Troponin C (TNNC1) gene therapy using AAV vectors

Abstract

Provided herein is a gene therapy for TNNC1 (Troponin C)-associated cardiomyopathy, for example, using an adeno-associated virus (AAV) vector. The promoter of the vector can be MHCK7 promoter or cardiac troponin T (hTNNT2) promoter. The capsid can be AAV9 or AAVrh.74 capsid or a functional variant thereof. Other promoters or capsids may be used. Further provided are methods of treatment, such as by intravenous, intracoronary, intracarotid or intracardiac administration of rAAV vector, as well as other compositions and methods. TIFF2024545507000048.tif136168

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 288,255, filed December 10, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text form in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is ROPA_026_01WO_SeqList_ST26.xml. The text file is approximately 152,263 bytes, was created on December 7, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0003] background Mutations in the TNNC1 gene are a major cause of cardiomyopathy. TNNC1, located at 3p21.1, mediates the regulation of myofilament Ca 2+ TNNC1 encodes cardiac troponin C, a calcium-binding subunit involved in sensing calcium and regulating contraction. Troponin C counteracts the inhibition of myosin-actin contractile interactions induced by troponin I-tropomyosin. Loss-of-function (LOF) mutations in TNNC1 result in Ca 2+ TNNC1 gain-of-function (GOF) mutations reduce Ca sensitivity and binding, leading to dilated cardiomyopathy (DCM). 2+ It increases sensitivity and binding, causing hypercontractility and hypertrophic cardiomyopathy (HCM).

[0004] Clinical manifestations of TNNC1 DCM include heart failure (e.g., mean ejection fraction (EF) less than 30%), left ventricular dilation, the need for heart transplantation, and risk of sudden cardiac death. The average age at onset of TNNC1 DCM is approximately 30 years, but it can occur earlier and in pediatric patients. Clinical manifestations of TNNC1 HCM include dyspnea, syncope, angina, arrhythmias, left ventricular hypertrophy (LVH), and left ventricular outflow tract obstruction (LVOTO).

[0005] There remains an unmet need in the art for the treatment of TNNC1 DCM, TNNC1 HCM, and other cardiomyopathies associated with mutations in TNNC1. The compositions and methods disclosed herein address this need. Summary of the Invention

[0006] overview The present invention relates generally to gene therapy for diseases or disorders, such as cardiac diseases or disorders, using vectors expressing TNNC1 or functional variants thereof.

[0007] Various other aspects and embodiments are disclosed in the following detailed description. The present invention is limited only by the scope of the appended claims. [Brief description of the drawings]

[0008] [Figure 1] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 57. Underlined is the expression cassette (SEQ ID NO: 63). [Diagram 2] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 58. Underlined is the expression cassette (SEQ ID NO: 64). [Diagram 3]A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 59. Underlined is the expression cassette (SEQ ID NO: 65). [Figure 4] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 60. Underlined is the expression cassette (SEQ ID NO: 66). [Diagram 5] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 61. Underlined is the expression cassette (SEQ ID NO: 67). [Figure 6] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 62. Underlined is the expression cassette (SEQ ID NO: 68). [Figure 7] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 57. The MHCK7 promoter described herein is labeled "Enhancer / MHCK7" in the diagram. [Figure 8] A diagram illustrating a non-limiting example of a vector genome is shown. The complete polynucleotide sequence of the vector genome is SEQ ID NO:58. [Figure 9] Western blot (WB) of human TnC protein expression in transduced CHO-Lec2 cells. WB shows human TnC (upper panel) or loading control GAPDH (lower panel). Lane 1 is AAV9-MHCK7-TNNC1 (transduced at MOI 3E5), lane 2 is AAVrh.74-MHCK7-TNNC1 (transduced at MOI 3E5), lane 3 is AAV9-hTnT-TNNC1 (transduced at MOI 3E6), lane 4 is AAVrh.74-hTnT-TNNC1 (transduced at MOI 3E6), lane 5 is non-transduced control. [Figure 10] Figure 10A and Figure 10B show Kaplan-Meier survival curves for D73N+ / - treated mice (n = 7-11 / group). Figure 10A: shows survival of male treated mice, and Figure 10B: shows survival of females. All AAV-injected animals lived significantly longer than D73N+ / - controls injected with formulation buffer (FB). FB contains phosphate buffered saline (PBS) with 0.01% Pluronic F-68 and no AAV. [Figure 11] Figures 11A and 11B show bar graphs illustrating that significant attenuation of disease-related end-diastolic diameter increase was observed in all male AAV-injected groups, with the greatest effect observed in the AAV9-MHCK7 group compared to D73N+ / - control animals injected with formulation buffer (FB) (Figure 11A). In female mice, a clear but non-significant effect was observed, most notably in the AAV9-hTnT-TNNC1 group (Figure 11B). Statistical analysis (one-way ANOVA) followed by Tukey's multiple comparison test was performed (*p≦0.05, **p≦0.01, ***p≦0.001, ****p<0.0001). [Figure 12] Figures 12A and 12B show bar graphs illustrating that a significant attenuation of disease-related end-systolic diameter increase (Figure 12A) was observed in male mice treated with AAV9-MHCK7-TNNC1 and AAVrh.74-TNNC1 compared to FB-injected D73N+ / - controls. A clear but non-significant effect was observed in female mice, most notably in the AAV9-hTnT-TNNC1-injected group (Figure 12B). Statistical analysis (one-way ANOVA) followed by Tukey's multiple comparison test was performed (*p≦0.05, ****p<0.0001). [Figure 13] Figures 13A and 13B show bar graphs illustrating that animals injected with AAV9-MHCK7-TNNC1 showed a significant attenuation of disease-associated dilated cardiomyopathy progression as revealed by a greater ejection fraction (%) compared to D73N+ / - control male mice injected with FB (Figure 13A). Statistical analysis (one-way ANOVA) followed by Tukey's multiple comparison test was performed (*p≦0.05). [Figure 14] Figure 14A and Figure 14B show bar graphs illustrating the normal progression of dilated cardiomyopathy as manifested by a greater fractional shortening (%) in animals injected with AAV9-MHCK7-TNNC1 compared to D73N+ / - control male mice injected with FB (Figure 14A). Statistical analysis (one-way ANOVA) followed by Tukey's multiple comparison test was performed (*p≦0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description of the Invention The present disclosure provides a gene therapy vector of TNNC1 that delivers a polynucleotide encoding TNNC1 or its functional variant, together with a method of use, as well as other compositions and methods. In some embodiments, the promoter is MHCK7 promoter. In some embodiments, the AAV vector is AAV9 vector. In some embodiments, the promoter is MHCK7 promoter and the AAV vector is AAV9 vector. In some embodiments, the promoter is MHCK7 promoter and the AAV vector is AAVrh.74 vector. In some embodiments, the promoter is hTNNT2 promoter. In some embodiments, the AAV vector is AAV9 vector. In some embodiments, the promoter is hTNNT2 promoter and the AAV vector is AAV9 vector. In some embodiments, the promoter is hTNNT2 promoter and the AAV vector is AAVrh.74 vector.

[0010] The present disclosure further provides a method for treating a disease or disorder in a subject by administering a gene therapy vector of the present disclosure. In a preferred embodiment, the disease or disorder is TNNC1 DCM or TNNC1 HCM.

[0011] According to the present invention, polynucleotides encoding TNNC1 or functional variants thereof can be used to generate gene therapy vectors. The resulting vectors can be used to treat diseases or disorders, such as TNNC1 DCM, TNNC1 HCM, or others.

[0012] definition The section headings are for organizational purposes only and are not to be construed as limiting the subject matter described to any particular aspect or embodiment.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will take precedence.In addition, the materials, methods and examples described herein are illustrative only and are not intended to be limiting.

[0014] All publications and patents mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.In case of conflict, this application, including any definitions herein, shall prevail.However, the mention of any references, articles, publications, patents, patent publications and patent applications cited in this specification is not intended to be, and should not be interpreted as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0015] In this description, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value in the recited range, and fractions thereof, where appropriate (e.g., tenths and hundredths of integers, etc.). The term "about", when immediately preceding a number or number, means that the number or number is within a range of plus or minus 10%. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components, unless otherwise indicated. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0016] As used throughout this disclosure, sequence "identity" can be determined by using a standalone executable BLAST engine program (bl2seq) to blast two sequences, which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety). When used in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" refers to the number or percentage of residues that are the same in a sequence of interest and a reference sequence. This percentage can be calculated by optimally aligning the sequence of interest with the reference sequence; comparing the two sequences over the entire length of the reference sequence; determining the number of positions where identical amino acid residues or nucleic acid bases occur in both sequences to obtain the number of matched positions; dividing the number of matched positions by the total number of positions in the reference sequence, adjusted for the number of gap positions introduced in the reference sequence during the generation of the alignment; and multiplying the result by 100 to obtain the percentage of sequence identity. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity calculation can be performed manually or by the BLAST algorithm.

[0017] As used herein, "AAV vector" or "rAAV vector" refers to a recombinant vector that contains one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a plasmid that encodes and expresses the rep and cap gene products. Alternatively, AAV vectors can be packaged into infectious particles using host cells that are stably engineered to express the rep and cap genes.

[0018] As used herein, "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle that is composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.As used herein, when a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene that is delivered to mammalian cells), it is usually referred to as "AAV vector particle" or simply "AAV vector".Therefore, the production of AAV vector particle necessarily includes the production of AAV vector, since the vector is contained within the AAV vector particle.

[0019] As used herein, "promoter" refers to a polynucleotide sequence capable of promoting the initiation of RNA transcription from a polynucleotide in a eukaryotic cell.

[0020] As used herein, "vector genome" refers to a polynucleotide sequence packaged by a vector (e.g., a rAAV virion), including flanking sequences (in AAV, inverted terminal repeats). The terms "expression cassette" and "polynucleotide cassette" refer to the portion of a vector genome between the flanking ITR sequences. "Expression cassette" means that the vector genome contains at least one gene encoding a gene product operably linked to elements that drive expression (e.g., a promoter), including any regulatory and / or enhancer elements. "Polynucleotide cassette" refers to the portion of a vector genome that contains at least one gene encoding a gene product operably linked to elements that drive expression (e.g., a promoter), including any regulatory and / or enhancer elements.

[0021] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk of or suffering from a disease, disorder, or condition that is suitable for treatment or improvement using a recombinant gene therapy vector or gene editing system disclosed herein. A patient or subject in need may be, for example, a patient or subject diagnosed with a heart-related disorder. The subject may have a mutation in the TNNC1 gene, or a deletion of all or part of the TNNC1 gene, or a deletion of a gene regulatory sequence, causing abnormal expression of the TNNC1 protein. "Subject" and "patient" are used interchangeably herein. The subject treated by the methods described herein may be an adult or a child. The age of the subject may vary.

[0022] As used herein, the terms "variant" or "functional variant" refer interchangeably to a protein that has one or more amino acid substitutions, insertions or deletions compared to a parent protein that retains one or more desired activities of the parent protein.

[0023] As used herein, "gene disruption" refers to partial or complete loss of function or abnormal activity in gene.For example, subject may suffer from gene disruption of expression or function in TNNC1 gene, which reduces the expression of TNNC1 protein or causes loss or abnormality of function in at least some cells (e.g., cardiac cells) of subject."Gene disruption" also refers to gene change that leads to gain-of-function mutation, for example, gain-of-function mutation of TNNC1 protein.

[0024] As used herein, "treat" refers to improve one or more symptoms of disease or disorder.The term "prevent" refers to delay or stop the onset of one or more symptoms of disease or disorder, or slow down the progression of TNNC1-related disease or disorder, such as TNNC1 DCM and / or TNNC1 HCM.

[0025] TNNC1 protein or polynucleotide The present disclosure contemplates compositions and methods of use related to TNNC1 protein.Various mutations in TNNC1 are known to be associated with TNNC1 DCM or TNNC1 HCM.Examples of mutations associated with TNNC1 DCM or TNNC1 HCM include, but are not limited to, Y5H, A8V, L29Q, A31S, C84Y, E134D, D132N, D145E, I148V, G159D, G159R, M103I and / or any combination thereof.

[0026] The native sequence of human TNNC1 is shown below. The TNNC1 isoform has a sequence of 161 amino acid residues (GenBank NP_003271.1): TIFF2024545507000002.tif26165

[0027] In some embodiments, the TNNC1 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. In some embodiments, the TNNC1 protein is encoded by a polynucleotide comprising a sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2. In some embodiments, the TNNC1 protein does not have a mutation associated with a disease. In some embodiments, the TNNC1 protein is a wild-type or native TNNC1 protein, e.g., human TNNC1.

[0028] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding TNNC1 or a functional variant thereof, operably linked to a promoter. In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding TNNC1, operably linked to a promoter. In some embodiments, the TNNC1 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. The polynucleotide encoding TNNC1 is selected from the group consisting of: It may comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000003.tif40165.

[0029] Optionally, the polynucleotide sequence encoding the vector genome may include a Kozak sequence, including but not limited to GCCACCATGG (SEQ ID NO: 3). The Kozak sequence may overlap with the polynucleotide sequence encoding the TNNC1 protein or a functional variant thereof. For example, the vector genome may include the following: TIFF2024545507000004.tif41165 may contain a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000004.tif41165 (underlined Kozak).

[0030] In some embodiments, the Kozak sequence is as follows: TIFF2024545507000005.tif62128 or any one of these alternative Kozak sequences.

[0031] In some embodiments, the vector genome does not contain a Kozak sequence. The polynucleotide sequence may be codon optimized.

[0032] Vector genome The AAV virion of the present disclosure comprises a vector genome. The vector genome may comprise an expression cassette (or a polynucleotide cassette for gene editing applications that do not require expression of a polynucleotide sequence). Any suitable inverted terminal repeat (ITR) may be used. The ITR may be from the same serotype as the capsid or a different serotype (e.g., AAV2 ITR may be used).

[0033] In some embodiments, the 5' ITR comprises an AAV ITR. It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000006.tif26165.

[0034] In some embodiments, the 5' ITR comprises an AAV2 ITR. It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000007.tif31164.

[0035] In some embodiments, the 5' ITR comprises an AAV ITR. It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000008.tif31165.

[0036] In some embodiments, the 5' ITR comprises an AAV ITR. It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000009.tif30164.

[0037] In some embodiments, the 3' ITR comprises an AAV ITR. In some embodiments, the 5' ITR comprises the following: It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000010.tif31164.

[0038] In some embodiments, the 3' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises the following: It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000011.tif30165.

[0039] In some embodiments, the 3' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises the following: It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000012.tif19165.

[0040] In some embodiments, the 3' ITR comprises an AAV2 ITR. In some embodiments, the 5' ITR comprises the following: It comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000013.tif19164.

[0041] In some embodiments, the vector genome comprises, for example, Contains one or more filler sequences that are at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000014.tif57165.

[0042] promoter In some embodiments, the polynucleotide sequence encoding the TNNC1 protein or a functional variant thereof is operably linked to a promoter. In a preferred embodiment, the promoter is the MHCK7 promoter, which includes the enhancer / promoter region of mouse muscle creatine kinase (MCK) and the enhancer region of the α-myosin heavy chain gene. (Salva MZ et al., Mol. Ther. 15(2):320-9 (2007).)

[0043] The present disclosure contemplates the use of various promoters. Promoters useful in the embodiments of the present disclosure include, but are not limited to, cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, or promoter sequence consisting of CMV enhancer and part of chicken β-actin promoter and rabbit β-globin gene (CAG). In some cases, the promoter may be a synthetic promoter. Exemplary synthetic promoters are provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010).

[0044] In some embodiments, the polynucleotide sequence encoding the TNNC1 protein or its functional variant is operably linked to an inducible promoter. The inducible promoter can be configured to transcribe or not transcribe the polynucleotide sequence in response to the addition or accumulation of an agent, or in response to the removal, degradation or dilution of the agent. The agent can be a drug. The agent can be tetracycline or one of its derivatives, such as, but not limited to, doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically regulated promoter, a physically regulated promoter (i.e., a promoter that responds to the presence or absence of light or to low or high temperatures). Inducible promoters include heavy metal ion inducible promoters (such as mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and promoters from T7 phage that are active in the presence of T7 RNA polymerase. This list of inducible promoters is non-limiting.

[0045] In some cases, the promoter is a tissue-specific promoter, for example, a promoter that can drive expression in cardiac cells to a greater extent than in non-cardiac cells. In some embodiments, the tissue-specific promoter is selected from any of a variety of cardiac cell-specific promoters, including but not limited to desmin (Des), α-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (cTnC), cardiac troponin T (hTNNT2), muscle creatine kinase (CK), and their promoter / enhancer region combinations, such as MHCK7. In some cases, the promoter is a ubiquitous promoter. "Ubiquitous promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the ubiquitous promoter is any one of CMV, CAG, UBC, PGK, EF1-α, GAPDH, SV40, HBV, chicken β-actin, and human β-actin promoters.

[0046] In some embodiments, the promoter sequence is selected from Table 3. In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 21-35.

[0047] [Table 3] TIFF2024545507000016.tif224166TIFF2024545507000017.tif225166TIFF2024545507000018.tif225166TIFF2024545507000019.tif22416 6TIFF2024545507000020.tif219166TIFF2024545507000021.tif222166TIFF2024545507000022.tif219166TIFF2024545507000023.tif58166

[0048] Further illustrative examples of promoters are the SV40 late promoter from Simian Virus 40, the baculovirus polyhedron enhancer / promoter element, the herpes simplex virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including the LTR element. A wide variety of other promoters are known in the art and are publicly available, and the sequences of many such promoters are available in sequence databases such as the GenBank database.

[0049] Other Regulatory Elements In some cases, the vectors of the present disclosure further comprise one or more regulatory elements selected from the group consisting of an enhancer, an intron, a polyA signal, a 2A peptide coding sequence, a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), and a HPRE (hepatitis B virus post-transcriptional regulatory element).

[0050] In some embodiments, the vector comprises a CMV enhancer.

[0051] In certain embodiments, the vector comprises one or more enhancers. In particular embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a β-actin enhancer sequence, or an EF1-α enhancer sequence. The above sequences are known in the art. For example, the sequence of the CMV immediate early (IE) enhancer is as follows: The file is TIFF2024545507000024.tif52165.

[0052] In certain embodiments, the vector comprises an enhancer linked to the promoter. For example, the vector can comprise the MHCK7 promoter and enhancer. In certain embodiments, the MHCK7 promoter and enhancer comprises the following sequence: is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to TIFF2024545507000025.tif107165.

[0053] In certain embodiments, the vector comprises one or more introns. In particular embodiments, the intron is a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, an SV40 intron, or an EF1-α intron sequence.

[0054] In certain embodiments, the vector comprises a polyA sequence. In particular embodiments, the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence. In some embodiments, the polyA signal can be a bovine growth hormone polyadenylation signal (bGHpA).

[0055] In certain embodiments, the vector comprises one or more transcript stabilizing elements. In particular embodiments, the transcript stabilizing element is a WPRE sequence, a HPRE sequence, a scaffold attachment region, a 3' UTR or a 5' UTR. In particular embodiments, the vector comprises both a 5' UTR and a 3' UTR.

[0056] In some embodiments, the vector comprises a 5' untranslated region (UTR) selected from Table 4. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs 38-48.

[0057] [Table 4] TIFF2024545507000027.tif219166TIFF2024545507000028.tif223166TIFF2024545507000029.tif224166TIFF2024545507000030.tif59166

[0058] In some embodiments, the vector comprises a 3' untranslated region selected from Table 5. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 49-57.

[0059] [Table 5] TIFF2024545507000032.tif224166TIFF2024545507000033.tif225166TIFF2024545507000034.tif161166

[0060] In some embodiments, the vector comprises a polyadenylation (polyA) signal selected from Table 6. In some embodiments, the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs 52-56.

[0061] [Table 6]

[0062] Exemplary vector genomes are depicted in Figures 1-6 and provided as SEQ ID NOs: 57-62. Expression cassettes for each of the sequences underlined in Figures 1-6 are SEQ ID NOs: 63-68. In some embodiments, the vector genome comprises a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 57-62, optionally with or without ITR sequences, consists essentially of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 57-62, optionally with or without ITR sequences, or consists essentially of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 57-62, optionally with or without ITR sequences. 57 to 62. In some embodiments, the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 57-62. The present disclosure also contemplates expression cassettes of the exemplary vector genomes depicted in Figures 1-6 and sequences comprising these, such as those set forth in SEQ ID NOs: 57-62, but lacking the 5' and 3' ITRs, as well as variants thereof sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the foregoing.

[0063] In a preferred embodiment, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57. In a preferred embodiment, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 58. In a preferred embodiment, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 59. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 60. In preferred embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 61. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 62.

[0064] In a preferred embodiment, the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In a preferred embodiment, the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64. In a preferred embodiment, the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65. In some embodiments, the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66. In preferred embodiments, the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 67. In some embodiments, the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 68.

[0065] Adeno-associated viral vectors and uses thereof Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two approximately 145 nucleotide inverted terminal repeats (ITRs). There are several known variants of AAV, which are sometimes classified according to antigenic epitopes and are called serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45: 555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively, and the AAV-9 genome is provided in Gao et al., J. Virol., 78: The AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (designated p5, p19 and p40 for their relative map positions) drive the expression of two AAV internal reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotide positions 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins possess multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three associated capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0066] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and nondividing cells and persist essentially for the life of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA within a plasmid, making the construction of recombinant genomes feasible. Furthermore, signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, so that part or all of the internal ∼4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) may be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins may be provided in trans. Another important feature of AAV is that it is a highly stable and potent virus. It easily survives the conditions used to inactivate adenovirus (56°C to 65°C for several hours), mitigating the importance of cryopreservation of AAV. AAV can also be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0067] Gene delivery viral vectors useful in the practice of the present invention can be constructed using methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are constructed from polynucleotides that code for the transgene, appropriate regulatory factors, and elements required for the production of viral proteins, which mediate cell transduction. Such recombinant viruses can be produced by techniques well known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus helper vectors), 293 cells, etc. AAV vectors can be produced using a herpes virus-based system, as described in US2017 / 0218395A1. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94 / 19478, the entire contents of each of which are incorporated herein by reference.

[0068] AAV vectors useful in the practice of the present invention can be packaged into AAV virions (virus particles) using a variety of systems, including adenovirus-based and helper-free systems. Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27. Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):281-304; Deyle DR, Russell DW. Curr Opin Mol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther. (2001) 8(16):1248-54; and Duan et al. Mol Ther. (2001) 4(4):383-91. Helper-free systems include those described in U.S. Patent Nos. 6,004,797; 7,588,772; and 7,094,604;

[0069] The AAV DNA in the rAAV genome can be from any AAV variant or serotype from which recombinant virus can be derived, including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAVrhlO. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0070] In some cases, rAAVs contain self-complementary genomes. As defined herein, rAAVs containing "self-complementary" or "double-stranded" genomes refer to rAAVs engineered such that the coding region of the rAAV constitutes an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001). The present disclosure contemplates the use of rAAVs containing self-complementary genomes in some cases, because upon infection (such as transduction), rather than waiting for cell-mediated synthesis of the second strand of the rAAV genome, the two complementary halves of the scAAV assemble to form one double-stranded DNA (dsDNA) unit, ready for immediate replication and transcription. It will be appreciated that instead of the full coding capacity found in rAAV (4.7-6 kb), rAAV containing self-complementary genomes may only retain about half that amount (approximately 2.4 kb).

[0071] In other cases, the rAAV vector comprises a single-stranded genome. As defined herein, a "single-stranded" genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAV has a single-stranded DNA genome. Some have indicated that the rAAV should be a scAAV to achieve efficient transduction of cells. However, the present disclosure contemplates rAAV vectors that may have a single-stranded genome rather than a self-complementary genome, with the understanding that other genetic modifications of the rAAV vector may be beneficial to obtain optimal gene transcription in target cells. In some cases, the present disclosure relates to single-stranded rAAV vectors that can achieve efficient gene transfer to the anterior segment of the mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017).

[0072] In some cases, the rAAV vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10 or AAVrh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In some cases, the rAAV vector is of serotype AAV9. In some embodiments, the rAAV vector is of serotype AAV9 and comprises a single-stranded genome. In some embodiments, the rAAV vector is of serotype AAV9 and comprises a self-complementary genome. In some embodiments, the rAAV vector comprises an inverted terminal repeat (ITR) sequence of AAV2. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2 / 9 vector, an AAV-2 / 6 vector, or an AAV-2 / 8 vector.

[0073] The full-length and capsid gene sequences of most of the known AAVs are provided in US Pat. No. 8,524,446, which is incorporated herein in its entirety.

[0074] AAV vector may comprise wild-type AAV sequence, or may comprise one or more modifications to wild-type AAV sequence.In certain embodiments, AAV vector comprises one or more amino acid modifications, such as substitutions, deletions or insertions, in capsid protein, such as VP1, VP2 and / or VP3.In certain embodiments, the modifications provide reduced immunogenicity when AAV vector is provided to a subject.

[0075] The capsid protein of the rAAV may be modified to target the rAAV to a specific target tissue of interest, such as endothelial cells, more specifically endothelial tip cells, hi some embodiments, the rAAV is injected directly into the intraventricular lumen of the subject.

[0076] In some embodiments, the rAAV virion is an AAV2 rAAV virion. The capsid can be an AAV2 capsid or a functional variant thereof. In some embodiments, the AAV2 capsid is a reference AAV2 capsid, e.g., shares at least 98%, 99% or 100% identity with TIFF2024545507000036.tif104166.

[0077] In some embodiments, the rAAV virion is an AAV9 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV9 capsid is a reference AAV9 capsid, e.g., shares at least 98%, 99% or 100% identity with TIFF2024545507000037.tif104165.

[0078] In some embodiments, the rAAV virion is an AAV6 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV6 capsid is a reference AAV6 capsid, e.g., shares at least 98%, 99% or 100% identity with TIFF2024545507000038.tif104166.

[0079] In some embodiments, the rAAV virion is an AAVrh.10 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAVrh.10 capsid is a reference AAVrh.10 capsid, e.g., shares at least 98%, 99% or 100% identity with TIFF2024545507000039.tif103166.

[0080] In some embodiments, the capsid protein is encoded by a polynucleotide provided on a plasmid in trans to a transfer plasmid. The polynucleotide sequence of wild type AAVrh.74 cap is as follows:

[0081] AAVrh.74 capsid coding sequence: TIFF2024545507000040.tif150165TIFF2024545507000041.tif136165

[0082] The present disclosure further provides protein sequences of AAVrh.74 VP1, VP2 and VP3, including SEQ ID NOs: 74-76, respectively, and homologs or functional variants thereof.

[0083] AAVrh.74 VP1: TIFF2024545507000042.tif92165

[0084] AAVrh.74 VP2: TIFF2024545507000043.tif77165

[0085] AAVrh.74 VP3: TIFF2024545507000044.tif70165

[0086] In certain cases, the AAVrh.74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, the rAAV vector comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP1, e.g., as set forth in SEQ ID NO: 74, or a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP1, e.g., as set forth in SEQ ID NO: 74. or alternatively, the AAVrh.74 vector may be, for example, AAVrh.74 vector, which may consist essentially of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of VP1, or further, e.g., AAVrh.74 vector, as set forth in SEQ ID NO: 74. and polypeptides consisting of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, and more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of VP1.In some embodiments, the rAAV vector comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP2, e.g., as set forth in SEQ ID NO:75, or a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP2, e.g., as set forth in SEQ ID NO:75. or further, the AAVrh.74 vector may be, for example, AAVrh.74 vector, which may consist essentially of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of VP2, or further, e.g., AAVrh.74 vector, as set forth in SEQ ID NO: 75. and polypeptides consisting of a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, and more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of VP2.In some embodiments, the rAAV vector comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP3, e.g., as set forth in SEQ ID NO:76, or a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP3, e.g., as set forth in SEQ ID NO:76. or further, for example, AAVrh.74 as set forth in SEQ ID NO: 76, which is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of VP3. and polypeptides consisting of a sequence at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, and more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of VP3.

[0087] In some embodiments, the rAAV virion is an AAV-PHP.B rAAV virion or a neutrotrophic variant thereof, such as, but not limited to, those disclosed in International Patent Publication Nos. WO 2015 / 038958 A1 and W 2017 / 100671 A1. For example, the AAV capsid can include at least four consecutive amino acids from the sequence TLAVPFK (SEQ ID NO: 78) or KFPVALT (SEQ ID NO: 79), e.g., inserted between the sequence encoding amino acid numbers 588 and 589 of AAV9.

[0088] The capsid can be an AAV-PHP.B capsid or a functional variant thereof. In some embodiments, the AAV-PHP.B capsid is a reference AAV-PHP.B capsid, e.g., Shares at least 98%, 99% or 100% identity with TIFF2024545507000045.tif92165.

[0089] Additional AAV capsids for use in the rAAV virions of the present disclosure include those disclosed in Patent Publication Nos. WO 2009 / 012176 A2 and WO 2015 / 168666 A2.

[0090] Without being bound by theory, the inventors have determined that the AAV9 vector, the AAVrh.74 or the AAVrh.10 vector confers the desired cardiac tropism to the vector. Without being bound by theory, the inventors have further determined that the AAV9 vector, the AAVrh.74 or the AAVrh.10 vector may provide the desired specificity for cardiac cells.

[0091] Pharmaceutical Compositions and Kits In one aspect, the disclosure provides a pharmaceutical composition comprising a rAAV virion of the disclosure and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0092] For purposes of administration, e.g., administration by injection, a variety of solutions can be utilized, including sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent can be first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared, e.g., at 0.001% or 0.01%, in water suitably mixed with a surfactant, such as poloxamer 188. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, all sterile aqueous media utilized are readily available by standard techniques well known to those skilled in the art.

[0093] Pharmaceutical forms suitable for injectable use include, but are not limited to, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0094] Sterile injectable solution can be prepared by incorporating the required amount of rAAV in a suitable solvent with various other components as listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle that contains basic dispersion medium and other components as listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technique, which produces powder of active ingredient plus any additional desired components from its solution that has been previously sterilized and filtered.

[0095] In another aspect, the disclosure includes a kit comprising a rAAV virion of the disclosure and instructions for use.

[0096] How to use In one aspect, the present disclosure provides a method for increasing wild-type TNNC1 expression and / or activity in a cell, comprising contacting the cell with a rAAV of the present disclosure. In another aspect, the present disclosure provides a method for increasing wild-type TNNC1 expression and / or activity in a subject, comprising administering a rAAV of the present disclosure. In some embodiments, the cell and / or subject is deficient in TNNC1 messenger RNA or TNNC1 protein expression level and / or activity, and / or comprises a loss-of-function mutation of TNNC1. In certain embodiments, the cell and / or subject has a gain-of-function mutation of TNNC1. In certain embodiments, the cell and / or subject has a mutation selected from the group consisting of Y5H, A8V, L29Q, A31S, C84Y, E134D, D132N, D145E, I148V, G159D, G159R, or any combination thereof, compared to the human wild-type TNNC1 gene. The cell may be a cardiac cell, e.g., a cardiomyocyte.

[0097] In some embodiments, the method promotes survival of cardiac cells, e.g., cardiomyocyte cells, in cell culture and / or in vivo. In some embodiments, the method promotes and / or restores cardiac function.

[0098] In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more TNNC1 protein levels detectable in cardiac fibroblasts (CFs) of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more TNNC1 protein levels detectable in cardiomyocytes of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more TNNC1 protein levels detectable in smooth muscle cells (SMCs) of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more TNNC1 protein levels detectable in endothelial cells (ECs) of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more TNNC1 protein levels detectable in the epicardium of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more TNNC1 protein levels detectable in the myocardium of the subject's heart. In certain embodiments, treatment with rAAV virions results in at least 2-fold, at least 5-fold, at least 10-fold or more TNNC1 protein levels detectable in the endocardium of the subject's heart.

[0099] Treatment Method In another aspect, the present disclosure provides a method for treating disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the rAAV virion of the present disclosure.In some embodiments, the disease or disorder is a cardiac disease or disorder.Exemplary cardiac disorders include heart failure, TNNC1 DCM, TNNC1 HCM, arrhythmogenic right ventricular cardiomyopathy (ARVC), Brugada syndrome (BrS) and idiopathic ventricular fibrillation, left ventricular noncompaction cardiomyopathy, or restrictive cardiomyopathy, hypertrophic cardiomyopathy.In a preferred embodiment, the subject suffers from or is at risk of TNNC1-related cardiomyopathy (e.g., TNNC1 DCM or TNNC1 HCM).

[0100] AAV-mediated delivery of TNNC1 protein to the heart may extend lifespan and prevent or attenuate cardiac cell degeneration, heart failure, scarring or fibrosis, reduced ejection fraction, arrhythmias, exercise intolerance, angina (chest pain), dyspnea (shortness of breath), edema, left ventricular hypertrophy, left ventricular noncompaction, ventricular dilation, syncope, sudden cardiac death, exertional muscle pain and cramps. AAV-mediated delivery of TNNC1 protein to the heart may show improvement from the usual disease course as detected by the use of pathological electrocardiograms, echocardiography, cardiac CT, cardiac MRI, cardiac biopsy, reduction in paroxysmal ventricular arrhythmias, reduction in sudden cardiac death, and / or reduction in fibrosis and / or myofibrillar disarray in the myocardium and / or lack of further progression, or may prevent the usual disease course as detected by the use of pathological electrocardiograms, echocardiography, cardiac CT, cardiac MRI, cardiac biopsy, reduction in paroxysmal ventricular arrhythmias, reduction in sudden cardiac death, and / or reduction in fibrosis and / or myofibrillar disarray in the myocardium and / or lack of further progression. The disclosed method may prevent the decline, restoration, and / or increase in left ventricular ejection fraction (LVEF) and / or ejection fraction, percent factional shortening, left ventricular end systolic dimension (LVESD), and left ventricular end diastolic dimension (LVEDD), left ventricular outflow tract velocity time integral (LVOT VTI).

[0101] In certain embodiments, the disclosed methods result in an increase in wild-type TNNC1 protein expression in a subject (e.g., an increase of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 70%, or about 70% to about 100%). In certain embodiments, the disclosed methods result in an increase in the ratio of wild-type TNNC1 protein to mutant TNNC1 protein in a subject (e.g., an increase of about 5% to about 25%, about 25% to about 50%, about 50% to about 100%, or about 100% to about 200%).

[0102] The methods disclosed herein can provide efficient biodistribution in heart.They can lead to sustained expression in all or a significant portion of cardiac cells, such as cardiomyocytes.Notably, the methods disclosed herein can provide long-term expression of TNNC1 protein throughout the life of subject after AAV vector administration.

[0103] Combination therapy is also contemplated by the present invention. The combination of the method of the present invention with standard medical treatment (e.g., corticosteroids or topical decompression agents) is particularly contemplated, as is the combination with novel treatments. In some cases, subjects may be treated with a combination of steroids and / or immunosuppressants to suppress or reduce immune response to administration of rAAV as described herein.

[0104] In some embodiments, the AAV vector is about 1 x 10 AAV vector (vg) per kilogram of total subject body weight. 12 ~5×10 14 The AAV vector is administered at a dose of vector genome (vg) (vg / kg). In some embodiments, the AAV vector is administered at a dose of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 ~1×1014 In some embodiments, the AAV vector is administered at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5 × 10 vg / kg 12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5×10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 vg / kg or less than about 7 × 10 15 It is administered in doses of less than vg / kg.

[0105] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 7×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg or approximately 7 × 10 15 The drug is administered at a dose of 100 mg / kg.

[0106] In some embodiments, the AAV vector is 1 x 10 12vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 7×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg or 7 × 10 15 The drug is administered at a dose of 100 mg / kg.

[0107] In some embodiments, the AAV vector is about 1 x 10 AAV vector (vg) per kilogram of total subject body weight. 12 ~5×10 14 The AAV vector is administered systemically at a dose (vg / kg) of vector genome (vg). In some embodiments, the AAV vector is administered in an amount of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 ~1×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5×10 vg / kg 12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5×10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg14 Less than 5×10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 vg / kg or less than about 7 × 10 15 It is administered systemically in doses of less than vg / kg.

[0108] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 7×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg or approximately 7 × 10 15 It is administered systemically at a dose of 1000 mg / kg.

[0109] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 7×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg or 7 × 10 15 It is administered systemically at a dose of 1000 mg / kg.

[0110] In some embodiments, the AAV vector is about 1 x 10 AAV vector (vg) per kilogram of total subject body weight. 12 ~5×10 14 The vector genome (vg) is administered intravenously at a dose (vg / kg). In some embodiments, the AAV vector is administered at a dose of about 1×10 13 ~5×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 ~3×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 ~1×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 12 Less than 3×10 vg / kg 12 Less than 5×10 vg / kg 12 Less than 7×10 vg / kg 12 Less than 1×10 vg / kg 13 Less than 3×10 vg / kg 13 Less than 5×10 vg / kg 13 Less than 7×10 vg / kg 13 Less than 1×10 vg / kg 14 Less than 3×10 vg / kg 14 Less than 5×10 vg / kg 14 Less than 7×10 vg / kg 14 Less than 1×10 vg / kg 15 Less than 3×10 vg / kg 15 Less than 5×10 vg / kg 15 vg / kg or less than about 7 × 10 15 It is given intravenously in doses of less than vg / kg.

[0111] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×1012 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 7×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 7×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg or approximately 7 × 10 15 It is given intravenously at a dose of 1000 mg / kg.

[0112] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5×10 12 vg / kg, 7×10 12 vg / kg, 1×10 13 vg / kg, 3 × 10 13 vg / kg, 5×10 13 vg / kg, 7×10 13 vg / kg, 1×10 14 vg / kg, 3 × 10 14 vg / kg, 5×10 14 vg / kg, 7×10 14 vg / kg, 1×10 15 vg / kg, 3 × 10 15 vg / kg, 5×10 15 vg / kg or 7 × 10 15 It is given intravenously at a dose of 1000 mg / kg.

[0113] Evidence of functional improvement, clinical benefit or efficacy in patients may be evidenced by changes in New York Heart Association functional class (NYHA class), pathological electrocardiograms, echocardiograms, cardiac CT, cardiac MRI, cardiac biopsies, reduction in paroxysmal ventricular arrhythmias, reduction in sudden cardiac death, and / or reduction or absence of further development of myocardial fibrosis. Benefits may be observed in electrocardiographic features typically associated with dilated, hypertrophic, left ventricular noncompaction, or restrictive cardiomyopathy.

[0114] Administration of the Composition Administration of an effective dose of the composition may be by any route standard in the art, including, but not limited to, systemic administration, local administration, direct injection, intravenous administration, or intracardiac administration. In some cases, administration includes systemic injection, local injection, direct injection, intravenous injection, or intracardiac injection. Administration may be performed by cardiac catheterization.

[0115] In some embodiments, the present disclosure provides for local and systemic administration of an effective dose of rAAV and compositions of the present invention. For example, systemic administration can be administration into the circulatory system so that the entire body is affected. Systemic administration includes parenteral administration by injection, infusion or implantation. The routes of administration of the compositions disclosed herein include intravenous ("IV") administration, intraperitoneal ("IP") administration, intramuscular ("IM") administration, intralesional administration or subcutaneous ("SC") administration, or implantation of a sustained release device, such as a mini-osmotic pump, a depot formulation, and the like. In some embodiments, the method of the present disclosure includes administering the AAV vector of the present disclosure or a pharmaceutical composition thereof by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic or intracranial administration.

[0116] In particular, administration of the rAAV of the present invention can be accomplished using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal, including, but not limited to, injection into the heart.

[0117] In some embodiments, the methods of the disclosure include intracardiac delivery. Infusion can be performed using specialized cannulas, catheters, syringes / needles with an infusion pump. Administration can include delivery of an effective amount of rAAV virions, or pharmaceutical compositions containing rAAV virions, to the heart. These can be accomplished, for example, by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic, or intracranial administration. The compositions of the disclosure can also be administered intravenously.

[0118] The treatment methods disclosed herein may reduce and / or prevent one or more symptoms, including, but not limited to, ventricular hypertrophy, syncope, chest pain, left ventricular outflow tract obstruction, left ventricular dilation, reduced ejection fraction, systolic dysfunction, NYHA Class III-IV heart failure, ventricular tachycardia, exercise intolerance, and angina.

[0119] Effect of rAAV administration In some embodiments, administration of a rAAV of the present disclosure can have a beneficial effect on a subject. For example, administration of a rAAV of the present disclosure can increase the survival of a subject compared to a subject not administered a rAAV of the present disclosure.

[0120] In some embodiments, administration of a rAAV of the disclosure increases viability by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject not administered a rAAV of the disclosure.

[0121] In some embodiments, administration of a rAAV of the disclosure improves survival by 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 15%, 15% to 20%, 20% to 35%, or 40% to 50% compared to a subject not administered a rAAV of the disclosure. %, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 100%-200%, 200%-300%, 300%-400% or 400%-500% increase.

[0122] In some embodiments, administration of a rAAV of the disclosure abrogates the decline in ejection fraction in a subject compared to a subject not administered a rAAV of the disclosure. In some embodiments, administration of a rAAV of the disclosure abrogates the decline in ejection fraction by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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 at least about 100% compared to a subject not administered a rAAV of the disclosure.

[0123] In some embodiments, administration of a rAAV of the disclosure reduces or prevents a decrease in ejection fraction by 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, or 12% compared to a subject not administered a rAAV of the disclosure. Suppresses by 10%-15%, 15%-20%, 20%-35%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% or 95%-100%.

[0124] In some embodiments, administration of the rAAV of the present disclosure inhibits decline in left ventricular ejection fraction (LVEF), restores left ventricular ejection fraction (LVEF), and / or increases left ventricular ejection fraction (LVEF) in a subject compared to a subject not administered the rAAV of the present disclosure. In some embodiments, administration of a rAAV of the present disclosure abrogates the decline in ejection fraction by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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 at least about 100% compared to a subject not administered a rAAV of the present disclosure.

[0125] In some embodiments, administration of a rAAV of the present disclosure results in an improvement in lung function compared to a subject not administered a rAAV of the present disclosure, ranging from 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 15%, 15% to 20%, 20% to 35%, 25% to 30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% or 95%-100% to arrest the decline in left ventricular ejection fraction (LVEF), restore left ventricular ejection fraction (LVEF), and / or increase left ventricular ejection fraction (LVEF).

[0126] In some embodiments, administration of the rAAV of the present disclosure abrogates the decline, restoration and / or increase in left ventricular end systolic dimension (LVESD) in a subject compared to a subject not administered the rAAV of the present disclosure. In some embodiments, administration of a rAAV of the disclosure inhibits an increase in end diastolic diameter (EDD) in a subject by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%, compared to a subject not administered a rAAV of the disclosure.

[0127] In some embodiments, administration of a rAAV of the present disclosure provides an improvement in pulmonary function by 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 15%, 15% to 20%, 20% to 35%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50 ... % to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, 100% to 200%, 200% to 300%, 300% to 400% or 400% to 500% inhibit the decline, recovery and / or increase in left ventricular end systolic dimension (LVESD) in a subject.

[0128] In some embodiments, administration of a rAAV of the present disclosure abrogates the decline, restoration, and / or increase in left ventricular end diastolic dimension (LVEDD) in a subject compared to a subject not administered a rAAV of the present disclosure. In some embodiments, administration of a rAAV of the disclosure inhibits an increase in LVPW in a subject by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400% or at least about 500% compared to a subject not administered a rAAV of the disclosure.

[0129] In some embodiments, administration of a rAAV of the present disclosure provides an improvement in pulmonary function by 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 15%, 15% to 20%, 20% to 35%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50 ... % to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, 100% to 200%, 200% to 300%, 300% to 400% or 400% to 500% inhibit the decline, recovery and / or increase in left ventricular end diastolic dimension (LVEDD) in a subject.

[0130] In some embodiments, administration of the rAAV of the present disclosure abrogates the decline, restoration and / or increase in left ventricular outflow tract velocity time integral (LVOT VTI) in a subject compared to a subject not administered the rAAV of the present disclosure. In some embodiments, administration of a rAAV of the disclosure inhibits an increase in LVPW in a subject by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400% or at least about 500% compared to a subject not administered a rAAV of the disclosure.

[0131] In some embodiments, administration of a rAAV of the present disclosure provides an improvement in pulmonary function compared to a subject not administered a rAAV of the present disclosure, with an improvement of 1% to 90%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 15%, 15% to 20%, 20% to 35%, 25% to 30%, 30% to 40%, 35% to 50%, 30% to 50%, 35% to 60%, 35% to 70%, 35% to 80%, 35% to 90%, 35% to 10 ... 0%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 100%-200%, 200%-300%, 300%-400% or 400%-500% inhibit the decline, recovery and / or increase in left ventricular outflow tract velocity time integral (LVOT VTI) in a subject. EXAMPLES

[0132] Example 1: Preclinical bioactivity and efficacy The vectors exemplified in Figures 1-6 are tested. The AAV vectors or respective expression cassettes are tested in vitro using cultured cardiomyocytes (e.g., patient-derived induced pluripotent stem cell cardiomyocytes (iPSC-CMs) or primary cardiomyocytes harvested from animal models) or other cells suitable for transfection or transduction with these constructs. Expression of TNNC1 is assessed by ELISA, immunofluorescence, immunohistochemistry and Western blotting. Vector DNA is detected by PCR and TNNC1 transgene mRNA is detected by qRT-PCR. Cell-based studies utilizing mutant cardiomyocytes demonstrate the benefits of overexpressing the TNNC1 transgene (either after transduction with AAV vectors and / or transfection with vector plasmids) in the regulation of Ca. 2+ This is manifested by normalization of binding sensitivity / dissociation rates and the resulting normalization of contractile properties under stimulation.

[0133] Selected vectors will be tested in vivo using a mutant mouse model of cardiomyopathy. + / -The knock-in mouse model displays a severe DCM phenotype. This mouse model displays one or more DCM elements of the human disease. + / - The knock-in mice (described, for example, in McConnell et al. Front. Physiol. 2015; 6:242) have a mutation in the regulatory N domain of cardiac troponin C (cTnC), which inhibits Ca 2+ Increased dissociation rate, Ca 2+ The mechanism of this phenotype is a second Ca 2+ It is caused by the replacement of an acidic Asp (D) with a neutral Asn (N) at position X of the binding loop. D73N + / - Knock-in mice recapitulate the DCM phenotype beginning at 4 weeks, with increased left ventricular (LV) size, thinning walls, and fibrosis observed at 12 weeks. Further DCM phenotypes observed include left ventricular ejection fraction (LVEF) reduced to approximately 28% at 4 weeks, reduced fractional shortening (FS), impaired LV systolic function, and prolonged QRS and QT intervals. The mouse model shows reduced viability by 6 weeks, with a median survival of approximately 12 weeks and 100% mortality by 19 weeks.

[0134] An exemplary experimental design is shown in Table 7.

[0135] [Table 7]

[0136] Inducible I61Q + / - The knock-in mouse model displays a moderate DCM phenotype. This mouse model displays one or more DCM elements of the human disease. + / - The knock-in mice (described, for example, in Davis et al., Cell, 2016; 165(5):1147-1159) have a doxycycline-inducible cTnC cardiac single amino acid variant, I61Q + / - Knockin mice show reduced cardiac function, eccentric hypertrophy and LV dilatation 2+It recapitulates the DCM phenotype, including reduced binding and tension, increased diastolic LV cavity diameter, decreased septal thickness, increased cardiac mass, increased myocyte length-to-width ratio, and heart failure observed at approximately 6 weeks. This mouse model exhibits a 50% survival rate at approximately 3 months, 25-30% survival rate at 4 months, and 100% mortality by 8 months.

[0137] The benefits of AAV-mediated TNNC1 expression are demonstrated in mouse models by increased viability, mitigation of weight loss, mitigation of the normal progression of cardiomyopathy (e.g., TNNC1 DCM or TNNC1 HCM) observed on echocardiograms of the left and / or right ventricles (e.g., LVESD, LVEDD), mitigation of enlargement of right and / or left ventricle size, and / or mitigation of typical reductions in left ventricular ejection fraction and / or fractional shortening. Electrophysiological evidence of the functional benefits of AAV-mediated TNNC1 protein delivery is demonstrated by mitigation of disease-associated calcium dynamics disturbances in diseased cardiomyocytes, most notably in measurements of L-type calcium currents, sarcoplasmic reticulum calcium leak, diastolic calcium leak, and standard measurements of calcium transitions in diseased (e.g., TNNC1-deficient) cardiomyocytes, such as time to peak amplitude and relaxation time constants. For example, abnormal Ca in cardiomyocyte force generation is reduced. 2+ Sensitivity can approach that observed in healthy controls.

[0138] Histological analysis may reveal benefit by disease-associated myofiber disarray and / or fibrosis, hypertrophy, reduced appearance of apoptotic cells, reduced γH2AX marker of DNA damage, and reduced disease-associated changes in atrial volume and absolute cardiac size. Benefit may also be revealed by reduction or normalization of myocardial β-myosin heavy chain levels, B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and MYH7 levels compared to non-AAV-TNNC1-treated disease controls. Benefit may also be revealed by cardiac histopathological analysis.

[0139] Example 2: Preclinical transgene expression The expression cassettes exemplified in Figures 7 and 8 were tested. The AAV vector or each plasmid expression cassette was tested in vitro using cultured CHO-Lec2, a mutant cell line with a 70-90% deletion of sialic acid in its glycoproteins and gangliosides, making this cell more susceptible to AAV9 transduction. The subsequent expression of human cardiac troponin C transgene protein (TnC) in transduced CHO-Lec2 cells was assessed by Western blot (Figure 9).

[0140] The vector was also tested in vivo using a mutant mouse model of cardiomyopathy. + / - The knock-in mouse model exhibits a severe dilated cardiomyopathy (DCM) phenotype. This mouse model displays one or more DCM elements of the human disease. + / - The knock-in mice (described, for example, in McConnell et al. Front. Physiol. 2015; 6:242) have a mutation in the regulatory N domain of TNNC1, which inhibits Ca 2+ Increased dissociation rate, Ca 2+ The mechanism of this phenotype is a second Ca 2+ It is caused by the replacement of an acidic Asp (D) with a neutral Asn (N) at position X of the binding loop. D73N + / - Knock-in mice recapitulate the DCM phenotype beginning at 4 weeks of age, with increased left ventricular (LV) size and thinning of the ventricular wall, followed by fibrosis at 12 weeks of age. Additional markers of the DCM phenotype observed in this mouse model include left ventricular ejection fraction (LVEF) reduced to approximately 28% at 4 weeks, reduced fractional shortening (FS), impaired LV systolic function, and prolonged QRS and QT intervals. This mouse model also shows reduced viability, which can appear as early as 6 weeks of age, with a median survival of approximately 12 weeks and 100% mortality by 21-22 weeks (with variability that may be experimentally influenced by the level of stress induced by the frequency of handling the animals).

[0141] The experimental design is shown in Table 8.

[0142] [Table 8] FB = formulation buffer; WT = wild type; CON = control

[0143] The benefit of AAV-mediated human TnC protein expression was demonstrated by increased survival. All AAV-injected animals outperformed FB-injected D73N + / - D73N mice injected with FB survived significantly longer than controls (Figures 10A and 10B). + / - Reduced end diastolic diameter (EDd) in all male groups injected with AAV compared to controls (Figure 11A), as well as reduced EDd in D73N injected with FB. + / - A significant benefit of AAV-mediated human TnC overexpression on cardiac function was revealed, evidenced by a reduction in end systolic diameter (ESd) in males treated with AAV9-MHCK7-TNNC1 and AAVrh.74-MHCK7-TNNC1 compared to controls (Figure 12A). Attenuation of normal progression of dilated cardiomyopathy was observed in FB-injected D73N mice. + / - This was also evident by a significantly higher ejection fraction (FIG. 13A) and shortening fraction (FIG. 14A) in animals injected with AAV9-MHCK7-TNNC1 compared to controls. As expected, no significant changes in heart weight or heart rate were observed.

[0144] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheets are incorporated herein by reference in their entireties.

[0145] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A polynucleotide comprising an expression cassette and optionally flanking adeno-associated virus (AAV) inverted terminal repeats (ITRs), the polynucleotide comprising a polynucleotide sequence encoding troponin C1 cardiac type (TNNC1) or a functional variant thereof, operably linked to a promoter.

2. 2. The polynucleotide of claim 1, wherein the promoter is an MHCK7 promoter, a cardiac troponin T (hTNNT2) promoter, a cardiac-specific promoter, a muscle-specific promoter, a cardiomyocyte-specific promoter, or a ubiquitous promoter, optionally a CMV promoter or a CAG promoter. (i) the promoter is the MHCK7 promoter, which shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 21; or (ii) the promoter is an hTNNT2 promoter that shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 22; The polynucleotide of claim 2.

4. The expression cassette (i) comprising exon 1 of the cardiac troponin T (hTNNC1) gene, optionally including the hTNNT2 promoter and exon 1 together, which share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 23; and / or (ii) a polyA signal, optionally including a human growth hormone (hGH) polyA; and / or (iii) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), optionally including a WPRE(x); and / or (iv) contains a Kozak sequence; and / or (v) containing an SV40 intron; The polynucleotide of claim 1.

5. The polynucleotide of claim 1, wherein the TNNC1 or functional variant thereof is TNNC1, functional TNNC1, or human TNNC1. (i) comprising the TNNC1 polynucleotide sequence set forth in SEQ ID NO: 2; and / or (ii) TNNC1 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 1; The polynucleotide of claim 5. (i) the polynucleotide sequence encoding TNNC1 is a human TNNC1 polynucleotide; and / or (ii) the polynucleotide sequence encoding TNNC1 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2; The polynucleotide of claim 1. Claim 8: (i) comprising at least about 2.4 kb, at least about 2.5 kb, at least about 2.6 kb, at least about 2.7 kb, at least about 2.8 kb, at least about 3 kb, at least about 3.2 kb, at least about 3.4 kb, or at least about 3.6 kb; (ii) containing at most about 2.6 kb, at most about 2.7 kb, at most about 2.8 kb, at most about 3 kb, at most about 3.2 kb, at most about 3.4 kb, at most about 3.6 kb, at most about 3.8 kb, or at most about 4 kb; (iii) about 4.0 kb to 4.6 kb, about 4.0 kb to 4.5 kb, or about 4.0 kb to 4.4 kb; (iv) about 2.4 kb to 3.6 kb, about 2.5 kb to 3.5 kb, about 2.6 kb to 3.4 kb, about 2.7 kb to 3.3 kb, about 2.8 kb to 3.2 kb, or about 2.9 kb to 3.1 kb; or (v) about 2.4 kb, about 2.5 kb, about 2.8 kb, about 2.9 kb, about 3.5 kb, or about 3.6 kb; The polynucleotide of claim 1.

9. 2. The polynucleotide of claim 1, which shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 57-62.

10. (i) the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs), or (ii) the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs), and the ITRs are AAV2 ITRs and / or the ITRs share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 11-17; The polynucleotide of claim 1.

11. A gene therapy vector comprising the polynucleotide of claim 1.

12. (i) The gene therapy vector is a recombinant adeno-associated virus (rAAV) vector. (ii) the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector, and the rAAV vector is AAV9 or AAVrh.74 or a functional variant thereof; or (iii) the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector, and the rAAV vector is AAV9 or AAVrh.74 or a functional variant thereof, and the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 70 or 73; The vector of claim 11.

13. A pharmaceutical composition for treating and / or preventing a disease or disorder in a subject in need thereof, comprising the vector of claim 11.

14. (i) the disease or disorder is a cardiac disorder; a cardiomyopathy, optionally dilated cardiomyopathy, left ventricular noncompaction cardiomyopathy, restrictive cardiomyopathy, or hypertrophic cardiomyopathy; heart failure; or a cardiomyopathy associated with dysfunction of TNNC1; and / or (ii) the disease or disorder is characterized by altered calcium binding and / or is caused by a mutation in TNNC1; 14. The pharmaceutical composition of claim 13.

15. (i) The disease is heart failure and is characterized by a low ejection fraction, or (ii) The disease is heart failure characterized by a low ejection fraction, and the ejection fraction is 30% or less; 15. The pharmaceutical composition of claim 14.

16. 15. The pharmaceutical composition of claim 14, wherein the disorder is hypertrophic cardiomyopathy and is characterized by syncope, angina pectoris and / or mild left ventricular hypertrophy.

17. The pharmaceutical composition of claim 14, wherein the mutation is a gain-of-function mutation, a loss-of-function mutation, or a mutation selected from the group consisting of Y5H, A8V, L29Q, A31S, C84Y, E134D, D132N, D145E, I148V, G159D, and G159R compared to the human TNNC1 gene.

18. 14. The pharmaceutical composition of claim 13, wherein the subject is a mammal, a primate, or a human.

19. 14. The pharmaceutical composition of claim 13, wherein the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheterization.

20. administering the pharmaceutical composition (i) increases wild-type TNNC1 expression by about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 70%, or about 70% to about 100%; and / or (ii) increasing the ratio of wild-type TNNC1 to mutant TNNC1 by about 5% to about 25%, about 25% to about 50%, about 50% to about 100%, or about 100% to about 200%; 14. The pharmaceutical composition of claim 13. (i) an effective amount of the vector, or (ii) the vector having about 1×10 11 to about 1×10 14 vector genomes or the vector having about 1×10 11 to about 1×10 15 vector genomes; 14. The pharmaceutical composition of claim 13, comprising:

22. A pharmaceutical composition comprising the vector of claim 11.

23. 22. A kit comprising the vector of claim 11 or the pharmaceutical composition of claim 21 and optionally instructions for use.

Images