EA048262B1 - INCREASED EFFICIENCY OF VECTOR PACKAGING FOR GENE THERAPY OF HEART DISEASE - Google Patents

Abstract

The present invention relates to compositions and methods useful in the treatment of cardiac diseases. The disclosed compositions and methods are based on gene therapy comprising a recombinant AAV-based vector for delivering two or more transgenes to the heart of an individual, wherein the transgenes encode the S100A1 protein and an apoptosis inhibitor, cardiac apoptosis repressor with a caspase recruitment domain (cARC), respectively. In various embodiments, the compositions and methods disclosed herein comprise vectors comprising S100A1 and/or cARC cDNA sequences that are codon-optimized for expression in humans. In various embodiments, the compositions and methods disclosed herein comprise vectors with improved packaging efficiency. In some aspects, targeting multiple sources of one or more cardiac conditions can provide synergistic benefits during treatment.

Description

Link to related applications

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/130,109, filed December 23, 2020, which is hereby incorporated by reference in its entirety.

List of sequences

In accordance with 37 C.F.R. 1.52(e)(5), this disclosure incorporates a Sequence Listing (provided in electronic form as a .txt file named U1197.70172WO00-SEQ). The .txt file is dated December 14, 2021 and is 57,573 bytes in size. The Sequence Listing is hereby incorporated by reference in its entirety.

Prior art of the present invention

Cardiomyopathy is the second most common cause of cardiac disease in dogs, and the only therapeutic option is medical treatment of secondary signs. The prognosis for affected dogs depends on the stage of the disease and, in dogs, the breed. Cardiac function is critically dependent on calcium-dependent signaling. During cardiac function, impaired calcium channel function in cardiac cells contributes to calcium cycling abnormalities, further depressing cardiac function. Gene transfer strategies to reduce calcium cycling abnormalities have shown improvement in cardiac health in small and large animal models, as well as in human clinical trials.

Dilated cardiomyopathy (DCM) is the most common type of human cardiomyopathy, occurring primarily in adults between the ages of 20 and 60. DCM is also present in canines (e.g., dogs), particularly large breed dogs. In both humans and dogs, DCM is a disease of the heart muscle that affects the ventricles and atria of the heart, the lower and upper chambers of the heart, respectively, resulting in weak contractions and decreased pumping action. As DCM progresses, the heart chambers become enlarged, one or more valves may leak, and signs of chronic heart failure develop. In dogs, the cause of DCM is often unclear, but some breeds have an inherited (e.g., genetic) predisposition.

In humans, most forms of DCM are caused by a variety of conditions, including coronary artery disease, myocardial infarction, high blood pressure, diabetes, thyroid disease, viral hepatitis, and viral infection that causes inflammation of the heart muscle. Alcohol abuse, drug abuse, and certain drugs such as cocaine and amphetamines, as well as at least two drugs used to treat cancer (doxorubicin and daunorubicin), can also lead to DCM. In addition, there are a number of genetic forms of DCM, including, but not limited to, DCM associated with Duchenne and Becker muscular dystrophies. In some forms of Becker muscular dystrophy, as well as most cases of Duchenne muscular dystrophy, cardiomyopathy may ultimately limit the patient's survival.

Brief summary of the present invention

In humans, dilated cardiomyopathy is the most common type of cardiomyopathy and can result from a number of acquired as well as genetic diseases. As in dogs and other animal models, although the underlying causes of the disease are dysfunctional calcium metabolism, the ultimate progression of the disease is mediated by mitochondrial dysfunction and/or stretch-induced cardiomyocyte apoptosis. Although treatment of calcium metabolism alone may be effective in the early stages of the disease, addressing the interplay of calcium metabolism, mitochondrial dysfunction, and apoptosis will be necessary for the treatment of all forms of DCM and at all stages of disease progression.

Provided herein are improved gene delivery approaches for the treatment of subjects with cardiomyopathy, and in some embodiments, chronic heart failure. These approaches involve the expression of S100A1 to address calcium metabolism and the expression of ARC (an apoptosis repressor with a caspase recruitment domain) to block all sources of apoptosis and normalize mitochondrial function (see also U.S. Patent Publication No. 2021/0260215, incorporated herein by reference in its entirety). Expression of S100A1 and ARC transgenes using the disclosed self-complementary AAV vector approach occurs rapidly (i.e., within hours), which is critical to counteracting the effects of end-stage heart failure, and is restricted to the heart. These approaches thus target all three factors in the onset and progression of DCM, and thus should be applicable to any form of DCM at any stage of progression.

In addition, the present document provides transgene kits for delivering genes that encode the ARC protein and S100A1. These transgenes may contain cDNA sequences, and these sequences may be delivered and expressed using a recombinant adeno-associated virus (rAAV) vector system. The disclosed cDNA sequences may be delivered with any type of gene delivery vector, including, but not limited to, all forms of AAV, lentiviruses, liposomes, and exosomes.

- 1 048262

In particular embodiments, these sequences can be expressed using a self-complementary version of an AAV-based vector DNA that comprises i) a wild-type or optimized cDNA encoding human ARC, and ii) a wild-type or optimized cDNA encoding human S100A1. The ARC and s100A1 cDNA sequences can be arranged such that ARC comprises the first cDNA and S100A1 comprises the second cDNA in the 5' to 3' direction, or vice versa. In other embodiments, these cDNA sequences can be expressed using two promoters instead of a promoter and an IRES.

Expression of these cDNA sequences can be functionally controlled by the cardiac troponin T (cTnT) promoter located at the 5' end of the first cDNA and/or the internal ribosome entry site (IRES) located at the 5' end of the second cDNA. The cTnT promoter restricts expression of the two transgenes to cardiomyocytes when AAV is administered via the bloodstream to the heart and other tissues or by direct injection. The resulting ARC expression prevents apoptosis when expressed in cardiomyocytes and helps normalize mitochondrial membrane potential by reducing free radical formation and improving mitochondrial function. Expression of S100A1 results in improved calcium pumping into the sarcoplasmic reticulum (SR) of cardiomyocytes via the sarco/endoplasmic reticulum Ca ²⁺ -ATPase (SERCA) pump (e.g., SERCA pump, isoform 2a, or SERCA2a) and decreased calcium leakage from the SR via the ryanodine receptor channel. The combination normalizes calcium handling by cardiomyocytes and improves systolic and diastolic cardiac function.

The combined effects of the absence of apoptotic effects, improvement of mitochondrial function and improvement of calcium handling slow the progression of heart failure and improve cardiac function. The disclosed rAAV-based vectors can be effective in all forms of human cardiomyopathy and heart failure. Thus, the disclosed rAAV-based vectors can be delivered to a subject, such as a human suffering from a disease, disorder or condition involving poor cardiac function, such as human cardiomyopathy and heart failure.

Thus, in some aspects of the present disclosure, recombinant adeno-associated virus (rAAV) vectors are provided for delivering transgenes to the heart of a subject. In some embodiments, such rAAV vectors comprise at least two transgenes, one encoding S100 family proteins and one encoding an inhibitor of apoptosis. Such rAAV vectors may comprise, sequentially from 5' to 3', a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a promoter operably linked to the transgenes, and a second AAV inverted terminal repeat (ITR) sequence. In some embodiments, the two transgenes are operably linked to the same promoter. In other embodiments, each transgene is operably linked to a separate promoter. In some embodiments, the rAAV vector further comprises at least one polyadenylation signal (e.g., at the 3' position of two transgenes expressed from a single promoter, or at the 3' position of one or both transgenes expressed from different promoters). In aspects of the disclosure, therefore, there are provided recombinant adeno-associated virus (rAAV) nucleic acid vectors for delivering two or more transgenes to the heart of a subject, wherein said vectors comprise, from 5' to 3', a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, two or more transgenes and a promoter operably linked to the two or more transgenes, a polyadenylation signal and a second AAV inverted terminal repeat (ITR) sequence, wherein the two or more transgenes encode an S100 family protein and an inhibitor of apoptosis, respectively.

The transgenes of the present disclosure may encode an S100 family protein and/or an apoptosis inhibitor. For example, an S100 family protein may include cardiac calcium-binding protein S100 A1 (cS100A1), or a variant thereof. In another example, an apoptosis inhibitor may include cardiac repressor of apoptosis with a caspase recruitment domain (cARC), or a variant thereof.

In some embodiments, one or more transgenes of the present disclosure are naturally occurring or wild-type sequences. In some embodiments, one or more transgenes (e.g., a cARC transgene) are codon optimized for expression in humans.

In various embodiments, provided herein are rAAV-based vectors comprising a promoter, a polyadenylation (polyA) signal, and a polynucleotide comprising two or more transgenes. In some embodiments, the first transgene encodes an S100 family protein (e.g., cS100A1 or a variant thereof) and the second transgene encodes a cARC. In particular embodiments, the S100 family protein and cARC transgenes are derived from humans (e.g., homo sapiens). In particular embodiments, the S100 family protein and cARC transgenes are derived from canids (e.g., canis lupus familiaris).

In some embodiments, the first transgene (encoding a S100 family protein) of the polynucleotide comprises the nucleotide sequence shown as SEQ ID NO: 5. Al

- 2 048262 ternatively, the first transgene may comprise the nucleotide sequence presented as SEQ ID NO: 8. In some embodiments, the first transgene may comprise the nucleotide sequence presented as SEQ ID NO: 25. In some embodiments, the first transgene may comprise the nucleotide sequence presented as SEQ ID NO: 26.

Thus, the present document provides rAAV nucleic acid vectors for delivering two or more transgenes to the heart of a subject, wherein said vectors comprise, sequentially from 5' to 3', a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, two or more transgenes and a promoter operably linked to the two or more transgenes, a polyadenylation (polyA) signal and a second AAV inverted terminal repeat (ITR) sequence, wherein the two or more transgenes encode an S100 family protein and an inhibitor of apoptosis, respectively, and wherein the transgene encoding the S100 family protein comprises a sequence that is at least 95%, at least 98%, or at least 99.5% identical to any of the nucleotide sequences of SEQ ID NOs: 25 and 26.

In some embodiments, the transgene encoding the S100 family protein is positioned 5' relative to the transgene encoding the apoptosis inhibitor. In some embodiments, the transgene encoding the apoptosis inhibitor is positioned 5' relative to the transgene encoding the S100 family protein.

In some embodiments, the rAAV-based vector encodes a protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to the amino acid sequences of SEQ ID NO: 13 or 14. In some embodiments, the rAAV-based vector encodes a protein comprising the amino acid sequences of SEQ ID NO: 13 or 14. In some embodiments, the rAAV-based vector encodes a protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to the amino acid sequences of SEQ ID NO: 24 or 29. In some embodiments, the rAAV-based vector encodes a protein comprising the amino acid sequences of SEQ ID NO: 24 or 29.

In some embodiments, the first transgene (encoding a S100 family protein) may comprise a nucleotide sequence that is at least 90%, at least 95%, or at least 99.5% identical to any of the nucleotide sequences of SEQ ID NOs: 19-21. The first transgene may comprise a nucleotide sequence of any of SEQ ID NOs: 19-21. In particular embodiments, the first transgene may comprise a nucleotide sequence of SEQ ID NO: 21.

In some embodiments, the first transgene (encoding an S100 family protein) may comprise a nucleotide sequence that is at least 90%, at least 95%, or at least 99.5% identical to a nucleotide sequence of any of SEQ ID NOs: 25 and 26. In some embodiments, the first transgene encoding an S100 family protein comprises any of the nucleotide sequences set forth as SEQ ID NOs: 25 and 26.

In some embodiments, the first transgene encoding the S100 family protein comprises a sequence having a reduced guanosine (G) and cytosine (C) (G/C) content, relative to any of the nucleotide sequences of SEQ ID NO: 5, 8, or 19-20. In some embodiments, the G/C content is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% relative to any of the nucleotide sequences of SEQ ID NO: 5, 8, or 19-20. In some embodiments, the G/C content is reduced by about 1-15%, 110%, 1-5%, 2-10%, 3-15%, 3-10%, 5-15%, 5-10%, 5-13%, 7-15%, or 7-10% relative to any of the nucleotide sequences of SEQ ID NO: 5, 8, or 19-20.

In some embodiments, the apoptosis inhibitor is a cardiac repressor of apoptosis with a caspase recruitment domain (ARC) or a variant thereof. In some embodiments, the second transgene encoding the apoptosis inhibitor comprises a sequence that is at least 90%, at least 95%, or at least 99.5% identical to any nucleotide sequence of SEQ ID NO: 3, 6, 7, or 15-18. In some embodiments, the transgene encoding the apoptosis inhibitor comprises any nucleotide sequence of SEQ ID NO: 3, 6, 7, or 15-18.

In some embodiments, the polyA signal comprises a nucleotide sequence that is at least 90%, 92.5%, 95%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the polyA signal comprises the nucleotide sequence of SEQ ID NO: 28.

In some embodiments, an internal ribosome entry site (IRES) is present between two or more transgenes (e.g., between an S100A1 transgene and a cARC transgene). In some embodiments, a transgene encoding an S100 family protein is 5' relative to a transgene encoding an inhibitor of apoptosis. In other embodiments, a transgene encoding an inhibitor of apoptosis is 5' relative to a transgene encoding an S100 family protein.

- 3 048262

In some embodiments, the rAAV promoter is a cardiac tissue-restricted promoter. In some embodiments, the promoter is a cardiac tissue-restricted promoter selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). In some embodiments, the promoter is a cardiac tissue-restricted promoter derived from a gene selected from the group consisting of an α-myosin heavy chain gene, a 6-myosin heavy chain gene, a myosin light chain 2v gene, a myosin light chain 2a gene, a CARP gene, a cardiac α-actin gene, a cardiac m2-muscarinic acetylcholine receptor gene, ANF, a cardiac sarcoplasmic reticulum Ca-ATPase gene, and a skeletal α-actin gene; or is an artificial cardiac promoter derived from an MLC-2v gene. In some embodiments, the promoter is cTnT.

In some embodiments, the rAAV-based vector is single-stranded. In some embodiments, the rAAV-based vector is self-complementary.

In some embodiments, the rAAV-based vector comprises a nucleotide sequence that is at least 90%, at least 95%, or at least 99.5% identical to any of the nucleotide sequences of SEQ ID NOs: 22 and 23. In some embodiments, the rAAV-based vector comprises the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the rAAV-based vector comprises the nucleotide sequence of SEQ ID NO: 23.

Further provided herein are rAAV vectors comprising one or more transgenes (e.g., an S100A1 transgene and a cARC transgene) that have improved or enhanced packaging efficiency. In some embodiments, the transgenes are specific for homo sapiens or canis lupus familiaris. In some embodiments, the rAAV vectors have improved packaging efficiency due to their shorter length, such as by shortening the length of the polyA sequence. In some embodiments, the rAAV vectors have improved packaging efficiency due to a reduced guanine/cytosine content, such as a reduced G/C content, in one or both transgenes, relative to a wild-type transgene or a transgene codon-optimized for expression in a particular host organism. In some embodiments, the rAAV vectors have a reduced G/C content in the first transgene (e.g., a transgene encoding S100A1). Further provided are rAAV particles that have been packaged with any of these rAAV-based vectors. The increase in vector packaging efficiency can be measured by assessing the vector genome titer from the resulting rAAV particle composition packaged with the vector, or by any other method known in the art.

In some embodiments, the increased packaging efficiency of the disclosed vectors results in compositions comprising increased titers of rAAV particles relative to prior art vectors, such as increased from about two to about five times. In some embodiments, the increased packaging efficiency of the disclosed vectors results in compositions comprising titers of rAAV particles from 2x10 ¹³ to 5x10 ¹³ vector genome copies/mL (vg/mL).

Further provided herein are rAAV particles comprising rAAV-based vectors described herein encapsulated in AAV capsids. In some embodiments, the AAV capsid comprises a capsid protein derived from serotypes AAV1, AAV2, AAV3, AAV6, AAV8, AAVrh.74, AAVrh.10, AAV2/6, or AAV9. In some embodiments, the AAV capsid comprises a capsid protein derived from serotype AAVrh. 10. In some embodiments, the AAV capsid comprises a capsid protein derived from serotype AAVrh.74.

Other aspects of the present disclosure include compositions comprising rAAV particles as described herein. Such compositions can be administered to a subject for gene therapy of a cardiac disease. Thus, some aspects of the disclosure contemplate a method of treating a subject suffering from a cardiac disease, comprising administering to the subject a composition comprising rAAV particles, or an rAAV particle, as described herein. In some embodiments, the cardiac disease causes heart failure in the subject. In some embodiments, the cardiac disease is a cardiomyopathy. In other embodiments, the cardiac disease is hypertrophic cardiomyopathy or dilated cardiomyopathy. In other embodiments, the cardiac disease is acute ischemia. In some embodiments, the cardiac disease is a cardiomyopathy or acute ischemia.

In some embodiments, provided herein are methods comprising administering to a cell (such as a cell of a subject ex vivo or in vivo) a composition comprising rAAV particles or an rAAV particle as described herein. In some embodiments, provided herein are methods comprising administering in vitro or ex vivo a composition comprising rAAV particles or an rAAV particle as described herein.

- 4 048262

Thus, provided herein are methods for treating diseases, conditions or disorders of the heart, such as cardiomyopathy. In some aspects, provided herein are methods for treating a subject suffering from a cardiac disorder, comprising administering to the subject a composition comprising rAAV particles or an rAAV particle as described herein. Also contemplated are methods of administration in vitro and ex vivo. Also contemplated is the use of any composition comprising rAAV particles or an rAAV particle as described herein as a medicament.

The compositions of the present disclosure can be administered to a subject in various ways. In some embodiments, the composition is administered by injection into the heart of the subject or by intravascular injection into the coronary vessels of the subject. In some embodiments, administering the composition results in expression of transgenes (e.g., two or more transgenes) in the heart of the subject. In various embodiments, the step of administering the composition results in an improvement in cardiac function in the subject, such as an improvement in cardiac function in the subject for more than 10 months. In some embodiments, administering results in an improvement in cardiac function for more than 12 months, more than 14 months, more than 16 months, more than 17 months, more than 20 months, more than 22 months, or more than 24 months. In some embodiments, the step of administering results in an improvement in cardiac function in the subject for more than 10 months.

In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a pet. In some embodiments, the pet is a dog or a cat.

Brief description of the drawings

The following drawings form a part of the present specification and are included to further illustrate certain aspects of the present disclosure. The disclosure can be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals designate like elements:

Fig. 1 is a schematic of the AAV construct according to the disclosure. The first AAV inverted terminal repeat (ITR) is followed by the cardiac troponin T (cTnT) promoter, then a sequence encoding S100 calcium binding protein A1 (cS100A1), followed by an internal ribosome binding site (IRES), followed by a sequence encoding apoptosis repressor with caspase recruitment domain (cARC), followed by a polyadenylation (pA) sequence and a second AAV ITR.

Figure 2 shows diastolic MRI in a dog (named Calvin) with treated muscular dystrophy at baseline and several weeks after gene delivery. The data confirm stable or slightly improved cardiac remodeling with a modest reduction in left ventricular diastolic volume.

Figure 3 shows systolic MRI in a dog (named Calvin) with treated muscular dystrophy at baseline and several weeks after gene delivery. The data confirm stable or slightly improved left ventricular systolic function after treatment, with a slight decrease in systolic volume suggesting improved contractility and increased left ventricular cardiac output.

Figure 4 shows the ejection fraction, peak strain, and cardiac output of D2.mdx mice after treatment with AAVrh.10-S100A1/ARC. Over a 24-week period, mice treated with therapeutic AAV had better preservation of ejection fraction, strain development, and cardiac output compared to sham-injected mice.

Figure 5 shows the expression levels of S100A1 and ARC in mice treated with the recombinant AAVrh.10-S100A1/ARC vector and control mice. Protein analysis (Western blot) confirmed that the levels of both S100A1 and ARC were increased in treated tissues compared to control (sham) tissues.

Figure 6 shows cardiomyocyte histologies from control (left) and treated (right, S100A1.ARC) mice at 10- and 20-fold magnification. Cardiac histology data show that treated mice exhibited less DMD pathology, including less muscle fibrosis, compared to control hearts.

Figure 7 shows that the first of two dystrophin-deficient dogs (GRMD dogs), named Calvin, showed improved cardiac function (as measured by ejection fraction) following treatment with recombinant AAVrh.10-S100A1/ARC. Both injected dogs showed improvements in ejection fraction and other cardiac parameters following treatment, as measured by cardiac MRI and confirmed by echocardiography.

Figure 8 shows that a second GRMD dog, Sebastian, showed improved cardiac function after treatment with AAVrh.10-S100A1/ARC.

Figures 9A–9C show that AAV-S100A1/ARC treatment reduced serum creatine kinase (CK) levels and prevented muscle wasting in dogs with GRMD. Measurements of end-timber muscle mass

- 5,048,262 findings were analyzed using MRI for the area of both paws (Fig. 9A), maximum cross-sectional area (CSA) (Fig. 9B), and volume of both paws (Fig. 9C). The results show that skeletal muscle mass either increased or remained unchanged after cardiac therapy.

Figure 10 shows that circulating creatine kinase (CK) levels in skeletal muscle of GRMD subjects were reduced following AAVrh.10-S100A1/ARC injection, indicating a reduction in ongoing muscle damage.

Fig. 11 shows a sequence alignment between the codon-optimized canine ARC cDNA sequence and the codon-optimized human ARC cDNA sequence. The sequences correspond to SEQ ID NOs: 15, 16, and 6 from top to bottom.

Figure 12 shows the sequence alignment between the codon-optimized and native human ARC cDNA sequences. The sequences correspond to SEQ ID NOs: 17, 18, and 6 from top to bottom.

Figure 13 shows a sequence alignment between the codon-optimized and native human S100A1 cDNA sequences. The sequences correspond to SEQ ID NOs: 19, 5, and 8 from top to bottom.

Fig. 14 shows a sequence alignment between the codon-optimized canine S100A1 cDNA sequence and the codon-optimized human S100A1 cDNA sequence. The sequences correspond to SEQ ID NOs: 20, 8, and 21 from top to bottom.

Figure 15 shows the fractional shortening and ejection fraction in D2.mdx mice (n=12) at 10 months of age following AAVrh.10-S100A1/ARC treatment at 1 month of age. Both the fractional shortening and ejection fraction were not significantly different from those in wild-type D2 mice for AAVrh.10-S100A1/ARC-treated mice, whereas a significant reduction in function was observed in untreated D2.mdx mice.

Figure 16 shows the left ventricular volumes and diameters of D2.mdx mice (n=12) at 10 months of age following AAVrh.10-S100A1/ARC treatment at 1 month of age. The increase in volume and diameter during both diastole and systole is indicative of dilated cardiomyopathy. Both volume and diameter were not significantly different from wild-type D2 mice for AAVrh.10-S100A1/ARC-treated mice, whereas a significant increase in both parameters was observed in untreated D2.mdx mice.

Figure 17 shows the age at 50% survival of D2.mdx.sk_utrophin mice (n=12 per group at baseline) that began training at 6 weeks of age but were not treated until 6 months of age. The untreated control group had a median survival of 10 months. The AAVrh.10-ARC-treated group had a median survival of 14 months, the AAVrh.10-S100A1-treated group had a median survival of 16 months, and the AAVrh.10S100A1/ARC-treated group had a median survival of 20 months.

Figure 18 shows changes in ejection fraction with age in a population of untreated GRMD dogs (n=10) and changes in ejection fraction in individual GRMD dogs treated with the AAVrh.10-S100A1/ARC vector at the ages indicated by the arrows (between 3 and 7 months of age). In all four treated dogs, ejection fraction improved after treatment and remained stable thereafter.

Figure 19 shows the histology of the left ventricle (hematoxylin and eosin stain) of a healthy 1-year-old golden retriever (far left panel) and a dog with GRMD (WnM3/Calvin) treated with the AAVrh.10-S100A1/ARC vector at 7 months of age (far right panel). The treated dog died at 34 months of age from aspiration pneumonia, although its cardiac function was still within normal limits (Fig. 18). The middle three panels show the histology of the left ventricle in untreated dogs with GRMD at 8, 24, and 30 months of age. Severe and progressive fibrosis replacing muscle tissue is present (darker shade). Note that in the treated dog panels, on the far right, the histology is not noticeably different from that of a normal dog and is noticeably less fibrous and more intact than that of an untreated dog with GRMD.

Fig. 20 is a silver-stained SDS-PAGE gel demonstrating the purity of two rAAV-based vectors of the present invention. SEQ ID NO: 9 is shown on the left and SEQ ID NO: 22 is shown on the right.

Detailed disclosure of the present invention

The present invention relates to compositions and methods for gene therapy of cardiac diseases for the treatment of cardiac diseases, such as cardiomyopathy, in human and canine subjects. The methods of the present disclosure relate to the use of recombinant AAV (rAAV) particles for the simultaneous delivery and expression of two or more transgenes. The transgenes of the present disclosure encode at least two classes of proteins, each of which performs a specific function to address different aspects of cardiac diseases. One class of transgenes encode proteins that regulate calcium signaling in cardiomyocytes, such as the S100 family proteins. Another class of transgenes encode repressors of apoptosis. In some embodiments, the transgenes may encode cardiac calcium-binding protein A1 S100 (cS100A1) or a variant thereof and/or cardiac apoptosis repressor with caspase recruitment domain (cARC) or a variant thereof.

- 6 048262

The compositions and methods of the present disclosure are based at least in part on the synergistic action of two transgenes, such as transgenes encoding S100A1 and ARC, respectively, when delivered and expressed simultaneously in the heart of a subject. The S100A1 protein improves aspects of calcium handling, including normalization of sarcoplasmic reticulum calcium transients, which leads to normalization of contractile function. The ARC protein blocks apoptosis initiated by mitochondrial and non-mitochondrial mechanisms (such as stretch-induced apoptosis) and improves mitochondrial function. In other words, S100A1 and ARC act synergistically on two separate components of heart failure (calcium handling dysfunction and apoptosis), which leads to longer-lasting therapeutic results. Additionally, the compositions and methods of the present disclosure are effective at any stage of heart failure.

Additionally provided herein are methods for creating rAAV particles suitable for delivering transgenes, such as transgenes encoding S100A1 and/or ARC, or variants thereof, to the heart of a subject. Such rAAV particles may comprise a recombinant AAV genome comprising nucleic acid molecules encoding the transgenes, wherein the nucleic acid molecules are encapsidated by AAV capsid proteins. In some embodiments, the rAAV particles comprise a recombinant adeno-associated virus (rAAV) nucleic acid vector. The recombinant AAV genome is single-stranded DNA, which may further comprise sequence elements that facilitate integration of the AAV genome into the host genome and expression of the transgenes. For example, the recombinant AAV genome may comprise tissue-specific promoters to provide expression of the transgenes in target tissues or organs. Such rAAV particles may be used in a composition for the treatment of heart disease.

Thus, the present disclosure further provides rAAV-based vectors for delivering transgenes to the heart of a subject. In some embodiments, the disclosed rAAV-based vectors comprise at least two transgenes, one encoding an S100 family protein and one encoding an inhibitor of apoptosis. These rAAV-based vectors may comprise, sequentially from 5' to 3', a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a promoter operably linked to the transgenes, and a second AAV inverted terminal repeat (ITR) sequence. In some embodiments, the two transgenes are operably linked to the same promoter. In other embodiments, each transgene is operably linked to a separate promoter. In some embodiments, the rAAV-based vector also comprises at least one polyadenylation signal (e.g., at the 3' position of two transgenes expressed from a single promoter, or at the 3' position of one or both transgenes expressed from different promoters).

The disclosure further provides rAAV nucleic acid vectors for delivering two or more transgenes to the heart of a subject. In some embodiments, the rAAV vector comprises exactly two transgenes. In some embodiments, the rAAV vector comprises three transgenes. In some embodiments, the vector comprises from 5' to 3' a first AAV ITR sequence, two or more transgenes and a promoter operably linked to the two or more transgenes, a polyadenylation signal, and a second AAV ITR sequence. In particular embodiments, the two or more transgenes comprise a first transgene encoding an S100 family protein and a second transgene encoding an apoptosis inhibitor.

In some embodiments, the polyA signal comprises a bovine growth hormone (BGH) signal sequence. In some embodiments, the polyA signal comprises a variant BGH signal sequence. In some embodiments, the polyA signal comprises a nucleotide sequence that differs from the sequence of SEQ ID NO: 27 by 1, 2, 3, 4, 5, or more than 5 nucleotides. In some embodiments, the polyA signal comprises a nucleotide sequence that has at least 90%, 92.5%, 95%, 98%, or 99% identity to SEQ ID NO: 27. In some embodiments, the polyA signal comprises SEQ ID NO: 27 below. BGH polyA signal:

AGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT

GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGA

AATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCA

NO: 27)

In some embodiments, the polyA signal comprises a variant (e.g., a minimal variant and/or a synthetic variant) of the polyA signal. In some embodiments, the variant polyA signal comprises fewer nucleotides (e.g., it is shorter or smaller) than the BGH polyA signal (e.g., SEQ ID NO: 27). In some embodiments, the variant polyA signal comprises a sequence comprising the sequence AATAAA and a sequence rich in

- 7 048262

GT/T. In some embodiments, the AATAAA sequence and the GT/T rich sequence are separated by 22 nucleotides. In some embodiments, the AATAAA sequence and the GT/T rich sequence are separated by 23 nucleotides. In some embodiments, the polyA signal comprises a variant polyA signal as described in Levitt, et al. (1989), Genes & Dev. 3:1019-1025. In some embodiments, the variant polyA signal comprises a nucleotide sequence that has at least 90%, 92.5%, 95%, 98%, or 99% identity to SEQ ID NO: 28. In some embodiments, the polyA signal comprises a nucleotide sequence that differs from the sequence of SEQ ID NO: 28 by 1, 2, 3, 4, 5, or more than 5 nucleotides. In some embodiments, the variant polyA signal comprises SEQ ID NO: 28, set forth below. In some embodiments, the polyA signal comprises a sequence complementary to any of SEQ ID NO: 27 or 28.

PolyA signal option:

AATAAAAGATCTTXATTTT^

ID NO: 28)

A transgene, as used herein, refers to a gene or genetic material that has been transferred from one organism to another by natural means or by any of a number of genetic engineering techniques. A transgene may encode a protein or polypeptide of interest (e.g., S100A1, ARC) or express an RNA of interest (e.g., siRNA or miRNA). In some embodiments, a single rAAV-based vector may comprise a coding sequence for one or more transgenes. For example, a single rAAV-based vector may comprise a coding sequence for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 transgenes. In some embodiments, the rAAV-based vectors of the present disclosure comprise a coding sequence for both S100A1 and ARC, or variants thereof. In some embodiments, the rAAV-based vector further comprises a region encoding a Rep protein. The transgenes of the present disclosure encode two classes of proteins, each of which has a specific function for addressing different aspects of one or more cardiac diseases. One class of transgenes may encode a protein that regulates calcium signaling in cardiomyocytes, such as a protein of the S100 family. The other class of transgenes may encode repressors of apoptosis.

As used herein, the term "variant" refers to a nucleic acid that has characteristics that differ from a reference sequence (e.g., one that occurs in nature). For example, a variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 96% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a reference sequence (e.g., a wild-type nucleic acid). In some embodiments, the reference sequence may be a wild-type sequence or a naturally occurring sequence corresponding to the variant. For example, a transgene variant is a nucleic acid comprising one or more substitutions in the nucleotides of the transgene compared to its wild-type sequence. In some embodiments, the reference sequence may be a sequence that is codon optimized relative to the wild-type or natural sequence for expression in a particular subject or tissue and that, in the wild-type or codon-optimized configuration, corresponds to the variant. For example, a transgene variant is a nucleic acid comprising one or more substitutions in the nucleotides of the transgene compared to its codon-optimized optimized sequence. These substitutions may be silent; for example, they do not change the amino acid sequence of any encoded protein (or otherwise result in a variant amino acid sequence).

In some embodiments, provided herein are substitutions in one or more transgenes of the disclosed vectors that do not alter the amino acid sequence of the encoded protein. In some embodiments, such substitutions comprise replacing a guanine (G) or cytosine (C) present in the corresponding wild-type and/or codon-optimized nucleic acid sequence with another nucleotide (e.g., adenine (A) or tyrosine (T); or, for a G substitution, C; or, for a C substitution, G). Such substitutions reduce the number of G and/or C nucleotides present in a variant nucleic acid sequence (e.g., a variant transgene) relative to the wild-type (e.g., native, unmodified) and/or codon-optimized nucleic acid sequence. Thus, such substitutions result in a decrease in the GC content of the variant nucleic acid sequence relative to the corresponding post-

- 8 048 262 wild-type nucleic acid sequence and/or codon-optimized nucleic acid sequence. Reducing the GC content of the nucleic acid sequence may be desirable in some embodiments to increase the rate of DNA replication in virus production.

In some embodiments, the GC content of the variant human S100A1 nucleic acid sequence is reduced by about 10% relative to the codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8). In some embodiments, the GC content of the variant human S100A1 nucleic acid sequence is reduced by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% relative to a codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8). In some embodiments, the GC content of the variant human S100A1 nucleic acid sequence is reduced by 12% relative to a codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8). In some embodiments, the GC content of the variant human S100A1 nucleic acid sequence is reduced by 7% relative to the codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8).

In some embodiments, the G content of the variant human S100A1 nucleic acid sequence is reduced by about 5 G nucleotides relative to the number of G nucleotides present in the codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8). In some embodiments, the G content of the variant human S100A1 nucleic acid sequence is reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides relative to the number of G nucleotides present in the codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8). In some embodiments, the C content of the variant human S100A1 nucleic acid sequence is reduced by about 12 C nucleotides relative to the number of C nucleotides present in the codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8). In some embodiments, the C content of the variant human S100A1 nucleic acid sequence is reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 C nucleotides relative to the number of C nucleotides present in the codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8). In some embodiments, the GC content of the variant human S100A1 nucleic acid sequence is reduced by 5 G nucleotides and/or 12 C nucleotides relative to the number of G nucleotides and C nucleotides present in the codon-optimized human S100A1 nucleic acid sequence (e.g., SEQ ID NO: 8).

In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of about 50-55% (e.g., 50-55% of the nucleotides in the nucleic acid sequence are G or C nucleotides). In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of about 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, or 65-70%. In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of 50%. In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of 51%. In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of 52%. In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of 53%. In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of 54%. In some embodiments, the variant human S100A1 nucleic acid sequence has a GC content of 55%.

In some embodiments, the GC content of the variant canine S100A1 nucleic acid sequence is reduced by about 5% relative to a codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO:21). In some embodiments, the GC content of the variant canine S100A1 nucleic acid sequence is reduced by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% relative to a codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO:21). In some embodiments, the GC content of the variant canine S100A1 nucleic acid sequence is reduced by 13% relative to the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21). In some embodiments, the GC content of the variant canine S100A1 nucleic acid sequence is reduced by 8% relative to the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21). In

- 9 048262 In some embodiments, the GC content of the variant canine S100A1 nucleic acid sequence is reduced by 4% relative to the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21).

In some embodiments, the G content of the variant canine S100A1 nucleic acid sequence is reduced by about 4 G nucleotides relative to the number of G nucleotides present in the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21). In some embodiments, the G content of the variant canine S100A1 nucleic acid sequence is reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G nucleotides relative to the number of G nucleotides present in the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21). In some embodiments, the C content of the variant canine S100A1 nucleic acid sequence is reduced by about 5 C nucleotides relative to the number of C nucleotides present in the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21). In some embodiments, the C content of the variant canine S100A1 nucleic acid sequence is reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 C nucleotides relative to the number of C nucleotides present in the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21). In some embodiments, the GC content of the variant canine S100A1 nucleic acid sequence is reduced by 4 G nucleotides and/or 5 C nucleotides relative to the number of G and C nucleotides present in the codon-optimized canine S100A1 nucleic acid sequence (e.g., SEQ ID NO: 21).

In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of about 50-55% (e.g., 50-55% of the nucleotides in the nucleic acid sequence are G or C nucleotides). In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of about 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, or 65-70%. In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of 50%. In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of 51%. In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of 52%. In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of 53%. In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of 54%. In some embodiments, the variant canine S100A1 nucleic acid sequence has a GC content of 55%.

In some embodiments, wherein the rAAV-based vector comprises one or more variant transgenes (e.g., a first transgene encoding a variant S100A1 protein) with reduced GC content relative to a corresponding reference nucleic acid sequence (e.g., codon-optimized or wild-type), the rAAV-based vector will also have reduced GC content relative to an rAAV-based vector comprising two transgenes (e.g., a first transgene encoding a variant S100A1 protein and a second transgene encoding a variant ARC protein) that do not have reduced GC content. In some embodiments, the rAAV-based vector with reduced GC content is one of SEQ ID NOs: 22 or 23, and the corresponding rAAV-based vector that does not have reduced GC content is any of SEQ ID NOs: 9-12. In some embodiments, the rAAV vector with reduced GC content comprises a sequence that has 100% identity to SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, the rAAV vector with reduced GC content has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to SEQ ID NO: 22 or SEQ ID NO: 23.

In some embodiments, a variant transgene is a nucleic acid comprising one or more deletions in the nucleotides of the transgene compared to its wild-type sequence. These deletions may be silent; for example, they do not alter the amino acid sequence of any encoded protein (or otherwise result in a variant amino acid sequence). In some embodiments, deletions that do not alter the amino acid sequence of the encoded protein include deletions of one or more nucleotides in the polyadenylation (PA) signal of an rAAV-based vector compared to a corresponding rAAV-based vector that does not contain such deletions. Thus, such deletions

- 10 048262 result in a PA signal that contains fewer nucleotides (e.g., is smaller, shorter, or truncated) relative to a corresponding rAAV-based vector that does not contain such deletions. A deletion of nucleotides in the PA signal (e.g., a truncated PA signal) in a nucleic acid sequence (e.g., in an rAAV-based vector sequence) may thus be desirable in some embodiments to reduce the size of the overall self-complementary construct to less than 5 kb (including the ITR), which will improve packaging efficiency.

In some embodiments, an rAAV-based vector of the present invention comprises a truncated PA signal relative to a corresponding rAAV-based vector that does not comprise a deletion in the PA signal. In some embodiments, the truncated PA signal comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more than 200 nucleotides in the PA signal relative to a corresponding non-truncated rA signal. In some embodiments, the truncated PA signal comprises a deletion of 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, 70-80, 75-85, 80-90, 85-95, 90-100, 95-105, 100-110, 105-115, 105-115, 110-120, 115-125, 120-130, 125-135, 130-140, 135-145, 140-150, 145-155, 150-160, 155-165, 160-170, 165-175, 170-180, 175-185, 180-190, 185-195, 190-200, 195-205 or more than 200 nucleotides relative to the corresponding untruncated PA signal. In some embodiments, the truncated PA signal comprises a deletion of about 160 or about 170 nucleotides relative to the untruncated PA signal. In some embodiments, the truncated PA signal comprises a deletion of 166 nucleotides nucleotides relative to the untruncated PA signal. In some embodiments, the variant polyA signal comprises a nucleotide sequence that has at least 90%, 92.5%, 95%, 98%, or 99% identity to SEQ ID NO: 28. In some embodiments, the variant polyA signal comprises SEQ ID NO: 28.

In some embodiments, the rAAV-based vector comprising the truncated PA signal is one of SEQ ID NO: 22 or 23, and the rAAV-based vector that does not comprise the truncated PA signal is any of SEQ ID NO: 9-12. In some embodiments, the rAAV-based vector with a truncated PA signal comprises a sequence that has 100% identity to SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, the rAAV-based vector with a truncated PA signal has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to SEQ ID NO: 22 or SEQ ID NO: 23. In some embodiments, the rAAV-based vector has a nucleic acid sequence that has at least about 80% identity, 90% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, 99.5% identity, or 99.9% identity to SEQ ID NO: 22 or NO: 23, except for containing a domain that has a sequence of at least 99% or 100% identity to SEQ ID NO: 25 or 26. In some embodiments, the rAAV vector has a nucleic acid sequence that has at least about 80% identity, 90% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, 99.5% identity, or 99.9% identity to SEQ ID NO: 22 or 23, except for containing a domain that has a sequence of at least 99% or 100% identity to SEQ ID NO: 28.

Alternatively, these substitutions may result in a modification of the amino acid sequence of the encoded protein, resulting in the encoded protein having one or more amino acid substitutions (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, or 15-20 amino acid substitutions) relative to the wild-type protein sequence. These substitutions include chemical modifications as well as truncations. The term also encompasses functional fragments of the wild-type nucleic acid sequence. These modifications to the reference sequence may occur at the 5' or 3' ends of the reference sequence or anywhere between these positions, alternating either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

From a practical standpoint, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleotide sequence of a transgene or an encoded protein, can be routinely determined using known computer programs. A preferred method for determining the best overall match between a query sequence (e.g., a sequence of the present disclosure) and an individual sequence, also referred to as a global sequence alignment, can be determined using FASTDB or the blastn computer program, based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).

- 11 048262

In a sequence alignment, the query sequence and the individual sequence are either nucleotide sequences or amino acid sequences. The result of said global sequence alignment is expressed as a percent identity. The optimal parameters used in FASTDB amino acid alignment are: matrix=PAM 0, best match region=2, mismatch penalty^, joining penalty=20, randomization group length=0, threshold score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or the length of the individual amino acid sequence, whichever is shorter. Whether the nucleotides or polypeptides match/align is determined from the results of the sequence alignment in FASTDB. This percentage is then subtracted from the identity calculated by the above FASTDB program using the specified parameters to obtain the final percent identity. This final percent identity is used for the purposes of the present disclosure. For individual sequences that are truncated at the 5' and/or 3' ends relative to the query sequence, the percent identity is adjusted by counting the number of nucleotides of the query sequence located 5' or 3' relative to the query sequence that do not match/align with the corresponding nucleotides of the individual sequence, as a percentage of the total number of bases of the query sequence. Similar methods can be used to compare amino acid sequences.

Proteins of the S100 family that can be used in accordance with the present disclosure include, but are not limited to, S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7 (e.g., psoriasin), S100A8 (e.g., calgranulin A), S100A9 (e.g., calgranulin B), S100A10, S100A11, S100A12 (e.g., calgranulin C), S100A13, S100A14, S100A15 (e.g., koebnerisin), S100A16, S100B, S100P, and S100Z, or variants thereof.

In some embodiments, the S100 family protein may be calcium binding protein A1 S100 (S100A1). In some embodiments, S100A1 is cardiac S100A1 (cS100A1) or a variant thereof. The cS100A1 protein is a regulator of myocardial contractility. The levels of the cS100A1 protein are reduced in hypertrophied right ventricular tissue in a model of pulmonary hypertension. In addition, S100A1 is a regulator of the genetic program underlying cardiac hypertrophy, since S100A1 inhibits alpha 1-adrenergic stimulation of hypertrophy genes, including MYH7, ACTA1, and S100B.

In cardiomyocytes, S100A1 regulates the calcium-gated SR network, sarcomeric and mitochondrial functions through modulation of ryanodine receptor 2 (RYR2), sarco/endoplasmic reticulum Ca ²⁺ -ATPase (SERCA), titin and mitochondrial F1-ATPase activities. As a result, cardiomyocytes and hearts overexpressing S100A1 exhibit enhanced systolic and diastolic performance as a result of improved Ca ²⁺ transient amplitudes due to increased SR Ca ²⁺ loading and subsequent systolic Ca ²⁺ release, coupled with reduced diastolic Ca ²⁺ leak into the SR and enhanced Ca ²⁺ resequestration. At the same time, S100A1 increases mitochondrial high-energy phosphate production and thus coordinates energy supply with increased demand for adenosine 5'-triphosphate (ATP) due to increased Ca ²⁺ turnover in cardiomyocytes. Decreased S100A1 expression in cardiomyocytes is associated with decreased contractile function, confirming the pathophysiological significance of this protein.

In some embodiments, the polynucleotide sequence of the S100A1 cDNA (transgene) of any of the disclosed rAAV vectors has 100% identity to the naturally occurring S100A1 sequence. In some embodiments, the naturally occurring S100A1 sequence is from a human. In some embodiments, the naturally occurring S100A1 sequence is from a dog. In other embodiments, the S100A1 cDNA sequence has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the naturally occurring S100A1 sequence.

In some embodiments, the S100A1 cDNA sequence is codon optimized for expression in human cells. In some embodiments, the S100A1 cDNA (transgene) sequence comprises a sequence that has 100% identity to SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 25. In some embodiments, the rAAV vector comprises a S100A1 transgene that comprises SEQ ID NO: 8, which is a sequence codon optimized for expression in humans. In some embodiments, the S100A1 cDNA sequence is SEQ ID NO: 25, which is a sequence codon optimized for expression in humans and further has a reduced guanine and cytosine (G/C) content. In other embodiments,

- 12 048262 the S100A1 cDNA sequence has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 25.

In other embodiments, the S100A1 cDNA sequence is codon optimized for expression in canine cells. In some embodiments, the S100A1 cDNA (transgene) sequence comprises a sequence that has 100% identity to SEQ ID NO: 1, SEQ ID NO: 21, or SEQ ID NO: 26. In some embodiments, the rAAV vector comprises a S100A1 transgene that comprises SEQ ID NO: 21, which is a codon optimized sequence for expression in dogs. In some embodiments, the S100A1 cDNA sequence is SEQ ID NO: 26, which is a codon optimized sequence for expression in dogs and further has a reduced G/C content. In some embodiments, the rAAV vector comprises a S100A1 cDNA sequence that has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to SEQ ID NO: 1, SEQ ID NO: 21, or SEQ ID NO: 26.

Non-limiting examples of S100A1 cDNA sequences are provided below.

Native S100A1 (homo sapiens)

ATGGGCTCTGAGCTGGAGACGGCGATGGAGACCCTCATCAACGTGTTCCACG

CCCACTCGGGCAAAGAGGGGGACAAGTACAAGCTGAGCAAGAAGGAGCTGAAAGA GCTGCTGCAGACGGAGCTCTGGCTTCCTGGATGCCCAGAAGGATGTGGATGCTGT GGACAAGGTGGAAGGAGCTAGACGAGAATGGAGACGGGGAGGTGGACTTCCAG GAGTATGTGGTGCTTGTGGCTGCTCTCACAGTGGCCTGTAACAATTTCTTCTGGGAG

AACAGTTGA (SEQ ID NO: 5)

Optimized S100A1 (homo sapiens)

ATGGGCAGCGAGCTGGAGACCGCCATGGAGACCCTGATCAACGTGTTCCACG

CCCACAGCGGCAAGGAGGGCGACAAGTACAAGCTGAGCAAGAAGGAGCTGAAGGA

GCTGCTGCAGACCGAGCTGAGCGGCTTCCTGGACGCCCAGAAGGACGTGGACGCCG TGGACAAGGTGATGAAGGAGCTGGACGAGAACGGCGACGGCGAGGTGGACTTCCA GGAGTACGTGGTGCTGGTGGCCGCCCTGACCGTGGCCTGCAACAACTTCTTCTGGGA GAACAGCTGA (SEQ ID NO: 8)

Optimized S100A1 with reduced G/C content (homo sapiens)

ATGGGCTCTGAGCTGGAGACCGCCATGGAGACCCTGATCAATGTGTTCCACG

CCCACTCTGGCAAGGAGGGCGATAAGTACAAGCTGTCTAAGAAGGAGCTGAAGGAG CTGCTGCAGACCGAGCTGTCTGGCTTCCTGGATGCCCAGAAGGATGTGGATGCCGTG GATAAGGTGATGAAGGAGCTGGATGAGAATGGCGATGGCGAGGTGGATTTCCAGGA GTACGTGGTGCTGGTGGCCGCCCTGACCGTGGCCTGCAATAATTTCTTCTGGGAGAA TTCTTGA (SEQ Ш NO: 25)

The alignment of nucleotide sequences between the codon-optimized human S100A1 cDNA sequence and the native human S100A1 cDNA sequence (SEQ ID NOs: 8 and 5, respectively) is shown in Fig. 13.

For reference, some non-limiting examples of animal-derived S100A1 cDNA sequences are described below. A nucleotide sequence alignment between a codon-optimized canine S100A1 cDNA sequence and a codon-optimized human S100A1 cDNA sequence (SEQ ID NOs: 21 and 8, respectively) is shown in Fig. 14. SEQ ID NO: 26, which is a codon-optimized canine S100A1 sequence with reduced G/C content, is set forth below.

- 13 048262

Native S100A1 (canis lupus familiaris) (NCBI reference sequence: XM_005622816.2) ATGGGCTCTGAGCTGGAGACAGCGATGGAGACTCTCATCAATGTGTTCCATG

CCCACTCGGGCAAGGAGGGAAACAAGTACAAGCTGAGCAAGAAGGAGCTAAAGGA GCTGCTGCAGACTGAGCTCTCCGGCTTCCTGGACGCCCAGAAGGATGCGGATGCTGT GGACAAGGTGATGAAAGAGCTAGATGAGAATGGAGATGGGGAGGTGGACTTCCAG GAGTATGTGGTGCTGGTGGCTGCCCTCACAGTGGCCTGTAACAACTTCTTCTGGGAA

AACAGTTGA (SEQ ID NO: 1)

Optimized S100A1 (canis lupus familiaris)

ATGGGCAGCGAGCTGGAGACCGCCATGGAGACCCTGATCAACGTGTTCCACG

CCCACAGCGGCAAGGAGGGCAACAAGTACAAGCTGAGCAAGAAGGAGCTGAAGGA GCTGCTGCAGACCGAGCTGAGCGGCTTCCTGGACGCCCAGAAGGACGCCGACGCCG TGGACAAGGTGATGAAGGAGCTGGACGAGAACGGCGACGGCGAGGTGGACTTCCA GGAGTACGTGGTGCTGGTGGCCGCCCTGACCGTGGCCTGCAACAACTTCTTCTGGGA

GAACAGCTGA (SEQ ID NO: 21)

Optimized S100A1 with reduced G/C content (canis lupus familiaris)

ATGGGCTCTGAGCTGGAGACCGCCATGGAGACCCTGATCAATGTGTTCCACG

CCCACAGCGGCAAGGAGGGCAATAAGTACAAGCTGTCTAAGAAGGAGCTGAAGGA GCTGCTGCAGACCGAGCTGTCTGGCTTCCTGGACGCCCAGAAGGACGCCGACGCCG TGGACAAGGTGATGAAGGAGCTGGACGAGAATGGCGACGGCGAGGTGGACTTCCA

GGAGTACGTGGTGCTGGTGGCCGCCCTGACCGTGGCCTGCAATAATTTCTTCTGGGA

GAATTCTTGA (SEQ ID NO: 26)

Native S100A1 (felis catus) (NCBI reference sequence: XM_003999773.3)

ATGGGCTCAGAGCTGGAGACGGCGATGGAGACTCTCATCAACGTGTTCCACG

CCCACTCGGGCAAGGAGGGAGACAAGTACAAGCTGAGCAAGAAGGAGCTAAAAGA GCTGCTGCAGACCGAGCTCTCTGGCTTCCTGGACGCCCAGAAGGATGCCGACGCTGT GGACAAGGTGATGAAAGAGCTAGACGAGAATGGAGATGGGGAGGTGGACTTCCAA GAGTATGTGGTGCTGGTGGCTGCCCTCACAGTGGCCTGTAACAACTTTTTCTGGGAG AACAGTTGA (SEQ ID NO: 2).

Aspects of the present disclosure provide compositions and methods that involve delivering a transgene encoding an apoptosis inhibitor (e.g., an anti-apoptotic agent). Illustrative examples of apoptosis inhibitors include fink, p35, crmA, Bcl-2, Bcl-XL, Mcl-1, E1B-19K from adenovirus, as well as antagonists of pro-apoptotic agents (e.g., antisense sequences, ribozymes, antibodies, etc.). In some embodiments, the apoptosis inhibitor is a cardiac apoptosis repressor with a caspase recruitment domain (ARC) or a variant thereof. In other embodiments, the apoptosis inhibitor is a cardiac ARC or a variant thereof. In some embodiments, it may be desirable to deliver the S100 family protein and the apoptosis inhibitor separately. In some embodiments, a transgene encoding an S100 family protein is delivered simultaneously or sequentially with one or more small molecule inhibitors of apoptosis. Other putative small molecule inhibitors of apoptosis include c-Myc inhibitors, Bax inhibitors, p53 inhibitors, tBid inhibitors, caspase inhibitors, and inhibitors of pro-apoptotic BCL-2 family members.

cARC is an apoptotic regulatory protein that is expressed almost exclusively in myogenic cells. It contains a caspase recruitment domain (CARD) through which it blocks the activation of several initiator caspases. ARC also blocks caspase-independent events associated with apoptosis. Apoptosis induced by acute ischemia and subsequent ventricular remodeling is considered a mediator of heart failure. Although postischemic heart failure may have multiple causes, recent attention has been directed toward understanding the contribution of apoptosis or programmed cell death. Apoptosis is characterized by the preservation of mitochondrial and sarcolemmal membranes, condensation of nuclear chromatin, and phagocytosis by macrophages or neighboring cells without triggering an inflammatory response. Activation of apoptosis is known to occur through mechanisms involving caspases, a family of cysteine proteases,

- 14 048262 which are synthesized as inactive precursors and are proteolytically cleaved to the active form. ARC is able to block the activation of apoptosis by blocking caspases.

Thus, in some aspects, provided herein are rAAV vectors (or rAAV nucleic acid vectors) comprising polynucleotides that comprise a sequence that is at least 90%, at least 95%, or at least 99.5% identical to any one of SEQ ID NOs: 6-8, 16, 21, 25, and 26. In some embodiments, the polynucleotide sequence differs from the sequence of any one of SEQ ID NOs: 6-8, 16, 21, 25, and 26 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 nucleotides. In some embodiments, the polynucleotide sequence comprises regions of approximately 35, 50, 75, 100, 125, 150, 175, 200, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, or 285 contiguous nucleotides in common with any of SEQ ID NOs: 6-8, 16, 21, 25, and 26. In particular embodiments, any of the disclosed rAAV vectors comprises a polynucleotide that comprises any of SEQ ID NOs: 6-8, 16, 21, 25, and 26.

In some embodiments, the disclosed cARC and S100 transgene sequences comprise 5' or 3' truncations relative to the wild-type sequence or a codon-optimized sequence. In some embodiments, the disclosed transgene sequences comprise 5' or 3' truncations relative to SEQ ID NOs: 1, 3, 5-8, and 15-21. In some embodiments, the transgene comprises a nucleotide sequence that differs from the sequence of any of SEQ ID NOs: 1, 3, 5-8, and 15-21 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 nucleotides. In some embodiments, the transgene comprises regions of approximately 35, 50, 75, 100, 125, 150, 175, 200, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 300, 350, 400, 450, 500, 550, 575, or 600 contiguous nucleotides in common with any of SEQ ID NOs: 1, 3, 5-8, and 15-21.

In some embodiments, the polynucleotide sequence of the cardiac ARC cDNA (or transgene) from the disclosed rAAV vectors has 100% identity to a naturally occurring cARC sequence. In other embodiments, the cARC cDNA sequence has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to a naturally occurring cARC sequence. In some embodiments, the naturally occurring cARC sequence is from a human. In some embodiments, the naturally occurring cARC sequence is from a dog.

In particular embodiments, the cARC cDNA sequence is codon optimized for expression in human cells. In some embodiments, the cARC cDNA sequence (transgene) comprises a sequence that has 100% identity to SEQ ID NO: 6 or SEQ ID NO: 7. In other embodiments, the cARC cDNA sequence has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to SEQ ID NO: 6 or SEQ ID NO: 7.

In other embodiments, the cARC cDNA sequence is codon optimized for expression in canine cells. In some embodiments, the rAAV-based vector comprises a cARC cDNA sequence that has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to SEQ ID NO: 16. In some embodiments, the rAAV-based vector comprises SEQ ID NO: 16.

Non-limiting examples of cARC cDNA sequences are described below.

The native ARC (homo sapiens) cDNA sequence is shown in SEQ ID NO: 18.

- 15 048262

Optimized ARC (homo sapiens)

ATGGGCAACGCCCAGGAGCGGCCCAGCGAGACCATCGACCGGGAGCGGAAG

CGGCTGGTGGAGACCCTGCAGGCCGACAGCGGCCTGCTGCTGGACGCCCTGCTGGC CCGGGGCGTGCTGACCGGCCCCGAGTACGAGGCCCTGGACGCCCTGCCCGACGCCG AGCGGCGGGTGCGGCGGCTGCTGCTGCTGGTGCAGGGCAAGGGCGAGGCCGCCTGC CAGGAGCTGCTGCGGTGCGCCCAGCGGACCGCCGGCGCCCCCGACCCCGCCTGGGA CTGGCAGCACGTGGGCCCCGGCTACCGGGACCGGAGCTACGACCCCCCCTGCCCCG GCCACTGGACCCCCGAGGCCCCCGGCAGCGGCACCACCTGCCCCGGCCTGCCCCGG GCCAGCGACCCCGACGAGGCCGGCGGCCCCGAGGGCAGCGAGGCCGTGCAGAGCG GCACCCCCGAGGAGCCCGAGCCCGAGCTGGAGGCCGAGGCCAGCAAGGAGGCCGA GCCCGAGCCCGAGCCCGAGCCCGAGCTGGAGCCCGAGGCCGAGGCCGAGCCCGAG CCCGAGCTGGAGCCCGAGCCCGACCCCGAGCCCGAGCCCGACTTCGAGGAGCGGGA

CGAGAGCGAGGACAGCTGA (SEQ ID NO: 6)

Optimized ARC (homo sapiens)

ATGGGGAATGCCCAAGAAAGGCCTTCTGAGACTATAGACCGCGAGCGCAAG

AGGCTTGTAGAAACCTTGCAGGCGGACTCTGGTCTCTTGCTGGACGCTCTGCTTGCG CGGGGTGTTCTGACTGGACCGGAGTACGAAGCATTGGATGCCCTTCCTGATGCAGA GAGACGAGTTAGACGCCTGTTGCTTCTTGTGCAAGGCAAGGGTGAAGCCGCCTGTC AAGAGCTCCTGAGGTGTGCTCAACGAACCGCCGGGGGCCCAGATCCGGCATGGGAT TGGCAACATGTGGGGCCCGGCTATCGGGACCGGAGCTACGATCCACCATGCCCGGG TCATTGGACGCCGGAGGCTCCAGGATCTGGTACAACATGCCCAGGACTCCCAAGAG CCAGTGACCCCGATGAAGCTGGAGGCCCCGAGGGCAGTGAAGCCGTACAGAGCGGT ACCCCAGAAGAACCAGAACCGGAGCTGGAGGCTGAAGCTAGTAAAGAGGCGGAAC CTGAACCCGAACCGGAGCCTGAGCTCGAGCCAGAGGCTGAGGCCGAGCCAGAGCCT GAACTCGAACCCGAACCTGATCCAGAACCAGAGCCCGACTTCGAGGAACGGGATGA GTCAGAGGATTCTTGA (SEQ ID NO: 7).

The alignment of nucleotide sequences between the codon-optimized human ARC cDNA sequence and the native human ARC cDNA sequence (SEQ ID NOs: 6 and 18, respectively) is shown in Fig. 12.

For reference, some non-limiting examples of ARC cDNA sequences obtained from animals are provided below. The nucleotide sequence alignment between the codon-optimized canine ARC cDNA sequence and the codon-optimized human ARC cDNA sequence (SEQ ID NOs: 16 and 6, respectively) is shown in Fig. 11.

- 16 048262

Native ARC (canis lupus familiaris) (NCBI reference sequence: NM_001048121.1) ATGCAGGAAGCGCCAGCCGCGCTGCCCACGGAGCCGGGCCCCAGCCCCGTGC

CTGCCTTCCTCGGCAAGCTGTGGGCGCTGGTGGGCGACCCGGGGACCGACCACCTC

ATCCGCTGGAGCCCGAGCGGGACCAGTTTCCTCGTCAGCGACCAGAGCCGCTTCGCC

AAGGAAGTGCTGCCCAGTACTTCAAGCACAGCAACATGGCGAGCTTCGTGCGGGCA

GCTCAACATGTACGGTTTTCGGAAGGTGGTGAGCATCGAGCAGGGCGGCCTGCTCA

GGCCGGAGCGCGACCACGTCGAGTTCCAGCACCCGAGCTTCGTCCGCGGCCGAGAG

CAACTCCTGGAGCGCGTGCGGCGCAAGGTGCCCGCGCTGCGCAGCGACGACGGCCG

CTGGCGCCCCGAGGACCTGGGCCGGCTGCTGGGCGAGGTGCAGGCTTTGCGGGGAG

TGCAGGAGATCACCGAGGCGCGGCTGCGGGAGCTCAGGCAGCAGAACGAGATCTTA

TGGAGGGAGGTGGTGACTCTGCGGCAGAGCCACGTCAGCAGCATCGCGTCATTGG

CAAGCTGATCCAGTGCCTCTTTGGGCCACTTCAGACAGGGTCCAGCGGCGCAGGAG

CTAAGAGAAAGCTGTCTCTGATGCTGGATGAGGGGAGCTCATGCCCAACACCGGCC

AAATTCAACACCTGTCCTTTACCTGGTGCCCTCTTGCAGGATCCCTACTTTATCCAGT

CGCCCCTCCCAGAGACCACCTTGGGCCTCAGCAGCTCTCATAGGACCAGGGGCCCTA

TCATCTCTGACATCCATGAAGACTCTCCCTCCCCTGATGGGACCAGGCTTTCTCCTTC

CAGTGGTGGCAGGAGGGAGAAGGGCCTGGCACTGCTCAAAGAAGAGCCGGCCAGC

CCAGGGGGGGAAGGCGAGGCCGGGCTGGCCCTGGCCCCAAACGAGTGTGACTTCTG

CGTGACAGCCCCCCCCCCACTGTCCGTGGCTGTGGTGCAGGCCATCCTGGAAGGGA

AGGGGAACTTCAGCCCCGAGGGGCCCAGGAATGCCCAACAGCCTGAACCAAGGGGT

CCCAGGGAGGTACCTGACAGGGGGACTCTGGGCCTGGACAGGGGGGCACGAAGCC

CAGAGAATCTGCTGCCTCCCATGCTGCTTCGGGCCCCCCCTGAAAGTGTGGAGCCTG

CAGGGCCCCTGGATGTGCTGGGCCCCAGCCATCAAGGGCGAGAATGGACCCTGATG

GACTTGGACATGGAGCTGTCCCTGATGCAGCCCTTGGGTCCAGAGAGGAGTGAGAC

TGAGCTGGCGGTCAAGGGGTTAAATTCTCCGGGGCCAGGGAAGGACTCCACACTTG

GGGCACCACTCCTGCTCGATGTCCAAGCGGCTTTGGGAGGCCCAGCTCTCAGCCTTC

CTGGAGCTTTAACCATTTACAGCACCCCTGAGAGCCGAGCCAACTACCTAGGCCCAG

GGGCCAATCCCTCCCCCTGA (SEP ID NO: 3)

Optimized ARC (canis lupus familiaris)

ATGGGCAACAGCCAGGAGCGGCCCAGCGAGACCATCGACCGGGAGCGGAAG

CGGCTGGTGGAGACCCTGCAGGCCGACAGCGGCCTGCTGCTGGACGCCCTGCTGGC

CCGGGGCGTGCTGGCCGGCCCCGAGTACGAGGCCCTGGACGCCCTGCCCGACGCCG

AGCGGCGGGTGCGGCGGCTGCTGCTGCTGGTGCAGAGCAAGGGCGAGGCCGCCTGC

CAGGAGCTGCTGCTGTGCGCCCAGCGGACCGCCCGGGCCCCCGACCCCGCCTGGGA

CTGGCAGCACGTGGGCACCGGCTACCGGGAGCGGAGCTGGGACGCCGCCTGCGCCG

GCCACTGGACCCCCGAGGCCCCCGGCAGCAGCACCACCTGCCCCGAGCTGCCCCGG

GCCGCCGACTGCGGCGAGCCCGGCGCCCCCGGCGGCAGCGAGGCCGCCCAGAGCGG

CAGCCTGGAGGAGCCCGACCCCGAGCTGGAGGCCGGCGCCGAGCTGGAGAGCGAG

CCCCAGATGGACCTGGAGCCCGAGCCCGAGGCCGAGCCCGAGCCCGAGCTGGAGCG

GGAGCCCGAGCCCGAGCCCGAGCCCGACCTGGAGGCCGGCGACGAGAGCGAGGAC

AGCTGA (SEQ ID NO: 16)

- 17 048262

Native ARC (felis catus) (NCBI reference sequence: XM_006941587.2)

ATGGGCAATGCGCAGGAGCGGCCCTCAGAGACGATCGATCGCGAGCGGAAA

CGCCTAGTGGAGACGCTGCAGGACGACTCCGGGCTGCTGCTGGATGCACTGCTGGC GCGCGGCGTGCTCACCGGGCCTGAGTATGAGGCGTTGGACGCGCTGCCTGATGCCG AGCGCAGGGTGCGTCGCCTGCTGCTGCTGGTACAAAGCAAGGGCGAGGCCGCCTGC CAGGAGCTGCTGCACTGCGCCCAGCGTACTACGCGCGCCAGACCCGGCCTGGGA CTGGCAGCACGTGGGCACTGGCTACCGGGAACGCAGCTACGACTCTCCATGCCCTG GCCACTGGACGCCTGAGGCACCTGACTTGAGGACCGCTTGCCCCGAAACGCCCAGA GCTTCAGACTGCGACGAGGGCTGGGGTTTCAGGGGGCTCGGAGGCAGTATCCGGAAC CCTCGAGGAACTCGATCCGGAAGTGGAAGCTGAAGTCTCTGAAGGGGCTGAGCCAG AGCCAGAGCCAGAGCCCGACTTTGAGGCGGGTGATGAGTCTGAAGATTCC (SEQ ID NO: 4).

In other aspects, two or more transgenes from any of the disclosed rAAV vectors comprise a transgene encoding a dominant negative form of phospholamban (or dn-PLN). Phospholamban (PLN) is an endogenous inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) pump, which mediates ion reuptake into the sarcoplasmic reticulum (SR) of cardiomyocytes. The dominant negative form is a pseudophosphorylated form that competes for SERCA2a binding with native phospholamban and thereby reduces its inhibitory effect on SERCA2a (see Bish, et al. Hum Gen Ther. 2011; 22(8): 969-977, incorporated herein by reference). Thus, in some embodiments, the second transgene of the disclosed rAAV-based vectors encodes dn-PLN. In some embodiments, the disclosed rAAV-based vectors may comprise any of the disclosed S100A1 transgenes (e.g., a codon-optimized human or canine S100A1 sequence) and a dn-PLN transgene. In some embodiments, the disclosed rAAV-based vectors may encode, and thus deliver to a cell, an S100A1 protein and a dn-PLN protein.

In further aspects, any of the disclosed rAAV vectors may comprise three or more transgenes. In some embodiments, the rAAV vectors comprise three transgenes. In some embodiments, the third transgene encodes a dn-PLN sequence. The three transgenes may include a first transgene encoding canine S100A1 (e.g., a codon-optimized human or canine S100A1 sequence), a second transgene encoding a cARC (e.g., a codon-optimized human or canine cARC sequence), and a third transgene encoding a dn-PLN.

In some embodiments, the disclosed rAAV-based vectors do not encode a protein derived from a dog or cat. In some embodiments, none of the disclosed vectors comprise a native nucleotide sequence encoding S100A1 or ARC of a cat or dog. In some embodiments, the disclosed rAAV-based vectors do not comprise SEQ ID NO: 1 or 2. In some embodiments, the disclosed rAAV-based vectors do not comprise SEQ ID NO: 3 or 4 or any of SEQ ID NO: 1-4.

Recombinant AAV (rAAV)-based vectors.

Aspects of the present disclosure relate to recombinant AAV vectors that can be used for gene therapy of cardiac diseases. As used herein, the term vector can refer to a nucleic acid vector (e.g., a plasmid or a recombinant viral genome), a wild-type AAV genome, or a virus that contains a viral genome. In some embodiments, the term vector can refer to viral particles, such as AAV viral particles.

The wild-type AAV genome is composed of sense or antisense single-stranded deoxyribonucleic acid (ssDNA). The genome contains two inverted terminal repeats (ITRs), one at each end of the DNA strand, and two open reading frames (ORFs), rep and cap, between the ITRs. The Rep ORF contains four overlapping genes encoding the Rep protein, which is essential for the AAV life cycle. The Cap ORF contains overlapping genes encoding the capsid proteins: VP1, VP2, and VP3, which interact together to form the viral capsid. VP1, VP2, and VP3 are translated from a single mRNA transcript that can be spliced in two different ways. Either the longer or shorter intron can be excised, resulting in two mRNA isoforms: ~2.3 kb and ~2.6 kb. The capsid forms a non-enveloped icosahedral T-1 lattice from a supramolecular aggregate of approximately 60 individual capsid protein subunits capable of protecting the AAV genome. The mature AAV capsid consists of VP1, VP2, and VP3 (molecular weights of approximately 87, 73, and 62 kDa, respectively) in a ratio of approximately 1:1:10.

- 18 048262

Recombinant AAV (rAAV) particles may comprise a recombinant nucleic acid vector (hereinafter referred to as an rAAV vector) that may comprise at least: (a) one or more heterologous nucleic acid regions comprising a transgene; and (b) one or more regions comprising sequences that facilitate integration of the heterologous nucleic acid regions (not necessarily with one or more nucleic acid regions comprising a sequence that facilitates expression) into the genome of a subject. In some embodiments, sequences that facilitate integration of regions of heterologous nucleic acid (not necessarily with one or more regions of nucleic acid containing a sequence that facilitates expression) into the genome of a subject are inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking one or more regions of nucleic acid (e.g., regions of heterologous nucleic acid).

In certain embodiments, the ITR sequences flank a nucleic acid region comprising two or more transgenes and a promoter operably linked to the two or more transgenes and a polyadenylation (polyA) signal. In some embodiments, the ITR sequences flank a nucleic acid region consisting of two or more transgenes and a promoter operably linked to the two or more transgenes and a polyA signal.

In some embodiments, the rAAV nucleic acid vector comprises one or more transgenes comprising a sequence encoding a protein or polypeptide of interest operably linked to a promoter, wherein the one or more transgenes are flanked on each side by an ITR sequence. In some embodiments, the nucleic acid vector further comprises a region encoding a Rep protein as described herein, either within the region flanked by the ITR or outside the region, or a nucleic acid operably linked to a promoter. The ITR sequences may be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or may be derived from more than one serotype. In some embodiments, the ITR sequences are derived from serotypes AAV2 or AAV6. In some embodiments, the first serotype provided herein is not serotype AAV2 or AAV8. In some embodiments, the ITRs of the first serotype are derived from AAV3, AAV5, or AAV6. In some embodiments, the ITR sequences are derived from AAV2, AAV3, AAV5, or AAV6. In some embodiments, the ITR sequences are the same serotype as the capsid (e.g., AAV6 ITR sequences and AAV6 capsid, etc.). In some embodiments, the ITR sequences are derived from serotype AAVrh.10.

ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, et al. Proc Natl Acad Sci USA. 1996 Nov 26;93(24): 14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6: 201 © Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young, Jr., Toni Cathomen and Richard Jude Samulski, U.S. Patent Nos. 5,139,941 and 5,962,313, all of which are incorporated herein by reference). In some embodiments, rAAV comprises the backbone of plasmid pTR-UF-11, which is a plasmid containing the AAV2 ITR. This plasmid is commercially available from the American Type Culture Collection (ATCC MBA-331).

In some embodiments, the rAAV nucleic acid vectors of the present disclosure comprise both a cS100A1 transgene and a cARC transgene, for simultaneous delivery and expression of both a cS100A1 protein and a cARC protein in a subject. A transgene encoding an S100 family protein (e.g., cS100A1) can be positioned upstream of a transgene encoding an inhibitor of apoptosis (e.g., cARC) in the rAAV nucleic acid vectors described herein. Alternatively, a transgene encoding an inhibitor of apoptosis can be positioned upstream of a transgene encoding an S100 family protein in the rAAV nucleic acid vectors described herein.

Thus, in some embodiments, the rAAV-based vector comprises one or more regions comprising a sequence that facilitates expression of a transgene (e.g., a heterologous nucleic acid), such as expression control sequences operably linked to the nucleic acid. Many such sequences are known in the art. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, internal ribosome entry site (IRES) termination signals, and polyA signals. Any combination of such control sequences is contemplated herein (e.g., a promoter and a polyA signal). In some embodiments, rAAV-based vectors comprise a promoter that

- 19 048262 which is functionally linked to the coding sequence of transgenes and promotes the expression of transgenes.

A promoter, as used herein, refers to a nucleic acid control region in which the initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter directs the transcription of a nucleic acid sequence that it regulates, thus it is typically located within or near the transcription start site of a gene. A promoter can have a length of, for example, from 100 to 1000 nucleotides. In some embodiments, a promoter is operably linked to a nucleic acid or nucleic acid sequence (nucleotide sequence). A promoter is considered to be operably linked to a nucleic acid sequence that it regulates when the promoter is in the correct functional position and orientation relative to the sequence, such that the promoter regulates (e.g., to control (direct) the initiation of transcription and/or expression) of the sequence.

Promoters that can be used in accordance with the present disclosure can include any promoter that can drive the expression of transgenes in the heart of a subject. In some embodiments, the promoter can be a tissue-specific promoter. A tissue-specific promoter, as used herein, refers to promoters that can only function in a certain type of tissue, such as the heart. Thus, a tissue-specific promoter is not capable of driving the expression of transgenes in other types of tissue. In some embodiments, a promoter that can be used in accordance with the present disclosure is a cardiac tissue-restricted promoter. For example, the promoter is a cardiac tissue-restricted promoter selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT).

Alternatively, the promoter may be, without limitation, a promoter from one of the following genes: the alpha-myosin heavy chain gene, the b-myosin heavy chain gene, the myosin light chain 2v (MLC-2v) gene, the myosin light chain 2a gene, the CARP gene, the cardiac alpha-actin gene, the cardiac m2-muscarinic acetylcholine receptor gene, ANF, cardiac troponin C, cardiac troponin I, cardiac troponin T (cTnT), the cardiac sarcoplasmic reticulum Ca-ATPase gene, and the skeletal alpha-actin gene; or is an artificial cardiac promoter derived from the MLC-2v gene.

In some embodiments of the disclosed rAAV vectors, two or more transgenes are operably controlled by a single promoter. In other embodiments, each of the two or more transgenes is operably controlled by a separate promoter.

In some embodiments, the rAAV vectors of the present disclosure further comprise an internal ribosome entry site (IRES). An IRES is a nucleotide sequence that allows translation to be initiated in the middle of a messenger RNA (mRNA) sequence as part of a larger protein synthesis process. Typically, in eukaryotes, translation can only be initiated at the 5' end of an mRNA molecule because recognition of the 5' cap is required for assembly of the initiation complex. In some embodiments, the IRES is located between transgenes. In such embodiments, proteins encoded by different transgenes are translated individually (i.e., are not translated as a fusion protein).

In some embodiments, the rAAV vectors of the present disclosure further comprise a polyadenylation (PA) signal. Eukaryotic mRNA is typically transcribed as a precursor mRNA. The precursor mRNA is processed to form a mature mRNA, including the process of polyadenylation. The polyadenylation process begins when gene transcription is completed. The 3'-most segment of the newly formed precursor mRNA is first cleaved by a set of proteins. These proteins then synthesize a poly(A) tail at the 3' end of the RNA. The cleavage site typically contains a polyadenylation signal, such as AAUAAA. The poly(A) tail is important for nuclear export, translation, and stability of the mRNA.

In some embodiments, the rAAV-based vectors of the present disclosure comprise, at least sequentially from 5' to 3', a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a promoter operably linked to a first transgene, an IRES operably linked to a second transgene, a polyadenylation signal, and a second AAV inverted terminal repeat (ITR) sequence.

In some embodiments, the rAAV is circular. In some embodiments, the rAAV-based vector is linear. In some embodiments, the rAAV-based vector is single-stranded. In some embodiments, the rAAV-based vector is double-stranded. In some embodiments, the rAAV-based vector is a self-complementary rAAV-based vector. Any rAAV-based vector provided herein can be encapsidated by a viral capsid, such as an AAV6 capsid, or any other serotype (e.g., a serotype of the same serotype as the ITR sequences). In some embodiments, the rAAV-based vector comprises a sequence complementary to any of the sequences of the disclosed rAAV vectors. In some embodiments

- 20 048262 rAAV-based vector comprises a sequence complementary to any one of SEQ ID NOs: 9-12 and 22-23.

Certain rAAV-based vectors of the present disclosure are described below. The vectors illustrated below comprise the linearized plasmid sequences shown as SEQ ID NOs: 9-12 and 22-23. The vectors of the disclosure can comprise nucleotide sequences that have at least 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with the sequences of SEQ ID NOs: 9-12 and 22-23. These sequences are annotated in the following descriptions.

In some embodiments, any of the disclosed rAAV nucleic acid vector sequences comprises truncations at the 5' or 3' end relative to the sequence of any of SEQ ID NOs: 9-12, 22, and 23. In some embodiments, any of the rAAV nucleic acid vectors comprises a nucleotide sequence that differs from the sequence of one of SEQ ID NOs: 9-12, 22, and 23 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more than 18 nucleotides. In some embodiments, any of the disclosed rAAV nucleic acid vectors has a sequence comprising regions of approximately 200, 300, 400, 500, 600, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2300, 2350, 2400, 2450, 2500, 2550, or 2600 consecutive nucleotides in common with any of SEQ ID NOs: 9-12, 22, and 23.

- 21 048262 pAAVsc.cTnT.Opt.hARC_Opt.hS100A1 (homo sapiens) (SEQ ID NO: 9)

Ct2C2C2CtC2CtC2Ctcact2a22CC2CCC222Caaa2CCC222C2tC222C2aCCttt22tC2CCC22CCtca2t2a2C2a2C2 agcgcgcagagagggagfetagccatgctctaggaagatcaattcaattcacgcgtggaattcgcccttaacgggccccctcgaggtc gggataaaagcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccag ccctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggttttaaaatagcaaaactctgagg ccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaaatagcagct cacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaagcctct gccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccgaagctctagaggatccagccttaagg ctagagtacttaatacgactcactataggctagcgccacc/l TGGGGAA TGCCCAA GAAA GGCCTTCTGA GA CT A TA GA CCGCGA GCGCAA GA GGCTTGTA GAAA CCTTGCA GGCGGA CTCTGGTCTCTTG CTGGACGCTCTGCTTGCGCGGGGTGTTCTGACTGGACCGGAGTACGAAGCATTGGAT GCCCTTCCTGATGCAGAGAGACGAGTTAGACGCCTGTTGCTTCTTGTGCAAGGCAAGG GTGAAGCCGCCTGTCAAGAGCTCCTGAGGTGTGCTCAACGAACCGCCGGGGCGCCAG A TCCGGCA TGGGA TTGGCAA CA TGTGGGGCCCGGCTA TCGGGA CCGGA GCTA CGA TC CA CCA TGCCCGGGTCA TTGGA CGCCGGA GGCTCCA GGA TCTGGTA CAA CA TGCCCA G GACTCCCAAGAGCCAGTGACCCCGATGAAGCTGGAGGCCCCGAGGGCAGTGAAGCCG TACAGAGCGGTACCCCAGAAGAACCAGAACCGGAGCTGGAGGCTGAAGCTAGTAAAG AGGCGGAACCTGAACCCGAACCGGAGCCTGAGCTCGAGCCAGAGGCTGAGGCCGAGC CAGAGCCTGAACTCGAACCCGAACCTGATCCAGAACCAGAGCCCGACTTCGAGGAAC GGGA TGA GTCA GA GGA TTCTTGA actagtgcgtaccaggtcccctctccctcccccccccctaacgttactggccgaa gccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccct gtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaag cttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccac gtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagc gtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagt cgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataagcttgccacaaccttgggcca ccATGGGCAGCGAGCTGGAGACCGCCATGGGACCCTGATCAACGTNTTCCACG CCCACAGCGGCAAGGAGGGCGACAAGTACAAGCTGAGCAAGAAGGAGCTGAA GGAGCTGCTGCAGACCGAGCTGAGCGGCTTCCTGGACGCCCAGAAGGACGTN GACGCCGTNGACAAGGTNATGAAGGAGCTGGACGAGAACGGCGACGGCGAGG TNGACTTCCAGGAGTACGTNGTNCTGGTNGCCGCCCTGACCGTNGCCTGCAAC AACTTCTTCTGGGAGAACAGCTGAgcggccgcatcgataccgtcgactagagctcgctgatcagcctcgactgt gccttctagttgccagccMctgttglttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatga tagcaggcgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatgga gttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcct cagtgagcgagcgagcgcgcag

Description:

ITR.

cTnT promoter.

hARC_Opt.

IRES.

hS100A1_Opt.

PolyA BGH signal.

- 22 048262 pAAVsc.cTnT.Opt.hS100A1_Opt.hARC (homo sapiens) (SEQ ID NO: 10)

Ct2C2C2CtC2CtC2Ctcact2a22CC2CCCS22Caaa2CCC2S2C2tC222C2aCCttt22tC2CCC2eCCtca2t2a2Cm2C2 agcgcgcagagagggagfetagccatgctctaggaagatcaattcaattcacgcgtggaattcgcccttaacgggccccctcgaggtc gggataaaagcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccag ccctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgagg ccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaaatagcagct cacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaagcctct gccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccgaagctctagaggatccagccttaagg ctagagtacttaatacgactcactataggctagcgccaccATGGGCAGCGAGCTGGAGACCGCCATGGAG ACCCTGATCAACGTNTTCCACGCCCACAGCGGCAAGGAGGGCGACAAGTACAA GCTGAGCAAGAAGGAGCTGAAGGAGCTGCTGCAGACCGAGCTGAGCGGCTTC CTGGACGCCCAGAAGGACGTNGACGCCGTNGACAAGGTNATGAAGGAGCTGG ACGAGAACGGCGACGGCGAGGTNGACTTCCAGGAGTACGTNGTNCTGGTNGCC GCCCTGACCGTNGCCTGCAACAACTTCTTCTGGGAGAACAGCTGAactagtgcgtacca ggtcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatatt gccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgca aggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaacc ccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtg agttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatggg atctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttc ctttgaaaaacacgatgataagcttgccacaaccttgggccaccA TGGGGAA TGCCCAA GAAA GGCCTTCTGA G ACTATAGACCGCGAGCGCAAGAGGCTTGTAGAAACCTTGCAGGCGGACTCTGGTCTCT TGCTGGACGCTCTGCTTGCGCGGGGTGTTCTGACTGGACCGGAGTACGAAGCATTGG

ATGCCCTTCCTGATGCAGAGAGACGAGTTAGACGCCTGTTGCTTCTTGTGCAAGGCAA GGGTGAAGCCGCCTGTCAAGAGCTCCTGAGGTGTGCTCAACGAACCGCCGGGGCGCC AGATCCGGCATGGGATTGGCAACATGTGGGGCCCGGCTATCGGGACCGGAGCTACGA TCCA CCA TGCCCGGGTCA TTGGA CGCCGGAGGCTCCA GGA TCTGGTA CAA CA TGCCC AGGACTCCCAAGAGCCAGTGACCCCGATGAAGCTGGAGGCCCCGAGGGCAGTGAAGC CGTACAGAGCGGTACCCCAGAAGAACCAGAACCGGAGCTGGAGGCTGAAGCTAGTAA AGAGGCGGAACCTGAACCCGAACCGGAGCCTGAGCTCGAGCCAGAGGCTGAGGCCGA GCCAGGCCTGAACTCGAACCCGAACCTGATCCAGAACCAGAGCCCGACTTCGAGGA A CGGGA TGA GTCA GA GGA TTCTTGH gcggccgcatcgataccgtcgactagagctcgctgatcagcctcgactgtg ccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgag gaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaat agcaggcgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggag ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctc agtgagcgagcgagcgcgcag

Description:

ITR.

cTnT promoter.

hS100A1_Opt.

IRES.

hARC_Opt.

BGH polyA signal.

- 23 048262 pAAVsc.cTnT.hS100A1.hARC_opt (homo sapiens) (SEQ ID NO: 11)

Ct2C2C2CtC2CtCSCtcact2a22CC2CCC222Caaa2CCC2S2C2tC222C2aCCttt22tC2CCC22CCtca2t2a2C2a2C2 agcgcgcagagagggagfetagccatgctctaggaagatcaattcaattcacgcgtggaattcgcccttaacgggccccctcgaggtc gggataaaagcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccag ccctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggttttaaaatagcaaaactctgagg ccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaaatagcagct cacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaagcctct gccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccgaagctctagaggatccagccttaagg ctagagtacttaatacgactcactataggctagcgccaccatgggctctgagctggagacggcgatggagaccctcatcaacgtgttcc acgcccactcgggcaaagagggggacaagtacaagctgagcaagaaggagctgaaagagctgctgcagacggagctctctggc ttcctggatgcccagaaggatgtggatgctgtggacaaggtgatgaaggagctagacgagaatggagacggggaggtggacttc caggagtatgtggtgcttgtggctgctctcacagtggcctgtaacaatttcttctgggagaacagttgaactagtgcgtaccaggtccc ctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtc ttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtct gttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaacccccca cctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttgg atagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctga tctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaa aaacacga_tgata_agcttgccacaaccttgggccacc/l TGGGGAA TGCCCAA GA A A GGCCTTCTGA GA STA TAGACCGCGAGCGCAAGAGGCTTGTAGAAACCTTGCAGGCGGACTCTGGTCTCTTGCT GGACGCTCTGCTTGCGCGGGGTGTTCTGACTGGACCGGAGTACGAAGCATTGGATGC CCTTCCTGA TGCA GA GA GA CGA GTTA GA CGCCTGTTGCTTCTTGTGCAA GGCAA GGGT GAAGCCGCCTGTCAAGAGCTCCTGAGGTGTGCTCAACGAACCGCCGGGGCGCCAGAT CCGGCA TGGGA TTGGCAA CA TGTGGGGCCCGGCTA TCGGGA CCGGA GCTA CGA TCCA CCA TGCCCGGGTCA TTGGA CGCCGGA GGCTCCA GGA TCTGGTA CAA CA TGCCCA GGA CTCCCAA GA GCCA GTGA CCCCGA TGAA GCTGGAGGCCCCGA GGGCA GTGAA GCCGTA CAGAGCGGTACCCCAGAAGAACCAGAACCGGAGCTGGAGGCTGAAGCTAGTAAAGAG GCGGAACCTGAACCCGAACCGGAGCCTGAGCTCGAGCCAGAGGCTGAGGCCGAGCCA GAGCCTGAACTCGAACCCGAACCTGATCCAGAACCAGAGCCCGACTTCGAGGAACGG G4TG)4G'TC4G4G'G4TTC7TG4gcggccgcatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttcta gttgccagccatctgttgtttgcccctcccccgtgccttccttgaOcctggaaggtgccactcccactgtccMcctaataaaatgaggaaatt gcgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtsatggagttggc cactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtga gcgagcgagcgcgcag

Description:

ITR.

cTnT promoter.

hS100A1.

IRES.

hARC_Opt

PolyA BGH signal.

- 24 048262 pAAVsc.cTnT.hARC_Opt.hS100A1 (homo sapiens) (SEQ ID NO: 12)

CtSC2C2CtC2CtC2Ctcact2a22CC2CCC222Caaa2CCC222C2tC22SC2aCCttt22tC2CCC2SCCtca2t2a2C2a2CS agcgcgcagagaggggfetagccatgctctaggaagatcaattcaattcacgcgtggaattcgcccttaacgggccccctcgaggtc gggataaaagcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccag ccctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgagg ccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaaatagcagct cacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaagcctct gccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccgaagctctagaggatccagccttaagg ctagagtacttaatacgactcactataggctagcgccacc/1 TGGGGAA TGCCCAA GAAA GGCCTTCTGA GA CT A TA GA CCGCGA GCGCAA GA GGCTTGTA GAAA CCTTGCA GGCGGA CTCTGGTCTCTTG CTGGACGCTCTGCTTGCGCGGGGTGTTCTGACTGGACCGGAGTACGAAGCATTGGAT GCCCTTCCTGATGCAGAGAGACGAGTTAGACGCCTGTTGCTTCTTGTGCAAGGCAAGG GTGAAGCCGCCTGTCAAGAGCTCCTGAGGTGTGCTCAACGAACCGCCGGGGCGCCAG A TCCGGCA TGGGA TTGGCAA CA TGTGGGGCCCGGCTA TCGGGA CCGGA GCTA CGA TC CA CCA TGCCCGGGTCA TTGGA CGCCGGA GGCTCCA GGA TCTGGTA CAA CA TGCCCA G GACTCCCAAGAGCCAGTGACCCCGATGAAGCTGGAGGCCCCGAGGGCAGTGAAGCCG TACAGAGCGGTACCCCAGAAGAACCAGAACCGGAGCTGGAGGCTGAAGCTAGTAAAG AGGCGGAACCTGAACCCGAACCGGAGCCTGAGCTCGAGCCAGAGGCTGAGGCCGAGC CAGAGCCTGAACTCGAACCCGAACCTGATCCAGAACCAGAGCCCGACTTCGAGGAAC GGGA TGA GTCA GA GGA TTCTTGA actagtgcgtaccaggtcccctctccctcccccccccctaacgttactggccgaa gccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccct gtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaag cttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccac gtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagc gtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagt cgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataagcttgccacaaccttgggcca ccatgggctctgagctggagacggcgatggagaccctcatcaacgtgttccacgcccactcgggcaaagagggggacaagtaca agctgagcaagaaggagctgaaagagctgctgcagacggagctctctggcttcctggatgcccagaaggatgtggatgctgtgga caaggtgatgaaggagctagacgagaatggagacggggaggtggacttccaggagtatgtggtgcttgtggctgctctcacagtg gcctgtaacaatttcttctgggagaacagttgagcggccgcatcgataccgtcgactagagctcgctgatcagcctcgactgtgccttcta gttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaatt gcgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggc cactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtga gcgagcgagcgcgcag

Description:

ITR.

cTnT promoter.

hARC_Opt.

IRES.

hS100A1.

PolyA BGH signal.

- 25 048262 pAAVsc.cTnT.hARC-Opt_IRES_hS100A1-Opt_v2 (homo sapiens) (SEQ ID NO: 22) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgag cgcgcagagagggagtgtagccatgctctaggaagatcaattcaattcacgcgtggaattcgcccttaacgggccccccctcgaggtcgg gataaaagcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagcc ctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgaggcc acacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccactgctgccaaaatagcagctca caagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaagcctctgc cccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccgaagctctctagaggatccagccttaaggcta

CATCGACCGGGAGCGGAAGCGGCTGGTGGAGACCCTGCAGGCCGACAGCGGCCTGCT GCTGGACGCCCTGCTGGCCCGGGGCGTGCTGACCGGCCCCGAGTACGAGGCCCTGGA CGCCCTGCCCGACGCCGAGCGGCGGGTGCGGCGGCTGCTGCTGCTGGTGCAGGGCA A GGGCGA GGCCGCCTGCCA GGA GCTGCTGCGGTGCGCCCA GCGGA CCGCCGGCGCC CCCGACCCCGCCTGGGACTGGCAGCACGTGGGCCCCGGCTACCGGGACCGGAGCTAC GACCCCCCCTGCCCCGGCCACTGGACCCCCGAGGCCCCCGGCAGCGGCACCACCTGC CCCGGCCTGCCCCGGGCCAGCGACCCCGACGAGGCCGGCGGCCCCGAGGGCAGCGA GGCCGTGCAGAGCGGCACCCCCGAGGAGCCCGAGCCCGAGCTGGAGGCCGAGGCCA GCAAGGAGGCCGAGCCCGAGCCCGAGCCCGAGCCCGAGCTGGAGCCCGAGGCCGAG GCCGAGCCCGAGCCCGAGCTGGAGCCCGAGCCCGACCCCGAGCCCGAGCCCGACTTC GAGGAGCGGGACGAGAGCGAGGACAGCTGATGKAC^GCGT^^CCCCTCTCCGT CCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTG TCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACC TGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAAT GCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACA AACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGT GCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCC CAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGC GTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCCATTGTATGGGATCTGA TCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTA GGCCCCCCGAACCACGGGGACGTGGTTTTTCCTTTGAAAAACACGATGATAAGCTTGC CACAACCttgggccaccATGGGCtctGAGCTGGAGACCGCCATGGAGACCCTGATCAAt GTGTTCCACGCCCACtctGGCAAGGAGGGCGAtAAGTACAAGCTGtctAAGAAGGA GCTGAAGGAGCTGCTGCAGACCGAGCTGtctGGCTTCCTGGAtGCCCAGAAGGAt GTGGAtGCCGTGGAtAAGGTGATGAAGGAGCTGGAtGAGAAtGGCGAtGGCGAGG TGGAtTTCCAGGAGTACGTGGTGCTGGTGGCCGCCCTGACCGTGGCCTGCAAtA AtTTCTTCTGGGAAAttctTGAtgagcggccgcAATAAAAGATCTTTATTTTCATTAGATC TGTGTGTTGGTTTTTTGTGTGatcgataccgtcgactacgtagataagtagcatggcgggttaatcattaactacaaggaa cccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggcttt gcccgggcggcctcagtgagcgagcgagcgcgcag

Description: ITR.

hARC-Opt IRES promoter.

hS100A1-Optv2 Synthetic polyA.

- 26 048262 pAAVsc.cTnT.cARC-Opt_IRES_cS100A1-Opt_v2 (amis lupus familiaris) (SEQ ID NO: 23) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgag cgcgcagagagggagtgtagccatgctctaggaagatcaattcaattcacgcgtggaattcgcccttaacgggccccccctcgaggtcgg gataaaagcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagcc ctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgaggcc acacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccactgctgccaaaatagcagctca caagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaagcctctgc cccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccgaagctctctagaggatccagccttaaggcta gagtacttaatacgactcactataggCT AGCgccaccA TGGGCAA CA GCCA GGA GCGGCCCA GCGA GA C CATCGACCGGGAGCGGAAGCGGCTGGTGGAGACCCTGCAGGCCGACAGCGGCCTGCT GCTGGACGCCCTGCTGGCCCGGGGCGTGCTGGCCGGCCCCGAGTACGAGGCCCTGGA CGCCCTGCCCGA CGCCGA GCGGCGGGTGCGGCGGCTGCTGCTGCTGGTGCA GA GCAA GGGCGAGGCCGCCTGCCAGGAGCTGCTGCTGTGCGCCCAGCGGACCGCCCGGGCCC CCGACCCCGCCTGGGACTGGCAGCACGTGGGCACCGGCTACCGGGAGCGGAGCTGG

GACGCCGCCTGCGCCGGCCACTGGACCCCCGAGGCCCCCGGCAGCAGCACCACCTGC CCCGAGCTGCCCCGGGCCGCCGACTGCGGCGAGCCCGGCGCCCCCGGCGGCAGCGA GGCCGCCCAGAGCGGCAGCCTGGAGGAGCCCGACCCCGAGCTGGAGGCCGGCGCCG AGCTGGAGAGCGAGCCCCAGATGGACCTGGAGCCCGAGCCCGAGGCCGAGCCCGAG CCCGAGCTGGAGCGGGAGCCCGAGCCCGAGCCCGAGCCCGACCTGGAGGCCGGCGA CGAGAGCGAGGACAGCTGATGAACta^GCGTAccaggtCCCCTCTCCCTCCCCCCCCCCT AACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTA

TTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGCCCGGAAACCTGGCCCTGTCT TCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGT

TGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCT GTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGG CCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACG TTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACA AGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCT CGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGA ACCACGGGGGACGTGGTTTTTCCTTTGAAAAACACGATGATAAGCTTGCCACAACCttggg ccaccATGGGCtctGAGCTGGAGACCGCCATGGAGACCCTGATCAAtGTGTTCCACG

CCCACAGCGGCAAGGAGGGCAAtAAGTACAAGCTGtctAAGAAGGAGCTGAAGG AGCTGCTGCAGACCGAGCTGtctGGCTTCCTGGACGCCCAGAAGGACGCCGACG CCGTGGACAAGGTGATGAAGGAGCTGGACGAGAAtGGCGACGGCGAGGTGGAC TTCCAGGAGTACGTGGTGCTGGTGGCCGCCCTGACCGTGGCCTGCAAtAAtTTC TTCTGGGAGAAttcttgatgagcggccgcAATAAAAGATCTTTATTTTCATTAGATCTGTGTG TTGGTTTTTTGTGTGatcgataccgtcgactacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtg atggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggc ggcctcagtgagcgagcgagcgcgcag

Description: ITR.

cARC-Opt promoter.

IRES cS100A1-Opt_v2.

Synthetic polyA.

Recombinant AA V particles (rAA V).

- 27 048262

Further provided herein are rAAV virus particles or rAAV preparations comprising such particles. The rAAV particles comprise a viral capsid and an rAAV-based vector as described herein that is encapsulated by the viral capsid. Methods for producing rAAV particles are known in the art and are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158167; and U.S. Patent Publication Nos. US 2007/0015238 and US 2012/0322861, which are incorporated herein by reference; and plasmids and kits available from the ATCC and Cell Biolabs, Inc.). For example, a plasmid containing an rAAV-based vector can be combined with one or more helper plasmids, such as those containing a rep gene (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified region of VP3 as described herein), and transfected into a producer cell line such that the rAAV particle can be packaged and then purified.

The rAAV particles or particles in a rAAV preparation provided herein may be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). As used herein, the rAAV serotype of the rAAV particle corresponds to the serotype of the capsid protein of the recombinant virus. In some embodiments, the rAAV particle is rAAV6 or rAAV9. Non-limiting examples of derivatives and pseudotypes include AAVrh.10, AAVrh.74, AAV2/1, AAV2/5, AAV2/6, AAV2/8, AAV2/9, AAV2-AAV3 hybrid, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShHIO, AAV2(Y->F), AAV8(Y733F), AAV2.15, AAV2.4, AAVM41, AAV6(TM6), and AAVr3.45. Such AAV serotypes and derivatives/pseudotypes, as well as methods for producing such derivatives/pseudotypes, are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A1, Schaffer DV, Samulski RJ). In particular embodiments, the capsid of any of the rAAV particles disclosed herein is of serotype AAVrh.10. In some embodiments, the capsid is of serotype AAV2/6. In some embodiments, the rAAV particle is a pseudotyped rAAV particle that comprises (a) an rAAV-based vector comprising an ITR of one serotype (e.g., AAV2, AAV3) and (b) a capsid comprising capsid proteins derived from another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10). Methods for producing and using pseudotyped rAAV-based vectors are known in the art (see, e.g., Duan et al., J. Virol., 75: 7662-7671, 2001; Halbert et al., J. Virol., 74: 1524-1532, 2000; Zolotukhin et al., Methods, 28: 158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10: 3075-3081, 2001).

rAAV-based hemotherapy for cardiac diseases.

The present disclosure also relates to compositions comprising one or more of the disclosed rAAV particles or preparations. In some embodiments, the rAAV preparation comprises an rAAV particle comprising an rAAV-based vector comprising an ITR of a first serotype (e.g., AAV3, AAV5, AAV6, or AAV9) and a protein capsid encapsulating the rAAV-based vector. In some embodiments, the protein capsid is of a first serotype (e.g., AAV3, AAV5, AAV6, or AAV9). In some embodiments, the preparation has at least four-fold higher transduction efficiency (e.g., in human hepatocellular carcinoma cells such as Huh7) compared to a preparation prepared using an rAAV vector comprising an AAV2 ITR.

As described herein, such compositions may further comprise a pharmaceutical excipient, buffer or diluent, and may be formulated for administration to host cells ex vivo or in situ of an animal, in particular a human or a dog. Such compositions may further optionally comprise a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere or nanoparticle, or may be otherwise prepared for administration to cells, tissues, organs, the body of a human or dog in need thereof. Such compositions may be formulated for use in a variety of therapeutic applications, such as, for example, amelioration, prevention and/or treatment of conditions such as peptide deficiency, polypeptide deficiency, peptide overexpression, polypeptide overexpression, including, for example, conditions that lead to the diseases or disorders described herein.

The rAAV-based vectors, rAAV particles, or compositions comprising rAAV particles of the present disclosure can be used for gene therapy of cardiac diseases in a subject in need thereof (e.g., a human or a dog). Examples of cardiac diseases that can be treated using the methods and compositions of the present disclosure include, but are not limited to, cardiomyopathy and acute ischemia. In some embodiments, the cardiomyopathy is hypertrophic cardiomyopathy or dilated cardiomyopathy. Heart failure caused by cardiomyopathy or other cardiac diseases comprises two components, calcium metabolism disorder and apoptosis. The rAAV-based vectors, particles, and compositions,

- 28 048262 containing rAAV particles can be used to treat such heart failures when administered to a subject in need thereof, such as by intravascular administration into the coronary arteries and/or direct injection into the heart. The rAAV-based vectors, particles and compositions containing rAAV particles direct the simultaneous expression of cS100A1 protein and ARC protein in the subject's cardiomyocytes. S100A1 improves aspects of calcium metabolism, including normalization of sarcoplasmic reticulum calcium transients, which leads to normalization of contractile function. ARC will block apoptosis initiated by mitochondrial and non-mitochondrial mechanisms (e.g., stretch-induced apoptosis), as well as improve mitochondrial function. Thus, the synergistic benefits of the two proteins expressed by the transgenes of the present disclosure may lead to longer lasting therapeutic results by targeting both aspects of cardiomyopathy.

The amino acid sequences of the human S100A1 and ARC proteins are described below.

Human ARC protein:

MGNAQERPSETIDRERKRLVETLQADSGLLLDALLARGVLTGPEYEALDALPDA

ERRVRRLLLLVQGKGEAACQELLRCAQRTAGAPDPAWDWQHVGPGYRDRSYDPPCPG HWTPEAPGSGTTCPGLPRASDPDEAGGPEGSEAVQSGTPEEPEPELEAEASKEAEPEPEP EPELEPEEAEAEPEPELEPEPPDPEPEPDFEERDESEDS (SEQ Ш NO: 13)

Human S100A1 protein:

MGSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLDAQKDVDA

VDKVMKELDENGDGEVDFQEYVVLVAALTVACNNFFWENS (SEQ Ш NO: 14)

In some aspects, the disclosed rAAV-based vectors encode a protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to SEQ ID NO: 13 or 14. In some aspects, the disclosed rAAV-based vectors encode a first protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to SEQ ID NO: 13 and a second protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to SEQ ID NO: 14. In some embodiments, the rAAV-based vector encodes a protein that comprises SEQ ID NO: 13. In some embodiments, the rAAV-based vector encodes a protein that comprises SEQ ID NO: NO: 14. In particular embodiments, the rAAV-based vector encodes a first protein that comprises SEQ ID NO: 13 and a second protein that comprises SEQ ID NO: 14.

In some embodiments, any of the disclosed rAAV vectors encodes a first protein sequence that differs from the sequence of any of SEQ ID NOs: 13 or 14 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 amino acids. In some embodiments, any of the disclosed rAAV vectors encode proteins that are truncated by 1, 2, 3, or more than 3 amino acids at the N- or C-terminus relative to any of SEQ ID NOs: 13 or 14. In some embodiments, any of the disclosed rAAV vectors encode one or more protein sequences that have regions of 50, 75, 90, 93, 100, 125, 175, 200, or 205 amino acids in common with SEQ ID NOs: 13 or 14.

The amino acid sequences of the S100A1 and ARC proteins obtained from dogs are described below.

Dog ARC Protein:

MGNSQERPSETIDRERKRLVETLQADSGLLLDALLARGVLAGPEYEALDALPDA

ERRVRRLLLLVQSKGEAACQELLLCAQRTARAPPDPAWDWQHVGTGYRERSWDAACA GHWTPEAPGSSTTCPELPRAADCGEPGAPGGSEAAQSGSLEEPDPELEAGAELESEPQM DLEPEPEEAEPEPELEREPEPEPEPPDLEAGDESEDS (SEQ Ш NO: 24)

Canine S100A1 protein:

MGSELETAMETLINVFHAHSGKEGNKYKLSKKELKELLQTELSGFLDAQKDADA

VDKVMKELDENGDGEVDFQEYVVLVAALTVACNNFFWENS (SEQ ID NO: 29)

In some aspects, the disclosed rAAV-based vectors encode a protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to SEQ ID NO: 24 or 29. In some aspects, the disclosed rAAV-based vectors encode a first protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to SEQ ID NO: 24 and a second protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to SEQ ID NO: 29. In some embodiments, the rAAV-based vector encodes a protein that comprises SEQ ID NO: 24. In some embodiments, the rAAV-based vector encodes a protein that comprises SEQ ID NO: NO: 29. In particular embodiments,

- 29 048262 implementation vector based on rAAV encodes a first protein that contains SEQ ID NO: 24, and a second protein that contains SEQ ID NO: 29.

In some embodiments, any of the disclosed rAAV-based vectors encodes one or more protein sequences that differ from the sequence of any of SEQ ID NOs: 24 or 29 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 amino acids. In some embodiments, any of the disclosed rAAV vectors encodes one or more protein sequences that are truncated by 1, 2, 3, or more than 3 amino acids at the N- or C-terminus relative to any of SEQ ID NOs: 24 or 29. In some embodiments, any of the disclosed rAAV vectors encodes one or more protein sequences that have regions of approximately 50, 75, 90, 93, 100, 125, 150, 175, or 200 contiguous amino acids in common with SEQ ID NOs: 24 or 29.

Thus, other aspects of the present disclosure relate to administering the rAAV particles of the present disclosure to a subject in need thereof. In some embodiments, the amount of rAAV particles administered to the subject can be in the range of about 106 to about ¹⁰¹⁴ particles/mL, or about 103 to about ¹⁰¹³ particles/mL, or any value in between for either range, such as about ¹⁰⁶ , ¹⁰⁷ , 108, 109, ¹⁰¹⁰ , ¹⁰¹¹ , ¹⁰¹² , ¹⁰¹³ , or ¹⁰¹⁴ particles/mL. In some embodiments, the amount of rAAV particles administered to the subject may be in the range of about 106 to about ¹⁰¹⁴ vector genomes (vg)/mL, or 103 to ¹⁰¹⁵ vg/mL, or any intermediate value of either range, such as about 106, ¹⁰⁷ , ¹⁰⁸ , 109, ¹⁰¹⁰ , ¹⁰¹¹ , ¹⁰¹² , ¹⁰¹³ , or ¹⁰¹⁴ vg/mL. The rAAV particles may be administered as a single dose or divided into two or more administrations as may be necessary to achieve therapy for the particular disease or disorder being treated. In some embodiments, the subject is administered doses in the range of about 0.0001 mL to about 10 mL.

If desired, rAAV particles and rAAV-based vectors can be administered in combination with other agents, such as, for example, proteins or polypeptides or various pharmaceutically active agents, including one or more administrations of therapeutic polypeptides, biologically active fragments or variants thereof. In fact, there are virtually no restrictions on other components that can also be included, provided that the additional agents do not cause significant adverse effects when in contact with target cells or host tissues. The rAAV particles or preparations can thus be delivered together with various other pharmaceutically acceptable agents, as required in a particular case. Such compositions can be purified from host cells or other biological sources, or alternatively can be chemically synthesized as described herein.

Formulations containing pharmaceuticals, excipients and/or carrier solutions are well known to those skilled in the art, as are the development of dosing approaches and treatment regimens for using the specific compositions provided herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, intra-articular, intramuscular administration and formulation.

Typically, these formulations may contain at least about 0.1% of the therapeutic agent (e.g., an rAAV particle or preparation and/or an rAAV vector) or more, although the percentage of active ingredient/ingredients may, of course, vary and may, for convenience, be from about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation. Naturally, the amount of therapeutic agent/agents in each therapeutically useful composition may be formulated so that a suitable dosage is obtained in any given unit dose of the compound. Those skilled in the art will consider factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological factors when preparing such pharmaceutical formulations. Additionally, various dosages and treatment regimens may be desirable.

In certain circumstances, it is desirable to deliver rAAV particles or preparations and/or rAAV vectors as suitably formulated pharmaceutical compositions as provided herein either subcutaneously, intravascularly, intracardially, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection into one or more cells (e.g., cardiomyocytes and/or other cardiac cells), tissues, or organs. In some embodiments, rAAV particles or compositions comprising rAAV particles of the present disclosure are administered intravascularly into the coronary arteries. In other embodiments, the disclosed rAAV particles or compositions are administered by direct injection into the heart of a subject. Direct injection into the heart may involve injection into one or more tissues of the myocardium, the lining of the heart, or the skeletal muscle surrounding the heart, such as with a needle catheter.

Pharmaceutical formulations of compositions suitable for injection include aqueous solutions

- 30 048262 solutions or dispersions. In some embodiments, the composition is sterile and liquid in cases where there is the possibility of easy administration by syringe. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, vegetable oils or other pharmaceutically acceptable carriers, such as those that are generally recognized as safe (GRAS) by the Food and Drug Administration. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by the use of surfactants.

The term carrier refers to a vehicle, adjuvant, excipient or carrier with which the rAAV particle or preparation and/or rAAV vector is administered. Such pharmaceutical carriers may be mixtures of liquids such as water and oils, including petroleum oils such as mineral oil, vegetable oils such as peanut oil, soybean oil and sesame oil, animal oil or synthetic oil. Physiological solutions, aqueous dextrose solutions and glycerol solutions may also be used as liquid carriers.

For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first made isotonic with saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, intravitreal, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used will be known to those skilled in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous fluid or injected into the intended infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the individual being treated. The person responsible for administration always determines the appropriate dose for a particular person. In addition, for administration to humans, preparations must meet standards of sterility, pyrogenicity, and general safety and purity, as required, for example, by the FDA's Office of Biological Standards.

Sterile injectable solutions are prepared by incorporating rAAV particles or preparations, Rep proteins and/or rAAV vectors in the required amount of an appropriate solvent with several other ingredients listed above, as required, followed by filtered sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients in a sterile vehicle containing a basic dispersion medium and the other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying methods, which yield a powder of the active ingredient plus any desired additional component from a previously sterile-filtered solution thereof.

The amount of compositions with rAAV particles or preparations and/or rAAV vectors and the time of administration of such compositions will be within the purview of one skilled in the art having the benefit of the present invention. However, it is likely that the administration of a therapeutically effective amount of the compositions of the present invention can be achieved by a single administration, such as, for example, a single injection of a sufficient amount of infectious particles to provide a therapeutic effect to a patient undergoing such treatment. Alternatively, in some circumstances it may be desirable to provide multiple or sequential administration of compositions with rAAV particles or preparations and/or rAAV vectors, either over a relatively short or relatively long period of time, as can be determined by a physician supervising the administration of such compositions.

The compositions of the present invention may comprise rAAV particles or preparations and/or rAAV vectors, either alone or in a formulation with one or more additional active ingredients, which may be obtained from natural or recombinant sources or chemically synthesized. In some embodiments, the rAAV particles or preparations are administered in combination, either in the same composition or administered as part of the same treatment regimen, with a proteasome inhibitor, such as bortezomib or hydroxyurea.

To treat a disease, as used herein, means to reduce the incidence or severity of at least one sign or symptom of the disease or disorder experienced by a subject. The compositions described above are typically administered to a subject in an effective amount, i.e., an amount capable of providing a desired result. The desired result will depend on the active agent administered. For example, an effective amount of rAAV particles may be an amount of particles that are capable of carrying a heterologous nucleic acid in

- 31 048262 host organ, tissue or cell.

The toxicity and efficacy of the composition used in the present invention can be determined by standard pharmaceutical methods using either cells in culture or experimental animals to determine the LD ₅₀ (the dose lethal to 50% of the population). The dose ratio between toxicity and efficacy is a therapeutic index and can be expressed as the ratio LD ₅₀ /ED ₅₀ . Preferred compositions are those that have greater therapeutic indices. Although compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that minimizes the potential for harm from such side effects. The dosage of the compositions as described herein is generally in a range that includes the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used.

Other aspects of the present disclosure relate to methods and formulations for use in a subject, such as a human or non-human (e.g., a dog) subject, of a host cell in situ in the subject, or a host cell obtained from the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a companion animal. A companion animal, as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock, such as horses, cattle, pigs, sheep, goats, and chickens; and other animals, such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a miniature pig, or mini-pig. In some embodiments, the subject is a human. In some embodiments, the subject is a dog. In some embodiments, the dog is a large breed dog. As used herein, a large breed dog includes canines (e.g., dogs) weighing greater than 55 pounds at any age. Thus, as used herein, a large breed dog may refer to a specific breed of dog that would naturally weigh more than 55 pounds (e.g., Doberman Pinschers, Great Danes, Irish Wolfhounds, etc.), as well as to dogs of any breed (specific or unknown) that may weigh more than 55 pounds for other reasons (e.g., due to obesity).

In some embodiments, the subject has a heart disease or is suspected of having a heart disease that can be treated with gene therapy. In some embodiments, the subject has any stage of heart failure. In some embodiments, the heart failure is caused by cardiomyopathy. In some embodiments, the heart failure is caused by hypertrophic cardiomyopathy or dilated cardiomyopathy (DCM).

The following examples are intended to illustrate certain embodiments of the present disclosure and are not limiting. The entire contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this disclosure are hereby expressly incorporated by reference. In the event of a conflict, the present disclosure, including any definitions herein, will control.

Examples

Example 1: Therapeutic targeting of multiple aspects of heart failure.

In some aspects, the present disclosure provides compositions and methods that are suitable for treating one or more cardiac conditions (e.g., cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, heart failure, heart disease, etc.). In some embodiments, the compositions provided in the disclosure are administered intravascularly into the coronary arteries. In some embodiments, the compositions can be administered to a subject via multiple direct injections into the heart. An AAV construct that can be provided to a subject is depicted in Fig. 1. In certain embodiments, such an AAV construct is encapsidated with a recombinant AAV (e.g., AAVrh. 10 or AAV6) and comprises coding sequences for S100 calcium binding protein A1 (S100A1) and apoptosis repressor with caspase recruitment domain (ARC) to target two separate aspects of one or more cardiac conditions (e.g., cardiomyopathy). Both transgenes from the AAV construct in Fig. 1 are driven by the cardiac TnT promoter and will thus be expressed only in cardiomyocytes.

S100A1 improves aspects of calcium metabolism, including normalization of sarcoplasmic reticulum calcium transients, leading to normalization of contractile function. ARC blocks apoptosis initiated by mitochondrial and non-mitochondrial mechanisms (e.g., stretch-induced apoptosis) and also improves mitochondrial function. These two distinct components of heart failure (impaired calcium metabolism and apoptosis) are considered separately, but never together. Thus, the synergistic benefits of this approach provide therapeutic options that may lead to improved long-term outcomes. By targeting

- 32 048262 on both aspects of cardiomyopathy, the compositions and methods provided in the present invention can be used to treat several cardiac conditions (eg, hypertrophic or dilated cardiomyopathy) and will be useful at any stage of heart failure.

Example 2. Hemotherapy of dilated cardiomyopathy in dogs.

Dilated cardiomyopathy (DCM) is the second most common cause of acquired heart disease in dogs, most commonly affecting large breed dogs such as Doberman Pinschers, Great Danes, and Irish Wolfhounds. In humans, those with the disease have surgical options such as heart transplants and left ventricular assist devices. However, in veterinary medicine, the only therapeutic option is drug treatment of the signs associated with heart failure. The prognosis for an affected dog depends on the stage of the disease and the breed. For example, most Doberman Pinschers live less than 6 months after developing congestive heart failure (CHF). In contrast, other breeds such as Cocker Spaniels tend to survive longer. As the heart disease progresses, the channels that regulate calcium movement in heart cells become dysfunctional, disrupting the calcium cycling pattern, further disrupting the regulation of contraction and relaxation of the heart. Notably, calcium transport abnormalities have been found in dogs with DCM as well as in heart failure secondary to many different etiologies.

Gene transfer strategies have been developed to normalize calcium cycling abnormalities, improving the health of diseased hearts in small and large animal models with various forms of heart disease. In fact, human clinical trials of this therapeutic approach to cardiomyopathy are already underway, and preliminary results are encouraging. A pilot study is evaluating the efficacy of delivering a gene designed to normalize calcium metabolism in Doberman Pinschers with DCM and CHF. Doberman Pinschers are being used because DCM is common in this breed, and the disease tends to progress rapidly and uniformly in this breed once CHF develops. New therapies to combat DCM will have a significant impact on all dog breeds predisposed to this idiopathic disease, including Dobermans, Boxers, Great Danes, German Shepherds, Golden Retrievers, etc. Notably, previous studies of myocardial protein levels in lesions in dogs with the most common forms of natural heart disease (dogs with degenerative valve disease and DCM) showed that several protein levels (including S100A1) were abnormal (S100A1 was decreased).

These data suggest that gene delivery targeting S100A1 may effectively treat DCM as well as myocardial failure secondary to degenerative valve disease. Furthermore, apoptosis (programmed cell death) is more common in diseased myocardium and ARC is a potent and versatile inhibitor of apoptosis. Currently, the standard of care in veterinary heart failure is medical treatment of fluid overload and hyperemia. Gene delivery methods targeting abnormal myocardial regulatory molecules offer a mechanistic target that may allow the veterinarian to specifically treat the myocardial pathological process from the outset. Additionally, the cost associated with current vector manufacturing technologies and intramyocardial vector delivery makes the cost of this therapy affordable for many owners, with costs expected to decrease over time.

A minimally invasive gene transfer approach using AAV 2/6 resulted in transduction of >75% of myocardial cells in healthy dogs (see Bish LT, Sleeper MM, Brainard B, et al. Percutaneous transendocardial delivery of self-complementary adeno-associated virus 6 achieves global cardiac gene transfer in canines. Mol. Ther. 16, 1953-9 (2008)). Six healthy mongrel dogs were treated with an AAV2 or AAV6-based vector encoding a dominant-negative form of phospholamban (dn-PLN) (a pseudophosphorylated form that competes with native phospholamban, thereby reducing its inhibitory effect on SERCA2a) (n=4) or AAV2/6 dn-PLN and S100A1 (n=2). All dogs remained healthy with normal cardiovascular function for 2 years after treatment, indicating that the therapy did not cause myocarditis or significantly alter cardiac function, thus supporting the safety of this therapeutic approach. Cardiac function can be measured by ejection fraction or any other method known in the art.

In fact, over 40 normal and diseased dogs have been injected (see below) and the results to date indicate that the injectable route is well tolerated. Additionally, 20 random dogs at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania were sampled for rAAV2/6 antibodies and titers were found to be within the acceptable treatment range in 19 of the 20 dogs, indicating that pre-existing immune responses should exclude a significant proportion of candidates for therapy. To determine whether this therapeutic approach was effective in treating DCM, Portuguese Water Dogs with a severe form of rapidly progressive juvenile DCM were then treated. Notably, dogs injected with AAV2/6dn-PLN showed a marked reduction in phosphorylated PLN, supporting the potential

- 33 048262 the potential of this approach to normalize calcium cycling in this disease model. Furthermore, gene delivery with a vector containing both dn-PLN and S100A1 delayed the progression of CHF secondary to DCM to a greater extent than delivery of a vector containing dn-PLN alone. The combined vector delayed the onset of CHF by an average of 4 weeks compared to dn-PLN alone. For this reason, the combined vector approach is being used in a pilot study to determine whether gene therapy is effective in prolonging the survival of Dobermans with DCM and adult-onset CHF.

The study has a blinded, placebo-controlled design. Based on the last 12 treated Doberman Pinschers with DCM and CHF, the mean survival was 148 days (SD 160 days). Using a power of 0.8, an alpha (2-sided) of 0.05, and a case-to-control ratio of 1, a sample size of 13 dogs in each group was required to detect a difference in six-month survival. This estimate was determined using a parametric sample size test. Twenty-six Doberman Pinschers with DCM and controlled CHF were included in the study. To be included in the study, a dog had to have a circulating neutralizing antibody titer to AAV2/6 less than 1:20 and no non-cardiac disease. Additionally, dogs with concomitant congenital heart disease or evidence of primary mitral valve disease were excluded. At baseline (time of inclusion), antibody titer, complete blood count, and biochemistry were used for screening purposes. Dogs underwent a 3-minute electrocardiogram (ECG) and a full echocardiogram (ECHO), and owners completed a pre-validated quality of life questionnaire. ECG was assessed for interval duration and arrhythmia. ECHO included 2D, M-mode, and Doppler (including tissue Doppler). Chest radiographs were used until recovery (dogs were clinically compensated with a history of chronic heart failure).

Dogs meeting enrollment requirements were randomly assigned to receive either placebo (cardiac injection with saline) or gene therapy (cardiac injection with AAV2/6-ARC-s100a1). Standard medical treatment for DCM and congestive heart failure was continued throughout the study in all dogs (pimobendan, angiotensin inhibitor, and diuretic therapy). Saline, instead of empty capsid, was used as sham therapy so that control dogs could undergo gene delivery if the treatment group demonstrated significant improvement compared with the placebo group. ECG, echo, quality of life questionnaire, and laboratory tests were repeated at 2, 4, 6, 9, and 12 months post-therapy. Statistical analyses were performed at two-month intervals.

Figures 2 and 3 depict diastolic (relaxation) and systolic (contraction) data, respectively, in a treated dog with muscular dystrophy. The outlines of the endocardium and epicardium are visible in each figure. The data indicate stable or slightly improved function after treatment for several weeks, as shown in Table 1. Table 1 below shows the results of left ventricular mass (LVM [g]), end-diastolic volume (EDV [mL]), end-systolic volume (ESV [mL]), stroke volume (SV [mL]), ejection fraction (EF [%]), and minute volume (MV [L/min]) from data obtained at Time 1 (before treatment) and Time 2 (after treatment).

Table 1

Getting data MLZ [g] KDO [ml] KSO [ml] U O [ml] FV [%] MO [l/min] Time 2 91,395035 54,22289 24,595001 29,627889 54,640926 3,940509 Time 1 87,251524 57,471229 25,660014 31,811215 55,351548 3,117499

Example 3. Evaluation of dystrophy phenotypes after vector administration to mice and dogs.

Delivery of AAV genes to the heart from the self-complementary S100A1/ARC vector was evaluated in mouse and dog models of Duchenne muscular dystrophy (dystrophin deficiency). Previously, AAV8 (including several variants), AAV9, and AAVrh.10 serotypes were compared for their ability to infect dog hearts, with AAVrh.10 proving to be the most effective. For this reason, AAVrh. 10 was used for all experiments described in this example.

DBA/2J-derived Mdx (dystrophin-deficient) mice (D2.mdx) were injected at 4 weeks of age with a recombinant AAVrh.10-S100A1/ARC vector (hereafter referred to as therapeutic AAV) and sacrificed at 24 weeks. D2.mdx mice recapitulate several human characteristics of Duchenne muscular dystrophy, such as decreased leg muscle mass, myofiber atrophy, increased fibrosis and inflammation, and muscle weakness. Over this 24-week period, mice injected with therapeutic AAV had better preservation of ejection fraction, strain development, and cardiac output compared with sham-injected mice (see Fig. 4), as assessed by cardiac MRI. Protein analysis (Western blot) confirmed that levels of both S100A1 and

- 34 048262

ARCs were increased in treated tissues compared to controls (sham injection) (see Fig. 5). In addition, cardiac histology showed that treated hearts exhibited significantly less pathology compared to control hearts (see Fig. 6).

Two dogs with GRMD (dystrophin deficiency), a canine model for human Duchenne muscular dystrophy, were injected with a therapeutic vector at the time of the first decline in their cardiac ejection fraction via catheter delivery into the coronary arteries. Cardiac ejection fractions are a marker for the onset of cardiomyopathy. Earlier canine history findings indicated that once ejection fraction begins to decline, it continues to decline progressively over the next year (Figure 18). Dogs typically do not survive beyond 8-12 months after ejection fraction begins to decline steadily.

As shown in Fig. 7 and 8, both subjects showed improvement in ejection fraction and other cardiac parameters several months after treatment with AAVrh.10-S100A1/ARC, as measured by cardiac MRI and confirmed by echo measurements. Almost 12 months after treatment, the first subject showed a sustained ejection fraction within the normal range. Similarly, almost 7 months after treatment, the second subject showed a sustained normal ejection fraction.

Not only was cardiac function improved, but there was also a consistent improvement in exercise capacity in the dogs, as assessed qualitatively by filming the subjects during exercise. Consistent with this improved exercise capacity, MRI measurements of individual limbs showed that skeletal muscle mass was either increased or unchanged following AAV treatment (Figures 9A-9C). Additionally, circulating creatine kinase (CK) levels in skeletal muscle were reduced following treatment (Figure 10), indicating a reduction in ongoing muscle damage.

Example 4. Treatment of dogs with DCM.

Doberman Pinschers have the highest incidence of DCM of any dog breed. The genetic basis for this is known only for a subpopulation of dogs. This provides a large animal model of DCM and heart failure without any other genetic complications. Only dogs with late-stage heart failure were treated with the same AAV.S100A1.ARC vector used for dogs with GRMD. The goal of treatment was to both improve cardiac function and prolong survival.

To date, two Doberman Pinschers have been treated with AAVrh.10-S100A1/ARC via coronary artery catheter placement, with both dogs experiencing heart failure during treatment. Both dogs showed rapid improvement following treatment. Each dog was evaluated with two sets of echocardiography following treatment to assess structural and functional cardiac parameters including, but not limited to, ventricular volumes, wall thickness, chamber diameters in diastole and systole, and fractional shortening and ejection fraction.

The first dog was treated with an ejection fraction of only 10% and was therefore close to death at the time of treatment. Within 24 hours of treatment, the ejection fraction had improved to 25%. At the dog’s initial follow-up visit 4 months after treatment, the ejection fraction remained stable at 26%. At the second follow-up 6 months later, its ejection fraction was 32%. The dog died of congestive heart failure at 8 months of age. Thus, the treatment appeared to prolong life, but cardiac function was already too compromised at the time of treatment for long-term survival.

The second Doberman Pinscher treated had an ejection fraction of 32% prior to treatment, a fraction that is low but not immediately life-threatening. The dog's ejection fraction improved to 49% within 24 hours of treatment, which is within the normal range. At follow-up 4 months after treatment, the dog's ejection fraction was 52%, and at 8 months, the ejection fraction was 50%. Before returning for an annual check-up, the dog was diagnosed with lung cancer and died two months later. Its cause of death was not related to heart failure.

Based on the results obtained, treatment with AAVrh.10-S100A1/ARC can restore normal cardiac function in dogs.

Example 5. Evaluation of vectors containing human cDNA sequences.

Plasmids containing human-derived ARC (hARC) and S100A1 (hS100A1) cDNA sequences were constructed for evaluation in mice. Native human cARC and S100A1 genes were codon optimized for expression in human cells.

These plasmids contain the nucleotide sequences of SEQ ID NOS: 9-12. Each of these plasmids contains the cARC and S100A1 cDNA sequences operably controlled by the cTnT promoter, as well as an IRES between these two sequences. All four plasmids contain a codon-optimized human cARC (hARC) sequence. The plasmids shown in SEQ ID NOS: 9 and 10 contain a codon-optimized hS100A1 sequence; and the plasmids shown in SEQ ID NOS: 11 and 12 additionally contain the wild-type hS100A1 sequence.

Each of these four plasmids was cloned into the self-complementary vector AAVrh.10 and then

- 35 048262 were encapsidated into a rAAV particle. rAAVrh.10 particles containing these vectors were injected into mice.

The expression level of ARC and S100A1 in mice was assessed. Dystrophic phenotypes and cardiac function, including cardiac ejection fraction, were monitored and assessed.

Example 6. Long-term studies of mice and dogs.

Studies of mice with dystrophy.

AAV-based delivery of S100A1 and ARC genes to the heart results in preservation of cardiac function. In addition to the previously presented mouse studies (Figs. 4–6), two types of long-term studies were performed, the results of which are summarized in Figs. 15–17. Figs. 15 and 16 show data from dystrophin-deficient mice with severe fibrosis, the DBA/2J mouse (also referred to as D2), thus creating what is called the D2.mdx mouse. These mice were treated at 1 month of age with AAV containing a dual transgene cassette (S100A1 and ARC) functionally controlled by the cardiac troponin T (cTnT) promoter. Mice were kept in a sedentary environment until 10 months of age, at which time their cardiac fitness was assessed.

Superiority of AAV-based S100A1 and ARC gene delivery to the heart compared to each transgene alone.

In parallel with these studies, a survival study was also conducted in D2.mdx mice, which have a transgenic rescue of skeletal muscle only by expressing utrophin under the control of the skeletal muscle alpha-actin promoter (see Rafael JA, et al. Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice. Nat Genet. 19: 79-82, 1998), and which are designated D2.mdx.sk_utrophin mice. The goal was to generate a mouse that would have pure dilated cardiomyopathy, leading to heart failure and death essentially without any loading of the pulmonary or skeletal muscles. This allowed the mice to be exercised by housing them individually with running wheels. Exercise places additional load and stress on the heart, leading to an accelerated development of cardiomyopathy and heart failure. To better model the clinical situation in which individuals would not receive treatment until the onset of measurable cardiac disease, mice were treated at 6 months of age, when fibrosis is present and cardiac function abnormalities begin to emerge.

To assess the potential superiority of delivering S100A1+ARC together in the same construct versus delivering ARC or S100A1 alone, either no transgene (control) was delivered or an rAAV vector containing the same cardiac promoter as in the dual transgene construct was delivered, driving either the same C100A1 or ARC cDNA used in the dual cassette, but alone rather than in combination. Thus, the ability of either ARC alone, S100A1 alone, or the combination of S100A1 and ARC to extend survival in this heart failure model was compared.

As shown in Fig. 17, either transgene alone was able to significantly extend lifespan, ARC by 4 months and S100A1 by 5 months. However, the combination of transgenes, S100A1 and ARC, extended lifespan by 10 months, to 20 months of age. Wild-type D2 mice have a lifespan of only about 23 months, so this represents an extremely potent cardiac rescue. The combination of transgenes in the dual rAAV construct provides synergistic efficacy. Because vector administration extended lifespan of D2 mice to approximately that of wild-type mice and extended lifespan relative to vectors with only one transgene by 4-5 months, these results represent a statistically significant improvement relative to vector alone.

Studies of dogs with dystrophy.

A study was then conducted to extend the mouse treatment results to dogs with GRMD. The study, conducted in dogs with GRMD (four dogs to date), involves treatment with AAVrh.10-S100A1/ARC (the dog transgene sequence) when cardiac function, as assessed by ejection fraction, falls below levels characteristic of the natural history of an untreated dog. Frequent echocardiography and/or MRI were used to assess structural and functional cardiac parameters to monitor the dogs' cardiac status. The first dog treated (WnM3/Calvin) was followed for over 2 years, during which time its cardiac function improved and remained stable. This dog died of complications related to aspiration pneumonia at 34 months of age, but its cardiac function remained stable and within normal limits throughout this age. All other three dogs improved following initial treatment and have demonstrated stable cardiac function for over a year. These data are shown in Fig. 18. Calvin's death from pneumonia allowed us to evaluate his cardiac histology, shown in Fig. 19. Notably, the histology of the left ventricle (Fig. 19, far right column of histology sections) is virtually indistinguishable from that of a normal dog (Fig. 19, far left column of panels). In contrast, without treatment, the histology of the left ventricle is severely fibrotic at 8 months of age (one month after WnM3 treatment) (Fig. 19, second column of panels from the left) and even more fibrotic at two years of age (middle panels). This demonstrates nearly complete cardiac salvage in a large animal model of DCM that progresses to heart failure and death in

- 36 048262 aged one to two years. In this population, there has never been an untreated dog that survived beyond 30 months of age (Fig. 18), and the typical cause of death is heart failure.

Example 7. Immune response studies.

Four miniature pigs weighing 45-70 kg were treated with the rAAV vectors of this disclosure. Two pigs received a high dose of ^2x1014 genome copies, and the other two pigs received a lower dose of ^2x1013 genome copies. In each case, two-thirds of the virus was delivered to the left side of the heart and one-third of the dose was delivered to the right side. Pigs were screened for antibodies against AAVrh10 prior to study entry. Blood for this purpose was provided by Synchrony.

Blood was collected in a red-top tube, allowed to clot for approximately 30 minutes, and centrifuged at 1000xG for 15 minutes, and serum was aliquoted into 200 µl and frozen prior to screening. Serum was used for pre-existing antibody analysis.

Immunosuppression regimen.

Beginning on day 1 before gene transfer, subjects receive 1 mg/kg glucocorticoid (or prednisone equivalent) daily for 60 days after infusion. Beginning 60 days after gene transfer, a tapering dose of glucocorticoids is administered and liver enzymes are monitored for immune response. If an immune response occurs, the glucocorticoid dose and regimen are repeated at the discretion of the physician.

Throughout the administration of glucocorticoids, prophylactic antibiotics are administered as a precaution.

Delivery of rAAV particles to the pig heart.

The introducer is placed into the carotid or femoral artery.

A pigtail angiocatheter is advanced into the left ventricle to perform coronary angiography to identify the coronary vessels using digital fluoroscopy.

Coronary catheters (each) are flushed with heparinized saline containing 1 ^cm3 of the subject's blood and inserted into the aortic root and the left and right coronary orifices (Judkins size R or L depending on the size of the animal).

Adenosine is administered intravenously at a constant infusion rate (1 mg/kg/min). The vector is delivered into the coronary bed after 15 seconds of constant infusion of adenosine. The vector is administered over 510 seconds, and the constant infusion rate is continued for 30 seconds after completion of vector delivery.

2/3 of the vector is introduced into the left coronary artery and 1/3 of the vector is introduced into the right coronary artery.

Pig subjects were monitored with weekly blood draws (serum chemistry, complete blood count, AAVrh10 antibodies (performed by sponsor), ELISPOT response) for 2 months after AAV delivery. Two months after delivery, pigs were sacrificed and hearts and other tissues (see list below) were obtained (samples were taken for formalin fixation and fresh frozen) and tissues were available.

Necessary blood samples:

Serum chemistry - 1 test tube with a red cap.

Complete Blood Count Panel - 1 EDTA tube with purple cap.

AAVrh10 ELISA assay - 1 red cap tube.

ELISPOT at least 1 tube of 10 ml with EDTA with purple cap.

Post-mortem procedures.

Special procedures.

- 37 048262

Collection of fresh frozen tissues and formalin-fixed, paraffin-embedded tissues.

Muscle/Organ Reduction Left/Right Anterior tibialis muscle R./L TA Both Long extensor of the fingers R/LEDL Both Soleus muscle R/LSOL Both Biceps R/LBIC Both Triceps R/LTRI Both Heart HRT - Light LUNG - Kidney KIDNEY - Liver LIV -

Spleen SPLN - Sex gland GND - Pancreas PANC - Popliteal lymph node POPLN - Inguinal lymph node INGLN - Peritoneal lymph node MESLN - Femoral artery R/L FEM ART Both Sural artery R/LSUR ART Both Bladder BLADDER - Diaphragm DIA - Stomach STMCH - The brain BRAIN - Spinal cord SPN CORD - Nerve NERVE - Sural nerve SUR L/R Portal vein Portal Vein - Aorta Aorta - Inferior vena cava IVC - Hepatic portal vein HPV -

Example 8. Recombinant AAV-based vectors with increased packaging efficiency and high titers.

Recombinant AAV (rAAV) vectors were developed that (1) reduced the guanosine (G) and cytosine (C) content of the S100A1 transgene and (2) contained a different, smaller polyA signal than previously developed vectors (including those shown in SEQ ID NOS: 9-12). The S100A1 transgenes with reduced G/C content, which were previously codon optimized for expression in humans or dogs, are shown in SEQ ID NOS: 25 (human) and 26 (dog). The cARC transgenes, which were unchanged from previous iterations and were previously codon optimized for expression in humans or dogs, are shown in SEQ ID NOS: 6 (human), 7 (human), and 16 (dog). Another, smaller polyA signal used in the newly designed vectors is shown in SEQ ID NO: 28. The newly designed complete rAAV-based vectors are shown in SEQ ID NO: 22 (human) and 23 (dog).

- 38 048262

The newly developed rAAV vectors were optimized to improve packaging efficiency, resulting in higher vector titers and thus lower manufacturing costs. To assess whether the newly developed rAAV vectors exhibited these properties relative to similar rAAV vectors previously developed by the inventors, a blinded study was conducted in which DNA for a newly developed human rAAV vector with reduced G/C content in the S100A1 transgene and a smaller polyA signal (SEQ ID NO: 22) and a previously developed human rAAV vector (SEQ ID NO: 9) was provided to a third-party manufacturing laboratory. The third-party manufacturing laboratory was blinded to the differences that existed between the DNA constructs provided by the inventors. The manufacturing volume for each vector can be seen in Table 2.

Table 2

Vector name SEQ ID NO Titer (genome content/ml) Volume and aliquot (ml) Total genome content AAVrh.10.cTnT.cARC- Opt lRES cS 100 Al -Opt # 1 9 ¹³ 6.8 ml, 34 tubes, 34x200 µl 6.80x10 ¹³ AAVrh.10.cTnT.cARC- OptlREScS 100A1 -Opt_#2 22 2.70χ10 ¹³ 18 ml, 6 tubes, 6x3 ml 4.86x10 ¹⁴

The previously designed construct (SEQ ID NO: 9) exhibited a titer of 1.00 x 10 ¹³ genome copies per ml, similar to previous studies. Notably, however, the newly developed vector (SEQ ID NO: 22) showed a 2.7-fold improvement in titer and exhibited a titer of 2.7 x 10 ¹³ genome copies per ml. Furthermore, the purity of the rAAV-based vectors was unchanged (Fig. 20). Thus, the newly developed human rAAV-based vector with reduced G/C content in the S100A1 transgene and less polyA signal (SEQ ID NO: 22) shows a 23-fold improvement in packaging efficiency and titer compared to the previously developed human rAAV-based vector (SEQ ID NO: 9). Improving packaging efficiency and titer in rAAV production is important because such improvements significantly reduce the overall cost of rAAV production.

In some embodiments, a newly developed human rAAV vector with reduced G/C content in the S100A1 transgene and a reduced polyA signal (SEQ ID NO: 22) exhibits a 2- to 3-fold improvement in packaging efficiency and titer compared to a previously developed human rAAV vector (SEQ ID NO: 9), and no change in the functionality of the rAAV vector is observed.

Equivalents.

Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of many other means and/or structures for performing the function and/or obtaining the results and/or one or more advantages proposed herein, and each of such variations and/or modifications is considered to be within the scope of the embodiments of the invention proposed herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations herein are for descriptive purposes and that the actual parameters, dimensions, materials and/or configurations will depend on the particular application or applications for which the inventive concept is employed. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention proposed herein. It is therefore to be understood that these embodiments are presented by way of example only and that, within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The plurality of embodiments of the present disclosure pertain to each individual feature, system, article, material, kit, and/or method provided herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods do not conflict with each other, is included within the scope of the invention of the present disclosure.

All definitions as defined and used herein are to be understood as taking precedence over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of defined terms.

- 39 048262

All references, patents and patent applications cited herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may cover the entire document.

The indefinite articles a and an, as used herein in the description and in the claims, unless expressly stated otherwise, shall be understood to mean at least one.

The phrase and/or, as used herein in the description and in the claims, is to be understood to mean either or both of the elements so combined, i.e., elements that are in some instances jointly present and in other instances separately present. Multiple elements listed with and/or are to be construed the same, i.e., one or more elements so combined. Other elements other than the elements specifically identified by the and/or clause may not necessarily be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, reference to A and/or B, when used in expressions with non-limiting wording such as comprising, may refer, in one embodiment, to only A (not necessarily including elements other than B), in another embodiment, to only B (not necessarily including elements other than A), in yet another embodiment, to both A and B (not necessarily including other elements), etc.

As used herein in the description and claims, or shall be understood to have the same meaning as and/or as defined above. For example, when separating elements in a list, or or and/or shall be interpreted as inclusive, i.e. including at least one, but also including more than one element of the series or list, and optionally additional unspecified elements. Only terms that clearly indicate the contrary, such as only one of or exactly one of, or when used in a claim, consisting of, shall refer to the inclusion of exactly one element of the series or list of elements. In general, the term or, as used herein, shall be interpreted as meaning exclusive alternatives (i.e., one or the other, but not both) only when preceded by exclusivity terms, such as any, one of, only one of, or exactly one of. Consisting essentially of, when used in a claim, has its ordinary meaning as used in the field of patent law.

As used herein in the specification and claims, the phrase "at least one" referring to a list of one or more elements shall be understood to mean at least one element from any one or more selected elements in the list of elements, but not necessarily including at least one of any elements specifically listed in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether or not related to those specifically identified elements. Thus, as a non-limiting example, at least one of A and B (or, equivalently, at least one of A or B, or, equivalently, at least one of A and/or B) may refer, in one embodiment, to at least one, optionally including more than one, of A, without the presence of B (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one, B, without the presence of A (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one, A, and at least one, optionally including more than one, B (optionally including other elements); etc.

It should also be understood that, unless expressly stated otherwise, in any methods disclosed herein that involve more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions are listed.

In the claims, as well as in the description above, all transition phrases such as comprising, including, carrying, having, consisting, comprising, including, consisting of, and the like are to be understood as open-ended, i.e., including as non-limiting examples. Only the transition phrases consisting of and consisting essentially of shall be closed or semi-closed transition phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described herein using an open transition phrase (e.g., comprising) are also contemplated in alternative embodiments as consisting of and consisting essentially of the feature described in the open transition phrase. For example, if a disclosure describes a composition comprising A and B, the disclosure also contemplates alternative embodiments a composition consisting of A and B and a composition consisting essentially of A and B.

-

Claims (44)

CLAUSE OF THE INVENTION 1. A recombinant adeno-associated virus (rAAV) nucleic acid vector for delivering two or more transgenes to the heart of a subject, wherein said vector comprises sequentially from 5' to 3' a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, two or more transgenes and a promoter operably linked to the two or more transgenes, a polyadenylation (polyA) signal and a second AAV ITR sequence, wherein the two or more transgenes encode an S100 family protein and an apoptosis inhibitor, respectively, and wherein the transgene encoding the S100 family protein comprises a sequence that is at least 95% identical to any of the nucleotide sequences of SEQ ID NOs: 25 and 26, and wherein the apoptosis inhibitor is a cardiac repressor of apoptosis with a caspase recruitment domain (ARC) or a variant thereof. 2. The rAAV nucleic acid vector of claim 1, wherein the transgene encoding the S100 family protein comprises any of the nucleotide sequences of SEQ ID NO: 25 and 26. 3. The rAAV nucleic acid vector of claim 1 or 2, wherein the polyA signal comprises a nucleotide sequence that has at least 90%, 92.5%, 95%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO: 28. 4. A rAAV nucleic acid vector according to any one of claims 1 to 3, wherein the polyA signal comprises the nucleotide sequence of SEQ ID NO: 28. 5. A rAAV nucleic acid vector according to any one of claims 1 to 4, wherein the transgene encoding a S100 family protein comprises a sequence having a reduced guanosine (G) and cytosine (C) (G/C) content relative to any nucleotide sequence of SEQ ID NOs: 5, 8, 19 and 20. 6. The rAAV nucleic acid vector of claim 5, wherein the G/C content is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% relative to any of the nucleotide sequences of SEQ ID NOs: 5, 8, 19 and 20. 7. The rAAV nucleic acid vector of any one of claims 1 to 6, wherein the transgene encoding the apoptosis inhibitor comprises a sequence that is at least 90%, at least 95%, or at least 99.5% identical to any of the nucleotide sequences of SEQ ID NOs: 3, 6, 7, and 15 to 18. 8. A rAAV nucleic acid vector according to any one of claims 1 to 7, wherein the transgene encoding the apoptosis inhibitor comprises any of the nucleotide sequences of SEQ ID NOs: 3, 6, 7, and 15 to 18. 9. A rAAV nucleic acid vector according to any one of claims 1 to 8, wherein an internal ribosome entry site (IRES) is present between the transgene encoding the S100 family protein and the transgene encoding the apoptosis inhibitor. 10. A rAAV nucleic acid vector according to any one of claims 1 to 9, wherein the transgenes are specific for homo sapiens or canis lupus familiaris. 11. A rAAV nucleic acid vector according to any one of claims 1 to 10, wherein the promoter is a cardiac tissue-restricted promoter. 12. The rAAV nucleic acid vector of claim 11, wherein the promoter is a cardiac tissue-restricted promoter selected from the group consisting of cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). 13. The rAAV nucleic acid vector of claim 11 or 12, wherein the promoter is cTnT. 14. The rAAV nucleic acid vector of claim 11, wherein the promoter is a cardiac tissue-restricted promoter derived from a gene selected from the group consisting of the α-myosin heavy chain gene, the β-myosin heavy chain gene, the 2v myosin light chain gene, the 2a myosin light chain gene, the CARP gene, the cardiac α-actin gene, the cardiac m2-muscarinic acetylcholine receptor gene, ANF, the cardiac sarcoplasmic reticulum Ca-ATPase gene, and the skeletal α-actin gene, or is an artificial cardiac tissue promoter derived from the MLC-2v gene. 15. The rAAV nucleic acid vector according to any one of claims 1 to 14, wherein the rAAV nucleic acid vector is single-stranded. 16. The rAAV nucleic acid vector according to any one of claims 1 to 15, wherein the rAAV nucleic acid vector is self-complementary. 17. The rAAV nucleic acid vector of any one of claims 1 to 16, wherein the rAAV nucleic acid vector comprises a nucleotide sequence that is at least 90%, at least 95% or at least 99.5% identical to any of the nucleotide sequences of SEQ ID NOs: 22 and 23. 18. The rAAV nucleic acid vector of claim 17, wherein the rAAV nucleic acid vector comprises the nucleotide sequence of SEQ ID NO: 22. - 41 048262 19. The rAAV nucleic acid vector of claim 17, wherein the rAAV nucleic acid vector comprises the nucleotide sequence of SEQ ID NO: 23. 20. An rAAV particle comprising an rAAV nucleic acid vector according to any one of claims 1 to 19, enclosed in an AAV capsid. 21. The rAAV particle of claim 20, wherein the AAV capsid comprises a capsid protein derived from serotype AAV1, AAV2, AAV3, AAV6, AAV8, AAVrh.74, AAVrh.10, AAV2/6, or AAV9. 22. The rAAV particle of claim 20 or 21, wherein the AAV capsid comprises a capsid protein derived from the AAVrh.10 serotype. 23. A composition for treating a heart disease comprising an rAAV particle according to any one of claims 20 to 22. 24. Use of a composition according to claim 23 or an rAAV particle according to any one of claims 20-22 for treating a subject suffering from a heart disease. 25. The use according to claim 24, wherein the heart disease causes heart failure in the subject. 26. The use according to claim 24 or 25, wherein the heart disease is a cardiomyopathy. 27. The use according to any one of claims 24 to 26, wherein the heart disease is hypertrophic cardiomyopathy or dilated cardiomyopathy. 28. The use according to claim 24 or 25, wherein the heart disease is acute ischemia. 29. The use according to any one of claims 24 to 28, wherein the composition is administered by injection into the heart of the subject or by intravascular injection into the coronary arteries of the subject. 30. The use according to any one of claims 24 to 29, wherein said use results in the expression of two or more transgenes in the heart of the subject. 31. The use according to any one of claims 24 to 30, wherein the subject is a mammal. 32. The use according to claim 31, wherein the mammal is a human. 33. The use according to claim 31, wherein the mammal is a domestic animal. 34. The use according to claim 33, wherein the pet is a dog or a cat. 35. The use according to any one of claims 24 to 34, wherein said use results in an improvement in cardiac function in the subject. 36. The use of claim 35, wherein said use results in an improvement in cardiac function in the subject for more than 10 months. 37. A rAAV nucleic acid vector according to any one of claims 1 to 19, wherein the transgene encoding the S100 family protein is located 5' relative to the transgene encoding the apoptosis inhibitor. 38. A rAAV nucleic acid vector according to any one of claims 1 to 19, wherein the transgene encoding the apoptosis inhibitor is located 5' relative to the transgene encoding the S100 family protein. 39. The rAAV nucleic acid vector of any one of claims 1-19, 37 and 38, wherein the rAAV nucleic acid vector encodes a protein comprising an amino acid sequence that is at least 90%, at least 95% or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 13 or 14. 40. The rAAV nucleic acid vector according to any one of claims 1-19 and 37-39, wherein the rAAV nucleic acid vector encodes a protein comprising the amino acid sequence of SEQ ID NO: 13 or 14. 41. The rAAV nucleic acid vector of any one of claims 1-19 and 37-40, wherein the rAAV nucleic acid vector encodes a protein comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 24 or 29. 42. The rAAV nucleic acid vector according to any one of claims 1-19 and 37-41, wherein the rAAV nucleic acid vector encodes a protein comprising the amino acid sequence of SEQ ID NO: 24 or 29. 43. A recombinant adeno-associated virus (rAAV) nucleic acid vector for delivering two or more transgenes to the heart of a subject, wherein said vector comprises sequentially from 5' to 3' a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, two or more transgenes and a promoter operably linked to the two or more transgenes, a polyadenylation (polyA) signal and a second AAV ITR sequence, wherein the two or more transgenes encode an S100 family protein and an apoptosis inhibitor, respectively, and wherein the transgene encoding an S100 family protein comprises a sequence that is at least 98% identical to any of the nucleotide sequences of SEQ ID NOs: 25 and 26, and wherein the apoptosis inhibitor is a cardiac repressor of apoptosis with a caspase recruitment domain (ARC) or a variant thereof. 44. Vector based on nucleic acid of recombinant adeno-associated virus -