Gene therapy methods for treating mitral valve disease

Abstract

Provided herein are methods for treating mitral valve disease in a subject. The methods generally involve administering to the subject a gene therapy comprising a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFbR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21). Also provided are methods for treating mitral valve disease in a subject, comprising administering the gene therapy to the subject in combination with an additional therapeutic agent (e.g., pimobendan).

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 376,472, filed September 21, 2022, and U.S. Provisional Patent Application No. 63 / 503,318, filed May 19, 2023, the entire disclosures of which are incorporated herein by reference.

[0002] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically in ST.26 format and is incorporated herein by reference in its entirety (a copy of said ST.26 created on September 19, 2023 is entitled "203422_SL.XML" and is 106,831 bytes in size). [Background technology]

[0003] Mitral valve disease is a degenerative condition that, over time, leads to insufficient functioning of the mitral valve. The mitral valve normally functions as a seal between the left atrium and left ventricle. When the ventricle contracts, the mitral valve closes, preventing blood from flowing backward into the atrium, a phenomenon known as regurgitation. If left untreated, increased pressure in the atrium can occur as the left atrium enlarges to accommodate the additional blood from the regurgitation, ultimately leading to congestive heart failure due to fluid accumulation in the lungs.

[0004] In dogs, approximately 10% of animal visits to primary care have heart disease, and myxomatous MVD (MMVD) is the most common heart disease in dogs in many parts of the world. MMVD accounts for approximately 75% of cases of heart disease seen in dogs in North America. MMVD most commonly affects the mitral valve, but in at least 30% of cases, the tricuspid valve is also involved. The MMVD staging system represents four basic stages of heart disease and heart failure: Stage A identifies dogs at high risk for developing heart disease but with no current structural cardiac compromise; Stage B identifies dogs with structural heart disease but have never developed clinical signs caused by heart failure; Stage B1 refers to asymptomatic dogs with no or mild radiographic or echocardiographic evidence of cardiac remodeling in response to MMVD; Stage B2 refers to asymptomatic dogs with more advanced hemodynamically severe and long-standing mitral regurgitation sufficient to cause radiographic and echocardiographic evidence of left atrial and left ventricular enlargement; Stage C identifies dogs with either current or past clinical signs caused by MMVD; and Stage D identifies dogs with end-stage MMVD in which clinical signs of heart failure are refractory to standard treatment. Pimobendan is fully approved for the symptomatic management of mild, moderate, or severe congestive heart failure in dogs due to MMVD and was recently approved to treat dogs with preclinical MMVD who have a heart murmur and cardiac enlargement but are not yet in congestive heart failure. However, there is no treatment for congestive heart failure due to MMVD.

[0005] Therefore, there is a need in the art for improved methods for treating mitral valve disease. Summary of the Invention

[0006] The present disclosure provides methods for treating mitral valve disease in a subject. The methods generally involve administering to the subject a gene therapy comprising a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21). In certain embodiments, the gene therapy is administered in combination with an effective amount of pimobendan or to a subject currently undergoing treatment with pimobendan.

[0007] Accordingly, in one aspect, the disclosure provides a method of treating mitral valve disease in a subject, the method comprising administering to the subject a gene therapy comprising a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21), wherein the subject has a left atrial-to-aortic diameter ratio (LA / Ao) of about 1.6 to about 2.1. In some embodiments, the subject has an LA / Ao of about 1.6. In some embodiments, the subject has an LA / Ao of about 1.8. In some embodiments, the subject is a mammal.

[0008] In certain embodiments, the subject is a dog. In some embodiments, the subject is a breed selected from the group consisting of Cavalier King Charles Spaniel, Miniature Poodle, Shih Tzu, Maltese, Chihuahua, Cocker Spaniel, Miniature Schnauzer, Dachshund, Whippet, Pomeranian, and combinations thereof. In certain embodiments, the subject is a Cavalier King Charles Spaniel.

[0009] In certain embodiments, the mitral valve disease comprises one or more diseases or conditions selected from the group consisting of myxomatous mitral valve disease, mitral stenosis, mitral valve prolapse, and mitral regurgitation.

[0010] In certain embodiments, the method further includes determining the subject's L A / A o before administration of the gene therapy and determining the subject's L A / A o at a period after administration of the gene therapy, hi certain embodiments, the subject's L A / A o after administration of the gene therapy is decreased compared to the subject's L A / A o before administration of the gene therapy.

[0011] In certain embodiments, the subject has been administered an effective amount of pimobendan.

[0012] In certain embodiments, the method further comprises administering to the subject an effective amount of pimobendan.

[0013] In another aspect, the disclosure provides a method of treating mitral valve disease in a subject, the method comprising administering to the subject a gene therapy comprising: a first nucleic acid encoding soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21); and / or an effective amount of pimobendan.

[0014] In another aspect, the disclosure provides a method for treating mitral valve disease in a subject who has been administered an effective amount of pimobendan, the method comprising administering to the subject a gene therapy comprising a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

[0015] In certain embodiments, the effective amount of pimobendan is 0.25 mg / kg. In some embodiments, gene therapy and pimobendan are administered simultaneously. In certain embodiments, pimobendan is administered orally. In certain embodiments, pimobendan is administered twice daily. In certain embodiments, the effective amount of pimobendan is 0.25 mg / kg twice daily.

[0016] In another aspect, the disclosure provides a method for determining the likelihood of successful treatment in a subject with mitral valve disease with gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, comprising determining the subject's LA / Ao, wherein an LA / Ao of 2.1 or greater indicates a decreased likelihood of successful treatment with gene therapy, and an LA / Ao of about 1.6 to about 2.1 indicates an increased likelihood of successful treatment with gene therapy. In certain embodiments, successful treatment of mitral valve disease comprises a decrease in the subject's LA / Ao compared to the LA / Ao prior to gene therapy.

[0017] In another aspect, the present disclosure provides a method for identifying a subject having mitral valve disease suitable for treatment with gene therapy comprising determining the subject's LA / Ao, the method comprising determining the subject's LA / Ao from a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, wherein the subject is suitable for treatment with gene therapy if the subject has an LA / Ao of about 1.6 to about 2.1.

[0018] In certain embodiments, the subject to be treated is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the dog is a breed selected from the group consisting of Cavalier King Charles Spaniel, Miniature Poodle, Shih Tzu, Maltese, Chihuahua, Cocker Spaniel, Miniature Schnauzer, Dachshund, Whippet, Pomeranian, and combinations thereof. In some embodiments, the subject is a Cavalier King Charles Spaniel.

[0019] In certain embodiments, the mitral valve disease comprises one or more diseases or conditions selected from the group consisting of myxomatous mitral valve disease, mitral stenosis, mitral valve prolapse, and mitral regurgitation.

[0020] In certain embodiments, the gene therapy is administered intravenously.

[0021] In certain embodiments, the first nucleic acid comprises a first transcriptional regulatory element operably linked to a sTGFβR2 coding sequence. In some embodiments, the sTGFβR2 coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, or 24. In certain embodiments, the sTGFβR2 coding sequence further comprises a heterologous or native secretory signal sequence, wherein the signal sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, 5, or 6. In certain embodiments, the sTGFβR2 coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9, 10, 11, 12, or 25. In certain embodiments, the sTGFβR2 coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, 14, 15, or 26. In certain embodiments, the sTGFβR2 coding sequence further encodes a secretory signal sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 or 8. In certain embodiments, the sTGFβR2 coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, 17, 18, 19, or 27.

[0022] In certain embodiments, the second nucleic acid comprises a second transcriptional regulatory element operably linked to the FGF21 coding sequence. In some embodiments, the FGF21 coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28, 29, 30, or 31. In certain embodiments, the FGF21 coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32, 33, or 34.

[0023] In certain embodiments, the first and second transcriptional regulatory elements each comprise one or more ApoE binding sites and / or a hAAT promoter. In certain embodiments, the first and second transcriptional regulatory elements each comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 38, 39, 40, and / or 48.

[0024] In certain embodiments, the first and second nucleic acids each further comprise a post-transcriptional regulatory element. In certain embodiments, the post-transcriptional regulatory element comprises a polyadenylation signal and / or a WPRE sequence. In certain embodiments, the polyadenylation signal is an SV40 polyadenylation signal. In certain embodiments, the WPRE sequence is a WPRE3 sequence. In certain embodiments, the post-transcriptional regulatory element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 50, 51, or 70.

[0025] In certain embodiments, the first nucleic acid is contained within a first vector, and the second nucleic acid is contained within a second vector. In certain embodiments, the first vector and / or the second vector are each viral vectors, optionally each independently selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, and alphavirus. In certain embodiments, the first vector is an AAV vector contained within a first recombinant AAV (rAAV), the first rAAV comprising an AAV capsid comprising AAV capsid proteins and a first rAAV genome; and / or the second vector is an AAV vector contained within a second rAAV, the second rAAV comprising an AAV capsid comprising AAV capsid proteins and a second rAAV genome.

[0026] In certain embodiments, the gene therapy comprises a first recombinant AAV (rAAV) comprising a first rAAV genome comprising an AAV capsid comprising AAV capsid proteins and a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 26; and a second rAAV comprising a second rAAV genome comprising an AAV capsid comprising AAV capsid proteins and a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 33.

[0027] In certain embodiments, the first rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:66.

[0028] In certain embodiments, the first rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence and a 3' inverted terminal repeat (3' ITR) nucleotide sequence. In certain embodiments, the 5' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 58 or 59, and / or the 3' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60 or 61. In certain embodiments, the first rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:67.

[0029] In certain embodiments, the second rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68.

[0030] In certain embodiments, the second rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence and a 3' inverted terminal repeat (3' ITR) nucleotide sequence. In certain embodiments, the 5' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 58 or 59, and / or the 3' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60 or 61. In certain embodiments, the second rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:69.

[0031] In certain embodiments, the AAV capsid protein is derived from a Clade A, Clade B, Clade C, Clade D, Clade E, Clade F, Clade G, Clade H, Clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof. In certain embodiments, the AAV capsid protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 63, 64, and / or 65.

[0032] In certain embodiments, the first and second nucleic acids are contained within a vector, and optionally, the first and second nucleic acids are separated by a polycistronic element. In certain embodiments, the vector comprises a first nucleic acid comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:26; and a second nucleic acid comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:33. In certain embodiments, the first and second nucleic acids are separated by a polycistronic element. In certain embodiments, the polycistronic element is an IRES or 2A sequence. In certain embodiments, the polycistronic element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:55, 56, or 57.

[0033] In certain embodiments, the vector is an AAV vector contained within a recombinant AAV (rAAV), which comprises an AAV capsid comprising AAV capsid proteins; and an rAAV genome. In certain embodiments, the AAV capsid proteins are derived from Clade A, Clade B, Clade C, Clade D, Clade E, Clade F, Clade G, Clade H, Clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid proteins, or engineered variants thereof. In certain embodiments, the AAV capsid protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 63, 64, and / or 65. [Brief explanation of the drawings]

[0034] [Figure 1A]1A and 1B are graphs showing the expression levels of sTGFβR2 and FGF21, respectively, in subjects administered the indicated doses of AAV8-sTGFβR2 and AAV8-FGF21. [Figure 1B] 1A and 1B are graphs showing the expression levels of sTGFβR2 and FGF21, respectively, in subjects administered the indicated doses of AAV8-sTGFβR2 and AAV8-FGF21.

[0035] [Figure 2-1] 2 is a graph showing LA / Ao over time in subjects administered AAV8-sTGFβR2 and AAV8-FGF21. Each symbol represents an individual subject. [Figure 2-2] 2 is a graph showing LA / Ao over time in subjects administered AAV8-sTGFβR2 and AAV8-FGF21. Each symbol represents an individual subject.

[0036] [Figure 3] Figure 3 is a graph showing the mean change in LA / Ao over time from baseline in subjects receiving pimobendan with AAV8-sTGFβR2 and AAV8-FGF21 (Pimo+GT). Error bars represent the interquartile range. The dashed line represents the change in LA / Ao over time based on published data for subjects receiving pimobendan alone.

[0037] [Figure 4] Figure 4 is a graph showing the mean change from baseline in left ventricular fractional shortening (FS%) over time in subjects receiving pimobendan with AAV8-sTGFβR2 and AAV8-FGF21 (Pimo+GT). Error bars represent the interquartile range. The dashed line represents the change in fractional shortening over time based on published data from subjects receiving pimobendan alone.

[0038] [Figure 5]Figure 5 is a graph showing the proportion of subjects receiving pimobendan with AAV8-sTGFβR2 and AAV8-FGF21 (Pimo+GT) over time who have not yet reached the primary endpoint. This data is overlaid on published data for subjects receiving pimobendan alone or placebo. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present disclosure provides methods for treating mitral valve disease in a subject. The methods generally involve administering to the subject a gene therapy comprising a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21). In certain embodiments, the gene therapy is administered in combination with an effective amount of pimobendan or to a subject currently undergoing treatment with pimobendan.

[0040] The present disclosure is based on the discovery that certain populations of subjects (e.g., dogs) with mitral valve disease responded well to sTGFβR2 and FGF21 (sTGFβR2 / FGF21) gene therapy. Subjects with an initial left atrial-to-aortic diameter ratio (LA / Ao) of less than 2.1 who underwent sTGFβR2 / FGF21 gene therapy maintained or reduced their LA / Ao over time. The present disclosure is also based on the discovery that combining pimobendan, the current leading therapeutic agent for treating MVD that has been shown to delay the onset of congestive heart failure through a reduction in heart size, with sTGFβR2 / FGF21 gene therapy resulted in a synergistic restoration of left atrial enlargement in subjects (e.g., dogs) with MVD.

[0041] definition As used herein, the term "replication-deficient adeno-associated virus" refers to an AAV comprising a genome lacking the Rep and Cap genes.

[0042] As used herein, the term "recombinant AAV genome" or "rAAV genome" refers to a coding sequence operably linked to an exogenous transcriptional regulatory element that mediates the expression of the coding sequence when the rAAV genome is introduced into a cell. In certain embodiments, the rAAV genome is not integrated into the chromosomal DNA of the cell. Those skilled in the art will understand that the portion of the rAAV genome that includes the transcriptional regulatory element operably linked to the coding sequence can be in the sense or antisense direction relative to the direction of transcription of the coding sequence.

[0043] As used herein, the "percentage identity" between two nucleotide sequences or two amino acid sequences is calculated by multiplying the number of matches between a pair of aligned sequences by 100 and dividing by the length of the aligned region, including internal gaps. Identity scoring counts only perfect matches and does not consider the degree of similarity between amino acids. Only internal gaps are included in the length, and gaps at the ends of the sequences are not included.

[0044] As used herein, the term "coding sequence" refers to the portion of a complementary DNA (cDNA) that begins with an initiation codon and ends with a termination codon that encodes a polypeptide. A gene may have one or more coding sequences due to alternative splicing, alternative translation initiation, and variation within a population. A coding sequence may be either wild-type or codon-optimized.

[0045] As used herein, the term "codon optimization" refers to altering the coding sequence of a gene (e.g., by nucleotide substitution) without altering the amino acid sequence of the polypeptide encoded by the coding sequence. Such codon alterations are advantageous in that they may increase the translation efficiency of the coding sequence and / or prevent recombination with the corresponding sequence of an endogenous gene when the coding sequence is transduced into a cell.

[0046] As used herein, the term "transcriptional regulatory element" or "TRE" refers to a cis-acting nucleotide sequence, e.g., a DNA sequence that controls (e.g., regulates, increases, or decreases) the transcription of an operably linked nucleotide sequence by an RNA polymerase to form an RNA molecule. A TRE depends on one or more trans-acting molecules, such as transcription factors, to control transcription. Thus, a single TRE may control transcription differently when it is in contact with different trans-acting molecules, e.g., in different cell types. A TRE may contain one or more promoter elements and / or enhancer sequences. Those skilled in the art will understand that promoter and enhancer sequences within a gene may be positionally adjacent, and the term "promoter" may refer to a sequence that includes promoter elements and enhancer sequences. Thus, the term "promoter" does not exclude enhancer sequences within a sequence. The promoter and enhancer sequences need not be derived from the same gene or species, and the sequence of each promoter or enhancer sequence may be either identical or substantially identical to the corresponding endogenous sequence within a genome.

[0047] As used herein, the term "operably linked" is used to describe the connection between a TRE and a coding sequence to be transcribed. Typically, gene expression is placed under the control of a TRE, which includes one or more promoter and / or enhancer sequences. A coding sequence is "operably linked" to a TRE if transcription of the coding sequence is controlled or influenced by the TRE. The promoter and enhancer sequences of the TRE can be in any orientation and / or distance from the coding sequence, so long as the desired transcriptional activity is obtained. In certain embodiments, the TRE is upstream of the coding sequence.

[0048] As used herein, the term "polyadenylation signal" or "polyadenylation sequence" refers to a DNA sequence that, when transcribed into RNA, constitutes a polyadenylation signal sequence. The polyadenylation sequence can be natural (e.g., with respect to the coding sequence of a gene) or exogenous. The exogenous polyadenylation sequence can be a mammalian or viral polyadenylation sequence (e.g., an SV40 polyadenylation sequence).

[0049] As used herein, "exogenous polyadenylation sequence" refers to a polyadenylation sequence that is not identical or substantially identical to the endogenous polyadenylation sequence of the coding sequence of a gene. In certain embodiments, the exogenous polyadenylation sequence may be from the same species (e.g., human) or from a different species (e.g., virus).

[0050] As used herein, the term "effective amount" in the context of administering a viral vector (e.g., a recombinant AAV) to a subject refers to the amount of the viral vector that achieves a desired prophylactic or therapeutic effect in the context of administering the viral vector (e.g., a recombinant AAV) to a subject. In the context of administering a compound, an effective amount refers to the amount of the compound that achieves a desired prophylactic or therapeutic effect.

[0051] As used herein, the term "polynucleotide" in its broadest sense includes any compound and / or substance comprising a polymer of nucleotides linked through phosphodiester bonds.

[0052] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. In a "treatment" method, a polynucleotide is administered to a subject having or susceptible to a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disease or disorder, or the recurrence of a disease or symptom, or to prolong the subject's survival beyond that expected in the absence of such treatment.

[0053] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of therapy that achieves a desired prophylactic or therapeutic effect.

[0054] As used herein, the term "subject" includes any human or non-human animal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a dog. In certain embodiments, the canine subject is a breed such as a Cavalier King Charles Spaniel, Miniature Poodle, Shih Tzu, Maltese, Chihuahua, Cocker Spaniel, Miniature Schnauzer, Dachshund, Whippet, Pomeranian, or a mix thereof.

[0055] As used herein, the term "about" when used in connection with a numerical value is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0056] Polynucleotides, Vectors, and Compositions In one aspect, the methods disclosed herein utilize a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

[0057] In certain embodiments, the sTGFβR2 coding sequence encodes a polypeptide comprising all or substantially all of the extracellular portion of TGFβR2, and does not encode any transmembrane or intracellular aspects of TGFβR2. In certain embodiments, the sTGFβR2 coding sequence encodes a polypeptide comprising the extracellular portion of wild-type TGFβR2. In certain embodiments, the sTGFβR2 coding sequence encodes a polypeptide comprising the extracellular portion of a functional variant of TGFβR2. In certain embodiments, the sTGFβR2 coding sequence encodes human, mouse, or canine TGFβR2.

[0058] In certain embodiments, the sTGFβR2 coding sequence encodes a polypeptide comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, 14, or 15. In certain embodiments, the sTGFβR2 coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 2, or 3.

[0059] In certain embodiments, the sTGFβR2 coding sequence encodes a polypeptide comprising all or substantially all of the extracellular portion of TGFβR2, wherein the extracellular portion of TGFβR2 comprises a secretory signal sequence. In certain embodiments, the TGFβR2 secretory signal sequence is a native secretory signal sequence. In certain embodiments, the TGFβR2 secretory signal sequence is a heterologous secretory signal sequence. For example, a heterologous secretory signal sequence can be derived from a TGFβR2 secretory signal sequence of a different species. Exemplary TGFβR2 secretory signal sequences include, but are not limited to, secretory signal sequences derived from human, mouse, and canine TGFβR2. Thus, the sTGFβR2 coding sequence may further encode a heterologous or native secretory signal sequence. In certain embodiments, the heterologous or native secretory signal sequence comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7 or 8. In certain embodiments, the heterologous or native secretory signal sequence is encoded by a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4, 5, or 6.

[0060] In certain embodiments, the sTGFβR2 coding sequence encodes a polypeptide comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, or 19. In certain embodiments, the sTGFβR2 coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 9, 10, 11, or 12.

[0061] In certain embodiments, the sTGFβR2 coding sequence further encodes a peptide to achieve extended half-life. Such peptides include, but are not limited to, an IgG constant domain or fragment thereof (e.g., an Fc domain), human serum albumin (HSA), or an albumin-binding polypeptide. In certain embodiments, the sTGFβR2 coding sequence further encodes an Fc domain (referred to herein as an sTGFβR2-Fc coding sequence). Exemplary Fc domains include wild-type Fc domains derived from human, mouse, or canine IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 or 23. In certain embodiments, the Fc domain is encoded by a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 20 or 21.

[0062] In certain embodiments, the sTGFβR2-Fc coding sequence encoding the polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26 or 27. In certain embodiments, the sTGFβR2-Fc coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 24 or 25.

[0063] In certain embodiments, the FGF21 coding sequence encodes wild-type FGF21 or a functional variant thereof. In certain embodiments, the FGF21 coding sequence encodes human, mouse, or canine FGF21. In certain embodiments, the FGF21 coding sequence encoding a polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, 33, or 34. In certain embodiments, the FGF21 coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:28, 29, 30, or 31.

[0064] In certain embodiments, a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) is each operably linked to a first transcriptional regulatory element (TRE). In certain embodiments, a second nucleic acid encoding fibroblast growth factor 21 (FGF21) is operably linked to a second TRE. In certain embodiments, the first TRE and the second TRE are the same. In certain embodiments, the first TRE and the second TRE are different. In certain embodiments, the first TRE and the second TRE comprise one or more common elements. The first TRE and the second TRE can be active in any mammalian cell (e.g., human cell, canine cell).

[0065] In certain embodiments, the TRE is active in a broad range of mammalian cells. Such TREs include, but are not limited to, cytomegalovirus (CMV) promoters (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:41); CMV enhancer sequences collectively referred to as CAG promoters, CBA promoters, and splice acceptors from exon 3 of the rabbit beta globin gene (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:42). a human calmodulin 1 (CALM1) promoter (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:43); a chicken beta actin (CBA) promoter (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:44);CASI promoter (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:45); smCBA promoter (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:46); human elongation factor 1 (AL) promoter (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:46); The TRE may comprise a constitutive promoter and / or enhancer sequence, including the EF1α promoter (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 35, 36, or 37); the SV40 promoter; the human phosphoglycerate kinase (PGK1) promoter; the human ubiquitin C (Ubc) promoter; the human beta-actin promoter; the human neuron-specific enolase (ENO2) promoter; the human beta-glucuronidase (GUSB) promoter; and / or the human methyl-CpG-binding protein 2 (MeCP2) promoter. Any of these TREs or elements within these TREs may be combined in any order to drive efficient transcription.

[0066] Alternatively, the TRE may be a tissue-specific TRE, i.e., active in a particular tissue(s) and / or organ(s). A tissue-specific TRE comprises one or more tissue-specific promoter and / or enhancer sequences, and optionally one or more constitutive promoter and / or enhancer sequences. Those skilled in the art will understand that tissue-specific promoter and / or enhancer sequences can be isolated from genes specifically expressed in that tissue by methods well known in the art.

[0067] In certain embodiments, the TRE is liver-specific, ie, active in cells of the liver.Liver-specific TREs include those provided in the Liver-Specific Gene Promoter Database (LSPD, rulai.cshl.edu / LSPD / ); the human alpha-1-antitrypsin (hAAT) promoter (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 149%, at apolipoprotein E (ApoE) binding site (e.g., a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 39 or 40); nucleotide sequence); human albumin (hAlb) or minimal promoter; transthyretin (TTR) promoter or TTR-minimal promoter (TTRm); apolipoprotein A1 (APOA1) promoter or minimal promoter; complement factor B (CFB) promoter; ketohexokinase (KHK) promoter; hemopexin (HPX) promoter or minimal promoter; nicotinamide N-methyltransferase (NNMT) promoter or minimal promoter; (liver) carboxylesterase 1 (CES1) promoter or minimal promoter; protein C (PROC) promoter or minimal promoter; apolipoprotein C3 (APOC3) promoter or minimal promoter; mannan-binding lectin serine protease 2 (MASP2) promoter or minimal promoter; hepcidin antimicrobial peptide (HAMP) promoter or minimal promoter; and serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) promoter or minimal promoter.

[0068] The TRE can be an inducible promoter. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0069] In certain embodiments, the first nucleic acid and / or the second nucleic acid comprises two or more TREs, optionally including at least one of the TREs disclosed herein. Those skilled in the art will understand that any of these TREs can be combined in any order, and that a combination of a constitutive TRE and a tissue-specific TRE can drive efficient and tissue-specific transcription.

[0070] In certain embodiments, the TRE may further comprise an intron sequence. Such an intron may increase transgene expression, for example, by reducing transcriptional silencing and enhancing mRNA transport from the nucleus to the cytoplasm. The intron may comprise a natural intron sequence of sTGFβR2 or FGF21, an intron sequence from the same gene in a different species, an intron sequence from a different gene in the same species, and / or a synthetic intron sequence. Those skilled in the art will understand that synthetic intron sequences can be designed to mediate RNA splicing by introducing any consensus splicing motif known in the art (see, for example, Sibley et al. Nature Reviews Genetics. 2016, 17:407-21, the entire contents of which are incorporated herein by reference). Exemplary intron sequences are provided in Lu et al., Molecular Therapy. 2013, 21(5):954-63, and Lu et al., Hum. Gen Ther. 2017, 28(1):125-34, which are incorporated by reference in their entireties. Suitable intron sequences include, but are not limited to, minute virus of mice (MVM) introns (e.g., including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, e.g., the nucleotide sequence set forth in SEQ ID NO:47); beta-globin intron sequences (e.g., including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to, e.g., the nucleotide sequence set forth in SEQ ID NO:48); and SV40 intron sequences.

[0071] In certain embodiments, the first nucleic acid and / or the second nucleic acid further comprise a post-transcriptional regulatory element. The post-transcriptional regulatory element can be any sequence that effectively terminates transcription, and one of skill in the art will understand that such a sequence can be isolated from any gene expressed in a cell in which transcription of the coding sequence is desired.

[0072] In certain embodiments, the post-transcriptional regulatory element comprises a polyadenylation signal sequence. In certain embodiments, the polyadenylation signal sequence is identical or substantially identical to the endogenous polyadenylation sequence of the sTGFβR2 or FGF21 gene. In certain embodiments, the polyadenylation signal sequence is an exogenous polyadenylation signal sequence.In certain embodiments, the polyadenylation signal sequence is selected from the group consisting of an SV40 polyadenylation sequence (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:51); a bovine growth hormone polyadenylation sequence (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:52). a rabbit beta globin polyadenylation sequence (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:53); or a human growth hormone polyadenylation sequence (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:54).

[0073] In certain embodiments, the post-transcriptional regulatory element comprises a woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE). In some embodiments, the post-transcriptional regulatory element comprises a WPRE sequence (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 49); or a WPRE3 sequence (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 50 or 70).

[0074] The first nucleic acid and / or second nucleic acid described herein can be transcribed from an expression vector (e.g., a recombinant expression vector). In certain embodiments, the first nucleic acid is contained in a first vector and the second nucleic acid is contained in a second vector. In certain embodiments, the first nucleic acid and the second nucleic acid are contained in a single vector. When the first nucleic acid and the second nucleic acid are contained in a single vector, the first nucleic acid and the second nucleic acid can be separated by a polycistronic element.

[0075] In certain embodiments, the polycistronic element comprises a nucleotide sequence encoding an internal ribosome entry site (IRES). An IRES is an element that promotes direct internal ribosome entry to the start codon, e.g., ATG, of a protein-coding region, thereby resulting in cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art and include, but are not limited to, IRESs derived from viral or cellular mRNA sources, such as immunoglobulin heavy chain-binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs derived from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). In certain embodiments, the polycistronic element comprises a nucleotide sequence encoding a 2A sequence. A 2A sequence refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein, which dissociates into component proteins during translation. Various 2A sequences are known to those skilled in the art, including, but not limited to, members of the picornaviridae family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO), Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1); and caryoviruses, such as tyloviruses and encephalomyocarditis viruses. The 2A sequences from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. In certain embodiments, the polycistronic element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 55, 56, or 57.

[0076] In certain embodiments, the vector is a non-viral vector. Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministrings, and oligonucleotides (e.g., mRNA, naked DNA). In certain embodiments, the non-viral vector is a DNA plasmid vector. In certain embodiments, the non-viral vector is a transposon-based vector. In certain embodiments, the non-viral vector is a PiggyBac-based vector or a Sleeping Beauty-based vector.

[0077] In certain embodiments, the vector is a viral vector. The viral vector can be replication-competent or replication-incompetent. The viral vector can be integrating or non-integrating. Several virus-based systems have been developed for gene transfer into mammalian cells, and a suitable viral vector can be selected by those skilled in the art. Exemplary viral vectors include, but are not limited to, adenoviral vectors (e.g., adenovirus 5), adeno-associated viral (AAV) vectors (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9), retroviral vectors (e.g., MMSV, MSCV), lentiviral vectors (e.g., HIV-1, HIV-2), gamma retroviral vectors, herpesvirus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flavivirus (e.g., Kunjin, West Nile, Dengue virus), rhabdovirus vectors (e.g., rabies virus, VSV), measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors (e.g., coxsackievirus). In certain embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, and alphavirus.

[0078] In certain embodiments, the vector is an AAV vector. In certain embodiments, the vector is a single-stranded AAV. In certain embodiments, the vector is a self-complementary AAV.

[0079] In certain embodiments, the vector is an AAV vector contained within a recombinant AAV (rAAV). In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and the rAAV genome.

[0080] Capsid proteins from any capsid known in the art can be used in the rAAV compositions disclosed herein, including, but not limited to, capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotypes. The capsid protein can be derived from Clade A, Clade B, Clade C, Clade D, Clade E, Clade F, Clade G, Clade H, Clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid proteins, or engineered variants thereof. In certain embodiments, the capsid protein is derived from AAV8. In certain embodiments, the capsid protein is encoded by a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:62.In certain embodiments, the capsid protein comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of amino acids 1 to 738 of SEQ ID NO:63; at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 99%, or 100% sequence identity to the amino acid sequence of amino acids 138 to 738 of SEQ ID NO:63; an amino acid sequence having 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and / or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of amino acids 204 to 738 of SEQ ID NO: 63.In certain embodiments, the capsid protein comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:63; at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:64; an amino acid sequence having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:65; and / or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:65.

[0081] In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and an rAAV genome comprising a nucleic acid encoding sTGFβR2. In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and an rAAV genome comprising a nucleic acid encoding sTGFβR2-Fc. In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and an rAAV genome comprising a nucleic acid encoding an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and an rAAV genome comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:66.

[0082] In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and a rAAV genome comprising a nucleic acid encoding FGF21. In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and a rAAV genome comprising a nucleic acid encoding an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:33. In certain embodiments, the rAAV comprises an AAV capsid comprising AAV capsid proteins and an rAAV genome comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:67.

[0083] In certain embodiments, the rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence of the coding sequence and a 3' inverted terminal repeat (3' ITR) nucleotide sequence 3'. In certain embodiments, the rAAV genome comprises a 5' ITR5' of the TRE and a 3' ITR3' of the coding sequence. ITR sequences from any AAV serotype or variant thereof may be used in the rAAV genomes disclosed herein. The 5' ITR and 3' ITR may be derived from AAV of the same serotype or from AAV of different serotypes. Exemplary ITRs for use in the rAAV genomes disclosed herein are set forth in SEQ ID NOs: 58, 59, 60, and 61.

[0084] In certain embodiments, the 5' ITR or the 3' ITR is derived from AAV2. In certain embodiments, both the 5' ITR and the 3' ITR are derived from AAV2. In certain embodiments, the 5' ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 58 or 59. In certain embodiments, the 3' ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 60 or 61. In some embodiments, the 5' ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:59, and the 3' ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:61.

[0085] In certain embodiments, the rAAV genome comprises, from 5' to 3': a 5' ITR (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 59). a transcriptional regulatory element operably linked to a nucleic acid encoding sTGFβR2 or sTGFβR2-Fc (e.g., a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119 ...20%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at post-transcriptional regulatory elements (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:50, 51, or 70); and 3' ITRs (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:61).In certain embodiments, the rAAV genome comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:67.

[0086] In certain embodiments, the rAAV genome comprises, from 5' to 3': a 5' ITR (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 59); a transcriptional regulatory element operably linked to a nucleic acid encoding FGF21 (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 30, 38, 39, 40, 47, or 48); post-transcriptional regulatory elements (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:50, 51, or 70); and 3' ITRs (e.g., comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:61).In certain embodiments, the rAAV genome comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:69.

[0087] In certain embodiments, the rAAV comprises (a) an AAV capsid protein comprising the amino acid sequence of amino acids 1 to 738 of SEQ ID NO: 63, the amino acid sequence of amino acids 138 to 738 of SEQ ID NO: 63, and / or the amino acid sequence of amino acids 204 to 738 of SEQ ID NO: 63; and (b) an rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 66, 67, 68, or 69. In certain embodiments, the rAAV comprises (a) an AAV capsid protein comprising an rAAV genome comprising the amino acid sequence of amino acids 1 to 738 of SEQ ID NO: 63 and the nucleotide sequence set forth in any one of SEQ ID NOs: 66, 67, 68, or 69; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138 to 738 of SEQ ID NO: 63 and the nucleotide sequence set forth in any one of SEQ ID NOs: 66, 67, 68, or 69; and / or (c) an AAV capsid protein comprising the amino acid sequence of amino acids 204 to 738 of SEQ ID NO: 63 and the nucleotide sequence set forth in any one of SEQ ID NOs: 66, 67, 68, or 69;

[0088] In another aspect, the disclosure provides a polynucleotide comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 66, 67, 68, or 69.

[0089] The polynucleotide may comprise DNA, RNA, modified DNA, modified RNA, or a combination thereof. In certain embodiments, the polynucleotide is an expression vector. In certain embodiments, the polynucleotide is comprised in a viral vector. In certain embodiments, the polynucleotide is comprised in a plasmid vector.

[0090] In another aspect, the present disclosure provides a pharmaceutical composition comprising the rAAV disclosed herein together with a pharmaceutically acceptable excipient, adjuvant, diluent, vehicle, or carrier, or a combination thereof. A "pharmaceutically acceptable carrier" includes any material that, when combined with the active ingredient of the composition, allows the ingredient to retain its biological activity without eliciting a destructive physiological response, such as an unintended immune response. Pharmaceutically acceptable carriers include water, phosphate-buffered saline, emulsions such as oil / water emulsions, and wetting agents. Compositions containing such carriers are formulated by well-known conventional methods, such as those described in Remington's Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., 3rd ed. American Pharmaceutical Assoc.

[0091] Treatment method In another aspect, the disclosure provides methods for treating mitral valve disease in a subject. The methods generally involve administering to the subject an effective amount of a gene therapy comprising a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

[0092] In certain embodiments, a method of treating MVD in a subject comprises administering (a) a first rAAV comprising an AAV capsid comprising an AAV capsid protein and a first rAAV genome (e.g., comprising a nucleic acid encoding sTGFβR2); and (b) a second rAAV comprising an AAV capsid comprising an AAV capsid protein and a second rAAV genome (e.g., comprising a nucleic acid encoding FGF21). In certain embodiments, a method of treating MVD in a subject comprises administering (a) a first rAAV comprising an AAV capsid comprising an AAV capsid protein and a first rAAV genome comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:26; and (b) a second rAAV comprising an AAV capsid comprising an AAV capsid protein and a second rAAV genome comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:33. In certain embodiments, a method of treating MVD in a subject comprises administering (a) a first rAAV comprising an AAV capsid comprising an AAV capsid protein and a first rAAV genome comprising the nucleotide sequence set forth in SEQ ID NO: 66 or 68; and (b) a second rAAV comprising an AAV capsid comprising an AAV capsid protein and a second rAAV genome comprising the nucleotide sequence set forth in SEQ ID NO: 67 or 69.

[0093] In certain embodiments, a method of treating MVD in a subject comprises administering an rAAV comprising an AAV capsid comprising AAV capsid proteins, and a rAAV genome comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, wherein the first nucleic acid and the second nucleic acid are separated by a polycistronic element. In certain embodiments, a method of treating MVD in a subject comprises administering an rAAV comprising an AAV capsid comprising AAV capsid proteins, and a rAAV genome comprising a first nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:26 and a second nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:33, wherein the first nucleic acid and the second nucleic acid are separated by a polycistronic element.

[0094] In certain embodiments, the method for treating MVD in a subject further includes determining the subject's LA / Ao before administration of gene therapy and determining the subject's LA / Ao at a certain time period (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years) after administration of gene therapy. Methods for determining a subject's LA / Ao are known in the art. For example, LA / Ao can be measured from a right parasternal short-axis view at the base of the heart. Briefly, the internal short-axis diameter of the aorta can be measured along the commissure between the non-coronary aortic valve cusp and the right coronary valve cusp on a first frame after aortic valve closure. Next, the internal short-axis diameter of the LA can be measured in the same frame at a line extending parallel from the commissure between the non-coronary aortic valve cusp and the left coronary valve cusp to the distal edge of the left atrium.

[0095] Various methods for diagnosing MVD apart from LA / Ao measurement are known in the art, including, but not limited to, echocardiograms, electrocardiograms, chest x-rays, magnetic resonance imaging, exercise tests, stress tests, and / or cardiac catheterization. MVD staging systems generally refer to four basic groups: Stage A—At Risk: Risk factors for MVD are present; Stage B—Advanced: MVD is mild or moderate and there are no valvular symptoms; Stage C—Severe Asymptomatic: MVD is severe and there are no valvular symptoms; and Stage D—Severe Symptomatic: MVD is severe and causes symptoms. Stage B can be further divided into Stage B1 and Stage B2; Stage B1 is diagnosed when a heart murmur is detected but there is no radiographic or echocardiographic evidence of cardiac remodeling or remodeling not severe enough to meet the treatment criteria in current clinical trials; Stage B2 is diagnosed when a heart murmur is detected and there is radiographic or echocardiographic evidence of cardiac remodeling sufficient to meet the treatment criteria in current clinical trials. It is known in the art that an LA / Ao greater than 1.6 indicates MVD stage B2.

[0096] In certain embodiments, a method for treating MVD in a subject includes administering gene therapy to the subject, wherein the subject has an LA / Ao of about 1.6 to about 2.1, e.g., about 1.6, about 1.7, about 1.8, about 1.9, about 2, or about 2.1. In certain embodiments, the subject has an LA / Ao of about 1.6. In certain embodiments, the subject has an LA / Ao of about 1.8. In certain embodiments, the subject has an LA / Ao of about 2.1. In certain embodiments, the subject has an LA / Ao of less than about 2.1. Regardless of the dose, subjects with an initial LA / Ao of less than 2.1 have been found to respond to gene therapy over time and exhibit reversal of left atrial enlargement over time. Those skilled in the art will readily appreciate that measuring LA / Ao is only one method for diagnosing MVD. Therefore, those skilled in the art will be able to determine which measurements correspond to subjects with an initial LA / Ao of less than 2.1 when using other methods for diagnosing MVD. The present disclosure also contemplates methods for treating MVD in subjects with diagnostic measurements equivalent to subjects with an initial LA / Ao of less than 2.1 (e.g., a diagnosis equivalent to an initial LA / Ao of less than 2.1).

[0097] In certain embodiments, the method for treating MVD in a subject further comprises administering to the subject an effective amount of one or more additional therapeutic agents to treat MVD. Such additional therapeutic agents for treating MVD may include, but are not limited to, diuretics, anticoagulants (i.e., anticoagulants), and blood pressure medications. For example, in dogs, pimobendan is used to manage heart failure due to MMVD. Pimobendan is often used in combination with an ACE inhibitor such as enalapril or benazepril. In certain embodiments, the method for treating MVD in a subject further comprises administering to the subject an effective amount of pimobendan. Other additional therapeutic agents for treating MVD include furosemide, spironolactone (i.e., an aldosterone antagonist), and angiotensin-converting enzyme (ACE) inhibitors.

[0098] In certain embodiments, a method of treating MVD in a subject comprises administering to the subject (a) an effective amount of gene therapy comprising a first nucleic acid encoding soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21); and (b) an effective amount of pimobendan. In certain embodiments, a method of treating MVD in a subject comprises administering to the subject (a) a first rAAV comprising an AAV capsid comprising an AAV capsid protein and a first rAAV genome comprising a nucleic acid encoding sTGFβR2, and a second rAAV comprising an AAV capsid comprising an AAV capsid protein and a second rAAV genome comprising a nucleic acid encoding FGF21; and (b) an effective amount of pimobendan. In certain embodiments, a method of treating MVD in a subject comprises administering to the subject: (a) an rAAV comprising an AAV capsid comprising AAV capsid proteins, and a rAAV genome comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, wherein the first nucleic acid and the second nucleic acid are separated by a polycistronic element; and (b) an effective amount of pimobendan.

[0099] In dogs, pimobendan may be administered orally at a total daily dose of 0.23 mg / lb (0.5 mg / kg) body weight. In humans, pimobendan may be administered at a dose of 2.5 mg / day. Typically, the total daily dose is divided into two portions administered approximately 12 hours apart. In certain embodiments, the effective amount of pimobendan is about 0.05 to about 0.5 mg / kg. In some embodiments, the effective amount of pimobendan is about 0.05 to about 0.5 mg / kg, administered twice daily at a total daily dose of about 0.10 to about 1.0 mg / kg. In some embodiments, the effective amount of pimobendan is about 0.25 mg / kg, administered twice daily at a total daily dose of about 0.5 mg / kg. In some embodiments, the effective amount of pimobendan is 0.25 mg / kg, administered twice daily at a total daily dose of 0.5 mg / kg.

[0100] In certain embodiments, the effective amount of pimobendan is about 0.2 mg / kg to about 0.6 mg / kg body weight once daily. In certain embodiments, the effective amount of pimobendan is administered at about 0.2 mg / kg to about 0.6 mg / kg body weight per day. In certain embodiments, the effective amount of pimobendan is administered at about 0.2 mg / kg to about 0.5 mg / kg body weight per day. In certain embodiments, the daily dose of pimobendan is administered as two doses of about 0.1 mg / kg to about 0.3 mg / kg body weight. In certain embodiments, the daily dose of pimobendan is administered as two doses of about 0.1 mg / kg to about 0.3 mg / kg body weight every 12 hours. In certain embodiments, the daily dose of pimobendan is administered as two doses of 0.25 mg / kg body weight every 12 hours.

[0101] In certain embodiments, the additional therapeutic agent for treating MVD (e.g., pimobendan) is administered simultaneously with the gene therapy. In certain embodiments, the additional therapeutic agent for treating MVD (e.g., pimobendan) is administered at a different time than the gene therapy.

[0102] In certain embodiments, a method of treating MVD in a subject comprises administering an effective amount of gene therapy comprising a first nucleic acid encoding soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21) to a subject who has been administered an effective amount of a non-gene therapy therapeutic to treat MVD. In certain embodiments, a method of treating MVD in a subject comprises administering an effective amount of gene therapy comprising a first nucleic acid encoding soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21) to a subject who has been administered an effective amount of pimobendan.

[0103] In another aspect, the present disclosure provides a method for determining the likelihood of successful treatment of a subject with mitral valve disease with gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, comprising determining the subject's LA / Ao, wherein an LA / Ao of 2.1 or greater indicates a decreased likelihood of successful treatment with gene therapy, and an LA / Ao of about 1.6 to about 2.1 indicates an increased likelihood of successful treatment with gene therapy. In certain embodiments, successful treatment of MVD comprises a decrease in the subject's LA / Ao compared to the LA / Ao measured before gene therapy. In certain embodiments, successful treatment of MVD comprises a slower increase in the subject's LA / Ao over time compared to the level of increase in LA / Ao in subjects not receiving gene therapy. In certain embodiments, successful treatment of MVD comprises maintaining the subject's LA / Ao over time compared to the change in LA / Ao in subjects not receiving gene therapy.

[0104] In another aspect, the present disclosure provides a method for identifying a subject having mitral valve disease suitable for treatment with gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, comprising determining the subject's LA / Ao, wherein the subject is suitable for treatment with gene therapy if the subject has an LA / Ao of about 1.6 to about 2.1.

[0105] In certain embodiments, the mitral valve disease comprises one or more diseases or conditions selected from the group consisting of myxomatous mitral valve disease, mitral stenosis, mitral valve prolapse, and mitral regurgitation.

[0106] In certain embodiments, the gene therapy is administered to the subject intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally.

[0107] In certain embodiments, the subject is a member of any mammalian or non-mammalian species. Suitable subjects include, but are not limited to, humans, non-human primates, dogs, cats, ungulates (e.g., horses, cows, swine (e.g., pigs)), birds, rodents (e.g., rats, mice), and other subjects. In certain embodiments, the subject is a human. In certain embodiments, the subject is a dog. In certain embodiments, the subject is a breed selected from the group consisting of Cavalier King Charles Spaniel, Miniature Poodle, Shih Tzu, Maltese, Chihuahua, Cocker Spaniel, Miniature Schnauzer, Dachshund, Whippet, Pomeranian, and combinations thereof. In certain embodiments, the subject is a Cavalier King Charles Spaniel. [Example]

[0108] The following examples are offered by way of illustration and not by way of limitation. Example 1: Canine sTGFβR2 and FGF21 Recombinant AAV Vectors

[0109] This example provides canine sTGFβR2 and FGF21 recombinant adeno-associated virus (rAAV) vectors for expression of canine sTGFβR2 and FGF21 in cells transduced with the vectors (e.g., canine liver cells).

[0110] The rAAV-sTGFβR2 contains an rAAV genome including, from 5' to 3', the following genetic elements: a 5' ITR element, an apolipoprotein E (ApoE) binding site, a human alpha-1 antitrypsin (hAAT) promoter, a beta globin intron sequence, a canine sTGFβR2-Fc coding sequence, a WPRE3 sequence, an SV40 polyadenylation signal, and a 3' ITR element. The sequences of these elements are shown in Table 1. This vector is capable of expressing a canine sTGFβR2-Fc fusion protein in cells (e.g., hepatocytes) transduced with the vector.

[0111] rAAV-FGF21 contains an rAAV genome containing the following genetic elements from 5' to 3': 5' ITR elements, an apolipoprotein E (ApoE) binding site, a human alpha-1 antitrypsin (hAAT) promoter, a beta globin intron sequence, a canine FGF21 coding sequence, a WPRE3 sequence, an SV40 polyadenylation signal, and a 3' ITR element. The sequences of these elements are shown in Table 1. This vector is capable of expressing canine FGF21 protein in cells (e.g., hepatocytes) transduced with the vector. [Table 1]

[0112] The rAAV vectors disclosed herein can be packaged into AAV capsids, such as, but not limited to, AAV8 capsids. Generally, viral particles are produced using standard triple transfection of HEK293T cells and either iodixanol gradient purification, CsCl purification, or affinity and anion column purification. See, for example, Davidsohn et al. (2019) Proc. Natl. Acad. Sci. 116(47):23505-23511; and Nass et al. (2018) Mol. Ther. Methods Clin. Dev. 9:33-46. Packaged viral particles can be administered to wild-type animals or animals suffering from mitral valve disease. Example 2: sTGFβ2 and FGF21 protein expression in dogs with mitral valve disease

[0113] The effects of sTGFβR2 and FGF21 gene therapy were investigated in dogs with mitral valve disease. Dogs with stage B2 myxomatous mitral valve disease (MMVD) were recruited. Stage B2 MMVD refers to dogs with MMVD that have not yet developed signs of heart failure but have a moderate or high-pitched mitral valve murmur due to mitral valve leakage and cardiac hypertrophy.

[0114] The left atrial-to-aortic diameter ratio (LA / Ao) is the most commonly used method to assess left atrial (LA) size in dogs. Standard M-mode, 2D Doppler echocardiographic images, and video loops were recorded with continuous ECG monitoring for all measurements. LA / Ao was measured from the right parasternal short-axis view at the base of the heart as previously described. Briefly, the internal short-axis diameter of the aorta was measured along the commissure between the non-coronary aortic valve cusp and the right coronary valve cusp on the first frame after aortic valve closure. The internal short-axis diameter of the LA was measured in the same frame on a line extending parallel from the commissure between the non-coronary aortic valve cusp and the left coronary valve cusp to the distal edge of the left atrium. A normal LA / Ao was defined as <1.6. Stage B2 MMVD was defined as dogs with an LA / Ao of 1.7-3 and requiring treatment with pimobendan but not yet experiencing heart failure.

[0115] To examine protein expression from livers transduced with the rAAV-sTGFβR2 and rAAV-FGF21 vectors, dogs with stage B2 MMVD were intravenously administered rAAV-sTGFβR2 and rAAV-FGF21, each packaged in an AAV8 capsid (AAV8-sTGFβR2 and AAV8-FGF21, respectively; see Table 1 for sequences). AAV8-sTGFβR2 was administered at doses of 1E13, 3E13, or 5E13 vg / kg, and AAV8-FGF21 was administered at doses of 1E13 or 3E13 vg / kg. Virus titers were determined by ddPCR using gene-specific primers for the gene of interest (e.g., FGF21 or sTGFβR2).

[0116] The expression of sTGFβR2-Fc and FGF21 was measured by ELISA using antibodies against canine TGFβR2 and FGF21, respectively. Figures 1A and 1B show the expression levels of sTGFβR2 and FGF21 in treated dogs. As shown in Figure 1A, dogs administered 1E13, 3E13, or 5E13 vg / kg of AAV8-sTGFβR2 achieved stable, long-term expression of sTGFβR2 for 16, 16, and 32 months, respectively. Dogs administered either 1E13 or 3E13 vg / kg of AAV8-FGF21 also achieved stable, long-term expression of FGF21 for 16 and 32 months, respectively (Figure 1B). In Figure 1B, one dog in the 3E13 vg / kg dose cohort was a non-responder. Example 3: Gene therapy for mitral valve disease in dogs

[0117] Dogs with MVD typically experience enlargement of the left atrium of the heart due to damage caused by mitral valve dysfunction. LA / Ao has been shown to correlate with the progression of MVD, with a 0.1 increase in LA / Ao increasing the risk of progression to the next stage of MVD by approximately 11%.

[0118] To investigate whether sTGFβR2 and FGF21 gene therapy can treat canine MVD, dogs with stage B2 MMVD were administered AAV8-sTGFβR2 and AAV8-FGF21 at a dose of 1E13 vg / kg, respectively, or at a dose of 3E13 vg / kg, respectively. LA / Ao was measured over time in treated dogs. Figure 2 shows the LA / Ao measurements of dogs over time at various time points indicated. As shown in Figure 2, regardless of dose, dogs that responded to gene therapy over time and had an initial LA / Ao of less than 2.1 showed reversal of left atrial enlargement over time. In Figure 2, the dashed line indicates the entry criteria for an LA / Ao of 1.6, and the dotted line indicates an LA / Ao of 2.1, where all dogs responded to therapy.

[0119] To date, 17 dogs have been administered AAV8-sTGFβR2 and AAV8-FGF21, with no adverse safety events recorded, and four of these dogs have been on the study for more than two years. Example 4: Combination Treatment of Mitral Valve Disease in Dogs

[0120] Pimobendan is the current best-in-class drug labeled for use in dogs to manage congestive heart failure (CHF) due to dilated cardiomyopathy (DCM) or degenerative MVD. Because it would be unethical to withhold standard treatment in a clinical trial pilot study, dogs diagnosed with stage B2 MMVD and prescribed pimobendan were administered sTGFβR2 and FGF21 gene therapy. Echocardiograms were performed at baseline (0 months), 2 and 4 months post-treatment, and every 4 months up to 32 months post-treatment. Left atrial size was measured using echocardiograms in reference to the aortic and left ventricular fractional shortening (FS%). While pimobendan has limited ability to reverse pathological progression as measured by echocardiography, published data show a 0.08 reduction in LA / Ao after one month of pimobendan administration (Boswood et al., J Vet Intern Med, 2018, 32(1):72-85). A significant 0.3 reversal of left atrial enlargement was observed in dogs with MVD after administration of AAV8-sTGFβR2 and AAV8-FGF21 ("GT") (Figure 3), suggesting a 33% reduction in disease progression. Left atrial size was quantified using echocardiography by measuring the size of the left atrium relative to the aorta. As shown in Figure 3, dogs treated with pimobendan in combination with AAV8-sTGFβR2 and AAV8-FGF21 ("Pimo+GT") demonstrated a reduction in LA / Ao of approximately 0.3 over 32 months. In this study, 12 dogs were treated at the early time point and 3 dogs were treated at the final time point by subjective rolling admission. In contrast, based on published data reporting late LA / Ao values ​​(dashed line), the use of pimobendan alone can minimize progressive left atrial dilation by only 0.8 over 32 months (see, e.g., Nakamura et al. J. Vet. Intern. Med. 2017, 31(2):316-325).

[0121] Dogs treated with pimobendan in combination with AAV8-sTGFβR2 and AAV8-FGF21 ("Pimo+GT") were able to preserve cardiac function. Based on published data, using fractional shortening as a measure of cardiac contractility, treatment of dogs with MVD with pimobendan limits the decrease in contractility to approximately 5% over 28 months (dashed line in Figure 4; see, e.g., Nakamura et al. J. Vet. Intern. Med. 2017, 31(2):316-325). Therefore, even with current best-in-class medications, dogs with MVD typically exhibit a decrease in fractional shortening. As shown in Figure 4, dogs with MVD treated with Pimo+GT showed a 3% increase in fractional shortening over 28 months, indicating a reversal of disease progression.

[0122] Furthermore, MVD dogs treated with Pimo+GT demonstrated a longer delay in progression of more than 1.5 years compared with MVD dogs treated with standard care. As shown in Figure 5, MVD dogs treated with Pimo+GT demonstrated an increased time to progression of approximately 600 days compared with published data for MVD dogs treated with pimobendan alone and approximately 800 days compared with published data for MVD dogs treated with placebo. Based on published data, MVD dogs treated with pimobendan alone demonstrated an increased time to progression of more than approximately 200 days compared with MVD dogs treated with placebo. See, for example, Boswood et al., J Vet Intern Med, 2018, 32(1):72-85. Time to progression was measured based on the proportion of animals that had not yet reached the primary endpoint of onset of congestive heart failure, cardiac-related death, or euthanasia.

[0123] Importantly, no safety issues were reported in three dogs with MVD treated with AAV8-sTGFβR2 and AAV8-FGF21 therapy for more than 3 years. * * *

[0124] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be included within the scope of the appended claims.

[0125] All references (e.g., publications, or patents or patent applications) cited in this specification are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual reference (e.g., publication, or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0126] Other embodiments are within the scope of the following claims.

Claims

1. 1. A method of treating mitral valve disease in a subject, comprising administering to the subject a gene therapy comprising a first nucleic acid encoding a soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21), wherein the subject has a left atrial-to-aortic diameter ratio (LA / Ao) of about 1.6 to about 2.

1.

2. 10. The method of claim 1, wherein the subject has an LA / Ao of about 1.

6.

3. 10. The method of claim 1, wherein the subject has an LA / Ao of about 1.

8.

4. The method according to any one of claims 1 to 3, wherein the subject is a mammal.

5. The method of any one of claims 1 to 4, wherein the subject is a dog.

6. 6. The method of any one of claims 1 to 5, wherein the subject is a dog breed selected from the group consisting of Cavalier King Charles Spaniel, Miniature Poodle, Shih Tzu, Maltese, Chihuahua, Cocker Spaniel, Miniature Schnauzer, Dachshund, Whippet, Pomeranian, and combinations thereof.

7. The method of any one of claims 1 to 6, wherein the subject is a Cavalier King Charles Spaniel.

8. 8. The method of any one of claims 1 to 7, wherein the mitral valve disease comprises one or more diseases or conditions selected from the group consisting of myxomatous mitral valve disease, mitral stenosis, mitral valve prolapse, and mitral regurgitation.

9. 9. The method of any one of claims 1 to 8, further comprising determining the subject's LA / Ao prior to administration of gene therapy, and determining the subject's LA / Ao at a period after administration of gene therapy.

10. The method of any one of claims 1 to 9, wherein the LA / Ao of the subject after administration of the gene therapy is reduced compared to the LA / Ao of the subject before administration of the gene therapy.

11. The method of any one of claims 1 to 10, wherein the subject is administered an effective amount of pimobendan.

12. The method of any one of claims 1 to 10, further comprising administering to the subject an effective amount of pimobendan.

13. A method for treating mitral valve disease in a subject, comprising administering to the subject a gene therapy comprising a first nucleic acid encoding soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21); and / or an effective amount of pimobendan.

14. A method for treating mitral valve disease in a subject receiving an effective amount of pimobendan, comprising administering to the subject a gene therapy comprising a first nucleic acid encoding soluble transforming growth factor beta receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

15. The method according to any one of claims 11 to 14, wherein the effective amount of pimobendan is 0.25 mg / kg.

16. 14. The method of claim 12 or 13, wherein the gene therapy and pimobendan are administered simultaneously.

17. 17. The method of any one of claims 11 to 16, wherein the pimobendan is administered orally, and optionally, the effective amount of the pimobendan is 0.25 mg / kg twice daily.

18. 1. A method for determining the likelihood of successful treatment in a subject having mitral valve disease with gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, the method comprising determining an LA / Ao in the subject, wherein an LA / Ao of 2.1 or greater indicates a decreased likelihood of successful treatment with the gene therapy, and an LA / Ao of about 1.6 to about 2.1 indicates an increased likelihood of successful treatment with the gene therapy.

19. 20. The method of claim 18, wherein successful treatment of mitral valve disease comprises a decrease in the subject's LA / Ao compared to the LA / Ao before gene therapy.

20. 1. A method for identifying a subject having mitral valve disease suitable for treatment with gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, the method comprising determining the subject's LA / Ao, wherein the subject is suitable for treatment with the gene therapy if the subject has an LA / Ao of about 1.6 to about 2.

1.

21. The method of any one of claims 18 to 20, wherein the subject is a mammal.

22. The method of any one of claims 18 to 21, wherein the subject is a dog.

23. 23. The method of claim 22, wherein the dog is a breed selected from the group consisting of Cavalier King Charles Spaniel, Miniature Poodle, Shih Tzu, Maltese, Chihuahua, Cocker Spaniel, Miniature Schnauzer, Dachshund, Whippet, Pomeranian, and combinations thereof.

24. The method of any one of claims 18 to 23, wherein the subject is a Cavalier King Charles Spaniel.

25. 25. The method of any one of claims 18 to 24, wherein the mitral valve disease comprises one or more diseases or conditions selected from the group consisting of myxomatous mitral valve disease, mitral stenosis, mitral valve prolapse, and mitral regurgitation.

26. The method of any one of claims 1 to 25, wherein the gene therapy is administered intravenously.

27. 27. The method of any one of claims 1 to 26, wherein the first nucleic acid comprises a first transcriptional regulatory element operably linked to the sTGFβR2 coding sequence.

28. 28. The method of any one of claims 1 to 27, wherein the sTGFβR2 coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, or 24.

29. 29. The method of any one of claims 1 to 28, wherein the sTGFβR2 coding sequence further comprises a heterologous or native secretory signal sequence, wherein the signal sequence is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, 5, or 6.

30. 30. The method of any one of claims 1 to 29, wherein the sTGFβR2 coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9, 10, 11, 12, or 25.

31. 28. The method of any one of claims 1 to 27, wherein the sTGFβR2 coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, 14, 15, or 26.

32. 32. The method of claim 31 , wherein the sTGFβR2 coding sequence further encodes a secretory signal sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 or 8.

33. 33. The method of claim 31 or 32, wherein the sTGFβR2 coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, 17, 18, 19, or 27.

34. 34. The method of any one of claims 1 to 33, wherein the second nucleic acid comprises a second transcriptional regulatory element operably linked to the FGF21 coding sequence.

35. 35. The method of any one of claims 1 to 34, wherein the FGF21 coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28, 29, 30, or 31.

36. 36. The method of any one of claims 1 to 35, wherein the FGF21 coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32, 33, or 34.

37. 37. The method of any one of claims 34 to 36, wherein the first and second transcriptional regulatory elements each comprise one or more ApoE binding sites and / or a hAAT promoter.

38. 38. The method of any one of claims 34 to 37, wherein the first and second transcriptional regulatory elements each comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 38, 39, 40, and / or 48.

39. 39. The method of any one of claims 1 to 38, wherein the first and second nucleic acids each further comprise a post-transcriptional regulatory element.

40. 40. The method of claim 39, wherein the transcriptional regulatory element comprises a polyadenylation signal and / or a WPRE sequence.

41. 41. The method of claim 40, wherein the polyadenylation signal is an SV40 polyadenylation signal.

42. 41. The method of claim 40, wherein the WPRE sequence is a WPRE3 sequence.

43. 43. The method of any one of claims 39-42, wherein the post-transcriptional regulatory element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 50, 51, or 70.

44. 44. The method of any one of claims 1 to 43, wherein the first nucleic acid is contained in a first vector and the second nucleic acid is contained in a second vector.

45. 45. The method of claim 44, wherein the first vector and / or the second vector are each viral vectors, optionally each independently selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, and alphavirus.

46. 46. ​​The method of claim 44 or 45, the first vector is an AAV vector contained within a first recombinant AAV (rAAV), the first rAAV comprising an AAV capsid comprising AAV capsid proteins; and a first rAAV genome; and / or The method, wherein the second vector is an AAV vector contained within a second rAAV, the second rAAV comprising: an AAV capsid comprising AAV capsid proteins; and a second rAAV genome.

47. The gene therapy an AAV capsid comprising an AAV capsid protein; and a first recombinant AAV (rAAV) comprising a first rAAV genome comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:26; an AAV capsid comprising an AAV capsid protein; and 47. The method of any one of claims 1 to 46, comprising a second rAAV comprising a second rAAV genome comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:

33.

48. 48. The method of claim 46 or 47, wherein the first rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

66.

49. 49. The method of any one of claims 46-48, wherein the first rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence and a 3' inverted terminal repeat (3' ITR) nucleotide sequence.

50. 50. The method of any one of claims 46 to 49, wherein the 5' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 58 or 59, and / or the 3' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60 or 61.

51. 51. The method of any one of claims 46-50, wherein the first rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

67.

52. 52. The method of any one of claims 46-51, wherein the second rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

68.

53. 53. The method of any one of claims 46-52, wherein the second rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence and a 3' inverted terminal repeat (3' ITR) nucleotide sequence.

54. 54. The method of claim 53, wherein the 5' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 58 or 59, and / or the 3' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60 or 61.

55. 55. The method of any one of claims 46-54, wherein the second rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

69.

56. 56. The method of any one of claims 46-55, wherein the AAV capsid protein is derived from a Clade A, Clade B, Clade C, Clade D, Clade E, Clade F, Clade G, Clade H, Clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof.

57. 57. The method of any one of claims 46-56, wherein the AAV capsid protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 63, 64, and / or 65.

58. 44. The method of any one of claims 1 to 43, wherein the first and second nucleic acids are comprised in a vector, and optionally the first and second nucleic acids are separated by a polycistronic element.

59. The vector the first nucleic acid comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:26; and 59. The method of claim 58, comprising a second nucleic acid comprising a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO:

33.

60. 60. The method of claim 58 or 59, wherein the first and second nucleic acids are separated by a polycistronic element.

61. 61. The method of claim 60, wherein the polycistronic element is an IRES or 2A sequence.

62. 62. The method of claim 60 or 61, wherein the polycistronic element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 55, 56, or 57.

63. 63. The method of any one of claims 58 to 62, wherein the vector is an AAV vector contained within a recombinant AAV (rAAV), the rAAV comprising an AAV capsid comprising AAV capsid proteins; and a rAAV genome.

64. 64. The method of claim 63, wherein the AAV capsid protein is derived from a Clade A, Clade B, Clade C, Clade D, Clade E, Clade F, Clade G, Clade H, Clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof.

65. 65. The method of claim 63 or 64, wherein the AAV capsid protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 63, 64, and / or 65.

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