial, a yeast, an insect or a mammalian cell, or a human cell.
  • a means for delivering a nucleic acid provided herein into a broad range of cells including dividing and non-dividing cells.
  • the present disclosure may be employed to deliver a nucleic acid provided herein to a cell in vitro, e.g. to produce a polypeptide encoded by such a nucleic acid molecule in vitro or for ex vivo gene therapy.
  • nucleic acid molecule, vector construct, cells and methods/use of the present disclosure are additionally useful in a method of delivering a nucleic acid provided here into a host, typically a host suffering from cardiomyopathy.
  • a pharmaceutical composition comprising a nucleic acid or a vector provided herein and a pharmaceutically acceptable diluent, excipient, or carrier.
  • the pharmaceutical composition may comprise a transgene, vector construct, donor construct, viral vector or viral particle described herein, for example, the rAAV particle or population of rAAV particles described herein.
  • the pharmaceutical composition may further comprise a second therapeutic agent, or adjuvant, etc.
  • the composition is sterile if meant for parenteral administration.
  • the composition is free of infectious viruses and toxins.
  • the composition is stable for a suitable period of time under storage conditions.
  • pharmaceutically acceptable it is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.
  • a pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral particle or cell directly to a subject.
  • a carrier may be suitable for parenteral administration, which includes intravenous, intraperitoneal or intramuscular administration.
  • the carrier may be suitable for sublingual or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions provided herein is contemplated.
  • compositions i.e. formulations
  • AAV particles useful for administration to subjects suffering from a genetic disorder to deliver a gene of interest, e.g. a gene encoding a protein of interest.
  • the pharmaceutical formulations provided herein are liquid formulations that comprise recombinant AAV particles comprising any of the vector constructs disclosed herein. The concentration of recombinant AAV virions in the formulation may vary.
  • the AAV particle pharmaceutical formulation provided herein comprises one or more sterile pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and/or administration to subjects for the treatment of the genetic disorder.
  • the formulation comprising recombinant AAV particle further comprises one or more buffering agents.
  • the recombinant AAV particle formulation provided herein may comprise one or more isotonicity agents, such as sodium chloride.
  • isotonicity agents such as sodium chloride.
  • Other buffering agents and isotonicity agents known in the art are suitable and may be routinely employed for use in the formulations provided herein.
  • sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition.
  • the recombinant AAV particle formulations provided herein may comprise one or more surfactants, which may be non-ionic surfactants.
  • exemplary surfactants include ionic surfactants, non-ionic surfactants, and combinations thereof.
  • the surfactant can be, without limitation, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate) , sodium dodecylsulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc) , poloxamer 407, poloxamer 188 and the like, and combinations thereof.
  • the recombinant AAV particle formulations provided herein are typically sterile and stable and can be stored for extended periods of time without an unacceptable change in quality, potency, or purity.
  • the vector constructs or AAV particles described herein are administered to subjects in a dose effective to deliver the nucleic acid encoding cTnT to the heart muscle of a mammalian subject.
  • the subject is preferably a human, including a juvenile subject. Juvenile subjects may range in age from 0-2, 2-6, 2-10, 2-12, 2-15, 2-18, 12-18, or 0-18 years of age, for example.
  • Such methods include methods of expressing cTnT in heart of a mammalian subject comprising administering to the subject an effective amount of a composition comprising the vector construct described herein, the rAAV particle described herein, or the pharmaceutical composition described herein, thereby expressing cTnT in the heart tissue (e.g., myocardium, or myocardiocytes) of the subject.
  • a composition comprising the vector construct described herein, the rAAV particle described herein, or the pharmaceutical composition described herein, thereby expressing cTnT in the heart tissue (e.g., myocardium, or myocardiocytes) of the subject.
  • Such methods also include a method of treating an alteration in functional wild type cTnT in a mammalian subject by administering an amount of the vector construct, rAAV particle or pharmaceutical composition effective to increase or change the ratio of functional verus mutant cTnT in the heart tissue (e.g. cardiomyocytes) .
  • such methods increase levels of cTnT expression in the heart, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%compared to the levels without treatment, or to the levels seen in healthy humans.
  • the amount of the vector construct, rAAV particle or pharmaceutical composition effective to increase the level of non-mutated or properly functioning cTnT in heart tissue (e.g. cardiomyocytes) by at least about 2-fold.
  • the amount is effective to reduce signs and symptoms of cardiomyopathy, such as reduce cardiac hypertrophy (thickening) , reduce obstruction of cardiac blood flow, reduce cardiac rigidity or stiffness, improve cardiac relaxation, and/or improve heart contractile function.
  • the amount is effective to reduce symptoms of disease seen on echocardiography, such as systolic dysfunction, diastolic dysfunction, left ventricular dilation (e.g., dilated left ventricular end-diastolic diameter, LVEDD) , or atrial dilation, or ventricular hypertrophy (e.g. left ventricular posterior wall thickness, LVPWTs) .
  • the amount is effective to improve the ejection fraction seen on echocardiography.
  • the amount is effective to reduce sudden cardiac death; reduce frequency, duration or severity of end-stage heart failure; reduce plasma biomarkers (e.g., NT-proBNP, Troponin) ; reduce palpitations, chest pain, shortness of breath, fatigue, dizziness, fainting, and/or swelling in feet, ankles, legs or belly.
  • reduce plasma biomarkers e.g., NT-proBNP, Troponin
  • Such methods also include a method of treating, preventing or slowing progression of cardiomyopathy in a mammal, including hypertrophic cardiomyopathy, restrictive cardiomyopathy or dilated cardiomyopathy, comprising administering a therapeutically effective amount of the vector construct, rAAV particle or pharmaceutical composition.
  • the mammal may have a mutation in one or both alleles of the cTnT gene.
  • the rAAV particle may be delivered at a dose of about 1e9 to about 1e15 vg/kg in an aqueous suspension.
  • the administration of the vector construct, rAAV particle, or pharmaceutical composition may further comprise administration of prophylactic or therapeutic corticosteroid treatment, and/or may further include administration of a second therapeutic agent for treating cardiomyopathy including but not limited to angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) , beta blockers, aldosterone antagonists, calcium channel blockers, cardiac glycosides, vasodilators, human B-type natriuretic peptide, inotropic agents, neprilysin inhibitor, nitrates, and/or anti-arrhythmia drugs.
  • ACE angiotensin-converting enzyme
  • ARBs angiotensin II receptor blockers
  • beta blockers beta blockers
  • aldosterone antagonists calcium channel blockers
  • cardiac glycosides vasodilators
  • human B-type natriuretic peptide inotropic agents
  • neprilysin inhibitor nitrates,
  • the methods of treatment may, for example, restore contractile force, relative tension, calcium-activated tension, and/or relaxation time, in engineered heart tissue in vitro or in mammalian tissue in vivo.
  • Such methods for example, reduce heart size, reduce cardiothoracic ratio, reduce end diastolic or end systolic left ventricular diameter, reduce anterior or posterior wall thickness, increase or normalize ejection time, increase aortic peak flow velocity or aortic flow time, and/or decrease other symptoms of cardiac disease or disorder in the subject.
  • such methods reduce the frequency or severity of symptoms of the cardiac disease or disorder in the subject.
  • the prospective patient may be assessed for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that are capable of blocking cell transduction or otherwise reduce the overall efficiency of the treatment.
  • the prospective patient may be assessed for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that are capable of blocking cell transduction or otherwise reduce the overall efficiency of the therapeutic regimen.
  • anti-AAV capsid antibodies or anti-AAV neutralizing antibodies may be present in the serum of the prospective patient and may be directed against an AAV capsid of any serotype.
  • the serotype against which pre-existing antibodies are directed is AAV6.
  • the serotype against which pre-existing antibodies are directed is AAV9.
  • TI assays cell-based in vitro transduction inhibition (TI) assays, in vivo (e.g., in mice) TI assays, and ELISA-based detection of total anti-capsid antibodies (TAb) (see, e.g., Masat et al., Discov. Med., vol. 15, pp. 379-389 and Boutin et al., (2010) Hum. Gene Ther., vol. 21, pp. 704-712) .
  • TI assays may employ host cells into which an AAV-inducible reporter vector has been previously introduced.
  • the reporter vector may comprise an inducible reporter gene such as GFP, etc.
  • Anti-AAV capsid antibodies present in human serum that are capable of preventing/reducing host cell transduction would thereby reduce overall expression of the reporter gene in the system. Therefore, such assays may be employed to detect the presence of anti-AAV capsid antibodies in human serum that are capable of preventing/reducing cell transduction by the therapeutic AAV particle.
  • the assays to detect anti-AAV capsid antibodies may employ solid-phase-bound AAV capsid as a "capture agent" over which human serum is passed, thereby allowing anti-capsid antibodies present in the serum to bind to the solid-phase-bound capsid "capture agent" .
  • a "detection agent” may be employed to detect the presence of anti-capsid antibodies bound to the capture agent.
  • the detection agent may be an antibody, an AAV capsid, or the like, and may be detectably-labeled to aid in detection and quantitation of bound anti-capsid antibody.
  • the detection agent is labeled with ruthenium or a ruthenium-complex that may be detected using electrochemiluminescence techniques and equipment.
  • the same above-described methodology may be employed to assess and detect the generation of an anti-AAV capsid immune response in a patient previously treated with a therapeutic AAV virus of interest.
  • these techniques may be employed to assess the presence of anti-AAV capsid antibodies prior to treatment with a therapeutic AAV virus, they may also be employed to assess and measure the induction of an immune response against the administered therapeutic AAV virus after administration.
  • contemplated herein are methods that combine techniques for detecting anti-AAV capsid antibodies in human serum and administration of a therapeutic AAV virus for the treatment of cardiomyopathy, wherein the techniques for detecting anti-AAV capsid antibodies in human serum may be performed either prior to or after administration of the therapeutic AAV virus.
  • Recombinant AAV particles comprising AAV6 or AAV9 capsid and vector constructs encoding cTnT protein, e.g. vector constructs of SEQ ID NOs: 39-48, were produced in HEK293 cells.
  • the rAAVs were produced by packaging the TNNT2 transgenes in AAV6 or AAV9 capsid as described in Crosson et al., Mol Ther Methods Clin Dev., 10: 1-7 (2018) .
  • HEK293T or 293 VPC suspension cells were triple transfected with (a) helper plasmid, (b) a plasmid encoding Rep and Cap proteins of either AAV6 or AAV9, and (c) one of the various vector constructs encoding cTnT protein described herein.
  • Cells were harvested 72 hours later and AAV particles were purified through an iodixanol gradient and/or AAVX affinity column.
  • Virus titer was measured by droplet digital polymerase chain reaction (ddPCR) .
  • ddPCR droplet digital polymerase chain reaction
  • Vector constructs were prepared in Formats A, B, C, D and E that encode wild type cTnT protein sequence with a Flag tag N-terminal to exon 2 (SEQ ID NO: 44-48, respectively) .
  • the relative expression levels of hTNNT2 mRNA, exogenous (Flag-tagged) cTnT protein, and endogenous cTNT protein were evaluated in wild type (WT) iPSC-cardiomyocytes.
  • iPSC-CM Induced pluripotent stem cell differentiated cardiomyocytes
  • the medium was refreshed with RPMI1640 + 2%B27 minus insulin at day 5 for 48 hrs.
  • the medium was changed to RPMI1640 + 2%B27 (with insulin) for 72 hrs.
  • the cardiomyocytes then underwent two rounds of purification using glucose-deprived medium before being used for experiments at Day 30.
  • WT iPSC-CMs were treated with AAV6 particles comprising different vector constructs, with each construct delivered at different multiplicity of infections (MOIs) , for 6 days before harvest.
  • MOIs multiplicity of infections
  • Transgene mRNA levels were measured by qPCR.
  • Flag-tagged cardiac troponin T proteins and endogenous troponin T proteins were detected by western blot and quantified.
  • a vector construct containing a CBA hybrid intron 5’ to exon 2 (Construct A2; Format A, SEQ ID NO: 44) was compared to intronless vector construct (Construct E2; Format E, SEQ ID NO: 48) .
  • a dose-dependent increase of transgene mRNA levels was observed for each vector group.
  • the transgene mRNA levels of the intron-containing vector construct were much higher than the transgene mRNA levels of the intronless vector construct.
  • Figure 2A A dose-dependent increase of transgene protein levels and concomitant decrease in endogenous TNNT2 levels was also observed for each vector group.
  • the total cTNT protein levels were approximately the same between the vector groups, but there was a significant decrease in endogenous cTNT protein levels.
  • the intron-containing vector construct resulted in a greater reduction of endogenous cTNT protein levels. See Figure 2B.
  • Vector constructs containing different intron sequences and at different positions in the vector construct were compared (Formats A-D) .
  • a vector construct containing a CBA hybrid intron 5’ to exon 2 (Construct A2; Format A, SEQ ID NO: 44) was compared to vector constructs containing different chimeric intron sequences within the hTNNT2 coding sequence (Construct B2, Construct C2 and Construct D2; Formats B-D, SEQ ID NOs: 45-47, containing introns of SEQ ID NOs: 26-28 respectively, located between exons 2 and 3) .
  • a dose-dependent increase of transgene mRNA levels was observed for each vector group.
  • the vector construct of Format D (e.g., Construct D2) containing the intron of SEQ ID NO: 28 provided the highest transgene mRNA level per dose tested, compared to the other vector constructs. See Figure 3A. A dose-dependent increase of transgene protein levels and concomitant decrease in endogenous TNNT2 levels was also observed for each vector group. The exogenous Flag-tagged cTNT protein levels were highest for the vector construct of Format D containing the intron of SEQ ID NO: 28 compared to the other vector constructs. See Figure 3B.
  • the hTNNT2 transgene mRNA level was highest for the vector construct of Format D, containing the intron of SEQ ID NO: 28 and lowest for the intronless vector construct of Format E (at the same dose) , compared to the other vector constructs. All of the vector constructs with an intron located between exons 2 and 3 (Formats B-D) produced higher mRNA levels compared to the vector construct of Format A (intron upstream of exon 2) . See Figure 4B.
  • Flag-tagged exogenous cTnT protein levels were highest for the vector construct of Format D containing the intron of SEQ ID NO: 28; however, the level of Flag-tagged exogenous cTnT protein was lowest for the vector construct of Format A (intron upstream of exon 2) .
  • Levels of Flag-tagged exogenous cTnT protein levels for the various formats were: Format A ⁇ Format C ⁇ Format E ⁇ Format B ⁇ Format D. See Figures 4C and 4D.
  • the reduction of endogenous mouse cTnT was greatest for the intronless vector construct and the vector construct containing hTNNT2 intron 2 between exons 2 and 3 (Format B) .
  • Relative levels of reduction of endogenous mouse cTNT Format A ⁇ Format C ⁇ Format D ⁇ Format E ⁇ Format B. See Figures 4E and 4F.
  • Example 3 Evaluation of effect of AAV particles in a DCM mouse model
  • DCM dilated cardiomyopathy
  • a TNNT2-targeted knock-in murine model of Arg141Trp (R141W) mutation in the TNNT2 gene was generated based on Ramratnam et al., PLOS ONE: 1-23 (2016) , and mice heterozygous (TNNT2 R141W/+ ) and homozygous (TNNT2 R141W/R141W ) for the mutation recapitulated the human phenotype of developing left ventricular dilation and reduced contractility.
  • Echocardiography results suggested improvement of cardiac function in TNNT2 R141W/R141W mice 4 weeks after AAV9 vector treatment, with the vector construct of Format B showing greater improvements. See Figure 6A (ejection fraction) , Figure 6B (dilated left ventricular end-diastolic diameter, LVEDD) and Figure 6C (left ventricular posterior wall thickness, LVPWTs) .
  • Figure 6A ejection fraction
  • Figure 6B diastolic diameter
  • Figure 6C left ventricular posterior wall thickness, LVPWTs
  • Vector constructs were prepared with 15 different codon optimized sequences, operably linked to hTNNT2 promoter of SEQ ID NO: 18, packaged into AAV6 capsids, and administered at different MOIs to WT iPSC-CMs prepared as described in Example 2.
  • the cells were harvested after 7 days, transgene hTNNT2 mRNA levels were measured by qPCR, and Flag-tagged cTnT protein was detected by western blot and quantified.
  • Codon optimized sequences that produced transgene mRNA levels that are equivalent to or higher than the wild type codon sequence are: #3 (SEQ ID NO: 5) , #7 (SEQ ID NO: 9) , #8 (SEQ ID NO: 10) , #11 (SEQ ID NO: 12) , #13 (SEQ ID NO: 14) , #14 (SEQ ID NO: 15) . See Figures 7A (#1-8; SEQ ID NOs: 3-10) and 7B (#10-16; SEQ ID NOs: 11-17) .
  • Codon optimized sequences that produced transgene Flag-tagged protein at levels higher than the wild type codon sequence are: #8 (SEQ ID NO: 10) , #11 (SEQ ID NO: 12) , #12 (SEQ ID NO: 13) , #13 (SEQ ID NO: 14) , #14 (SEQ ID NO: 15) . See Figures 7C (#1-8; SEQ ID NOs: 3-10; lower MOI) and 7D (#1-8; SEQ ID NOs: 3-10; higher MOI) and 7E (#10-16; SEQ ID NOs: 11-17; lower MOI) , and 7F (#10-16; SEQ ID NOs: 11-17; higher MOI) .
  • Codon optimized sequences #8 (SEQ ID NO: 10) , #11 (SEQ ID NO: 12) and #13 (SEQ ID NO: 14) were selected for further testing in healthy mice in a Format A, packaged in an AAV9 capsid. Eight-week-old WT mice were treated with a single dose of these various recombinant AAV9 particles (WT hTNNT2 sequence, #8, #11 and #13) .
  • the WT hTNNT2 sequence vector construct was administered at two doses: one dose that was the same as for the other vector constructs and a higher dose.
  • Format B vector four-week-old TNNT2 R141W/R141W mice (Homo) were treated with Format B1 vector at four doses (dose 1-4: from low to high, among which doses 2 and 3 were identical to LD and HD above) . Echocardiography was performed on mice before AAV vector treatment (baseline) and 4 weeks (W4) post injection (P. I. ) . The impaired systolic function of Homo mice were dose-dependently rescued by Format B1 vector ( Figure 10A) . Necropsy and tissue collection were conducted 6 weeks after AAV vector injection.
  • Format B1 vector reduced HW/BW ratio of Homo mice in a dose-dependent manner ( Figure 10B) .
  • An additional long-term study was conducted to monitor the survival of Homo mice following Format B1 vector treatment.
  • Format B1 vector significantly improved the survival rate of Homo mice even at the lowest dosage of dose 1, with no observed death of Homo mice until 10-month-old at high dosages (dose 3 and dose 4, Figure 10C) .
  • a promoter screening study was performed in WT mice.
  • Three promoters Chicken ⁇ -Actin (CBA) promoter (SEQ ID NO: 109) , chicken TNNT2 promoter (cTnT413, SEQ ID NO: 102) , and human TNNT2 promoter (hTNNT2, SEQ ID NO: 21) were evaluated using fLuc2-T2A-eGFP as the transgene reporter.
  • the plasmids were packaged into AAV9 and the AAV vectors were injected into 6 to 8-week-old mice at a dose between LD and HD in Example 5 intravenously.
  • mice tissues including heart, gastrocnemius muscle (GM) and liver, were harvested and assessed for luciferase activity by making lysates with the Glo lysis buffer (Promega) and assaying with the Steady-Glo Assay System.
  • CBA promoter led to highest luciferase expression, closely followed by hTNNT2 promoter, and cTnT413 promoter was the lowest ( Figure 12A) .
  • hTNNT2 was the best with the high luciferase activity restricted to cardiomyocytes ( Figure 12B) .
  • hTNNT2 promoter human beta MHC (hbMHC) promoter (SEQ ID NO: 105) , rat beta MHC (rbMHC) promoter (SEQ ID NO: 106) and rat MLC-2 (rMLC) promoter (SEQ ID NO: 107) .
  • the plasmids were packaged into AAV9 and dosed 6 to 8-week-old mice at a dose of HD in Example 5 intravenously.
  • mice tissues including heart, triceps muscle (Tri) , gastrocnemius muscle (GM) , diaphragm muscle (Dia) , intercostal muscle (IM) and liver, were harvested and assessed for luciferase activity by making lysates with the Glo lysis buffer and assaying with the Steady-Glo Assay System.
  • hTNNT2 promoter led to highest luciferase expression compared with other promoters ( Figure 13A) .
  • tissue specificity hTNNT2 was the best with the high luciferase activity restricted to cardiomyocytes ( Figure 13B) .

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L'invention concerne des compositions de thérapie génique et des méthodes de traitement de la cardiomyopathie.
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