Gene therapy methods for treating mitral valve disease

Abstract

Provided herein are methods of treating a mitral valve disease in a subject. The method comprises administering to the subject a gene therapy comprising a first nucleic acid encoding soluble transforming growth factor beta receptor 2 (sTGF beta R2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF 21). Also provided are methods of treating a mitral valve disease in a subject comprising administering a gene therapy to the subject in combination with an additional therapeutic agent (e.g., pimobendan).

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 376,472, filed September 21, 2022, and U.S. Provisional Patent Application Serial No. 63 / 503,318, filed May 19, 2023, the entire disclosures of which are hereby incorporated by reference herein.

[0003] Reference Sequence Listing

[0004] This application contains a Sequence Listing that has been submitted electronically in accordance with 37 C.F.R. § 1.821 and 1.825 and is hereby incorporated by reference in its entirety (the copy of the Sequence Listing (203422_SL.XML) created on September 19, 2023, is named “203422_SL.XML” and is 106,831 bytes in size). Background of the Invention

[0005] Mitral valve disease is a degenerative condition that over time leads to mitral valve dysfunction. The mitral valve normally acts as a seal between the left atrium and the left ventricle such that when the ventricle contracts, the mitral valve closes and prevents blood from leaking back into the atrium, a phenomenon known as regurgitation. If left untreated, as the left atrium grows larger to accommodate the extra blood from the regurgitation, the pressure in the atrium increases, eventually leading to congestive heart failure due to fluid buildup in the lungs.

[0006] In canines, approximately 10% of canines presenting to primary care veterinary practices have heart disease, and among them, myxomatous mitral valve disease (MMVD) is the most common heart disease in canines in many parts of the world. MMVD accounts for approximately 75% of canine heart disease cases in North America. MMVD most commonly affects the mitral valve, but in at least 30% of cases, the tricuspid valve is also involved. The staging system for MMVD describes four basic stages of heart disease and heart failure: Stage A identifies canines at high risk of developing heart disease but currently without identifiable cardiac structural disorders; Stage B identifies canines with structural heart disease but who have never developed clinical signs caused by heart failure. Stage B1 refers to asymptomatic canines without radiographic or echocardiographic evidence of heart remodeling in response to MMVD to mild, and Stage B2 refers to asymptomatic canines with more severe mitral regurgitation that is hemodynamically severe and of long duration enough to cause radiographic and echocardiographic findings of left atrial and left ventricular enlargement; Stage C identifies canines with current or past clinical signs caused by MMVD; and Stage D identifies canines with end-stage MMVD, in which the clinical signs of heart failure are refractory to standard treatment. Pimobendan is fully approved for managing signs of mild, moderate, or severe congestive heart failure caused by MMVD in canines and has recently been approved for treating canines with heart murmurs and cardiac enlargement but not yet in congestive heart failure in the preclinical stage of MMVD. However, there is no cure for congestive heart failure caused by MMVD.

[0007] Accordingly, there is a need in the art for improved methods for treating mitral valve disease. SUMMARY OF THE INVENTION

[0008] The present disclosure provides methods for treating mitral valve disease in a subject. The methods generally comprise 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). In certain embodiments, the gene therapy is administered in combination with an effective amount of pimobendan or to a subject currently receiving treatment with pimobendan.

[0009] Accordingly, in one aspect, the present disclosure provides a method of treating a subject having mitral valve disease, 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), wherein the left atrium to aortic root ratio (LA / Ao) of the subject is from about 1.6 to about 2.1. In some embodiments, the subject's LA / Ao is about 1.6. In some embodiments, the subject's LA / Ao is about 1.8. In some embodiments, the subject is a mammal.

[0010] In certain embodiments, the subject is a canine. In some embodiments, the subject is a canine breed selected from: 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.

[0011] In certain embodiments, the mitral valve disease comprises one or more diseases or conditions selected from: myxomatous mitral valve disease, mitral stenosis, mitral valve prolapse, and mitral regurgitation.

[0012] In certain embodiments, the method further comprises determining the subject's LA / Ao prior to administering the gene therapy and determining the subject's LA / Ao over a period of time after administering the gene therapy. In certain embodiments, the subject's LA / Ao is decreased after administering the gene therapy as compared to the subject's LA / Ao prior to administering the gene therapy.

[0013] In certain embodiments, the subject has been administered an effective amount of pimobendan.

[0014] In certain embodiments, the method further comprises administering to the subject an effective amount of pimobendan.

[0015] In another aspect, the present disclosure provides a method of treating a subject having mitral valve disease, the method comprising administering to the subject a gene therapy and / or an effective amount of pimobendan, the 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).

[0016] In another aspect, the present disclosure provides a method of treating mitral valve disease in a subject that has received an effective amount of pimobendan, the method comprising administering to the subject a gene therapy comprising a first nucleic acid encoding soluble transforming growth factor β receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

[0017] In certain embodiments, the effective amount of pimobendan is 0.25 mg / kg. In some embodiments, the 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.

[0018] In another aspect, the present disclosure provides a method of determining the likelihood of successful treatment of a subject having mitral valve disease with a gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, the method comprising determining the LA / Ao of the subject, wherein an LA / Ao greater than 2.1 indicates a decreased likelihood of successful treatment with the gene therapy and an LA / Ao of from about 1.6 to about 2.1 indicates an increased likelihood of successful treatment with the gene therapy. In certain embodiments, successful treatment of mitral valve disease comprises a decrease in the LA / Ao of the subject compared to the LA / Ao prior to the gene therapy.

[0019] In another aspect, the present disclosure provides a method of identifying a subject having mitral valve disease suitable for treatment with a gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, the method comprising determining the LA / Ao of the subject, wherein if the LA / Ao of the subject is from about 1.6 to about 2.1, the subject is suitable for treatment with the gene therapy.

[0020] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a canine. In certain embodiments, the canine 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.

[0021] 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.

[0022] In certain embodiments, the gene therapy is administered intravenously.

[0023] In certain embodiments, the first nucleic acid comprises a first transcriptional regulatory element operably linked to an 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 secretion 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. 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 secretion 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.

[0024] In certain embodiments, the second nucleic acid comprises a second transcriptional regulatory element operably linked to an FGF21 coding sequence. In certain 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.

[0025] In certain embodiments, each of the first transcriptional regulatory element and the second transcriptional regulatory element comprises one or more ApoE binding sites and / or the hAAT promoter. In certain embodiments, each of the first transcriptional regulatory element and the second 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:38, 39, 40 and / or 48.

[0026] In certain embodiments, each of the first nucleic acid and the second nucleic acid further comprises 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 the SV40 polyadenylation signal. In certain embodiments, the WPRE sequence is the 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.

[0027] 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 a viral vector, optionally wherein each is independently selected from 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), wherein the first rAAV comprises: an AAV capsid containing an AAV capsid protein; and a first rAAV genome; and / or the second vector is an AAV vector contained within a second rAAV, wherein the second rAAV comprises: an AAV capsid containing an AAV capsid protein; and a second rAAV genome.

[0028] In certain embodiments, the gene therapy comprises: a first recombinant AAV (rAAV) comprising: an AAV capsid containing an AAV capsid protein; and a first rAAV genome comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:26; and a second rAAV comprising: an AAV capsid containing an AAV capsid protein; and a second rAAV genome comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:33.

[0029] 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.

[0030] 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 with 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 with 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 with SEQ ID NO: 67.

[0031] 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 with SEQ ID NO: 68.

[0032] 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 with 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 with 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 with SEQ ID NO: 69.

[0033] In certain embodiments, the AAV capsid protein is 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 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 NO: 63, 64, and / or 65.

[0034] In certain embodiments, the first nucleic acid and the second nucleic acid are contained within a vector, optionally wherein the first nucleic acid and the second nucleic acid 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 nucleic acid and the second nucleic acid are separated by a polycistronic element. In certain embodiments, the polycistronic element is an IRES or a 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.

[0035] In certain embodiments, the vector is an AAV vector contained within a recombinant AAV (rAAV), wherein the rAAV comprises: an AAV capsid comprising an AAV capsid protein; and an rAAV genome. In certain embodiments, the AAV capsid protein is 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 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 NO: 63, 64, and / or 65. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A and Figure 1B are graphs showing sTGFβR2 ( Figure 1A ) and FGF21 ( Figure 1B ) expression levels in subjects administered AAV8-sTGFβR2 and AAV8-FGF21 at indicated doses.

[0037] Figure 2 is a graph showing LA / Ao over time in subjects administered AAV8-sTGFβR2 and AAV8-FGF21. Each symbol represents an individual subject.

[0038] Figure 3 is a graph showing the mean change in LA / Ao relative to baseline over time in subjects administered pimobendan in combination with AAV8-sTGFβR2 and AAV8-FGF21 (Pimo+GT). Error bars represent the interquartile range. Dashed lines represent the change in LA / Ao over time based on published data from subjects administered pimobendan alone.

[0039] Figure 4 is a graph showing the mean change in percentage left ventricular fractional shortening (FS%) relative to baseline over time in subjects administered pimobendan in combination with AAV8-sTGFβR2 and AAV8-FGF21 (Pimo+GT). Error bars represent the interquartile range. Dashed lines represent the change in fractional shortening over time based on published data from subjects administered pimobendan alone.

[0040] Figure 5 is a graph showing the percentage of subjects administered pimobendan in combination with AAV8-sTGFβR2 and AAV8-FGF21 (Pimo+GT) who have not reached the primary endpoint over time. This data overlaps with published data from subjects administered pimobendan alone or placebo. DETAILED DESCRIPTION

[0041] The present disclosure provides methods of treating mitral valve disease in a subject. The methods generally comprise administering to the subject a gene therapy comprising a first nucleic acid encoding soluble transforming growth factor β 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 receiving pimobendan treatment.

[0042] The present disclosure is based on the discovery that a certain subject population (e.g., canines) with mitral valve disease responds well to sTGFβR2 and FGF21 (sTGFβR2 / FGF21) gene therapy. In subjects with an initial left atrial to aortic root ratio (LA / Ao) of less than 2.1 who receive sTGFβR2 / FGF21 gene therapy, the LA / Ao remains or decreases over time. The present invention is also based on the discovery that pimobendan in combination with sTGFβR2 / FGF21 gene therapy results in a synergistic reversal of left atrial enlargement in subjects (e.g., canines) with MVD. Pimobendan is currently the best-in-class drug for treating MVD and has been shown to delay the onset of congestive heart failure by reducing heart size.

[0043] Definitions

[0044] As used herein, the term "replication-deficient adeno-associated virus" refers to an AAV comprising a genome lacking the Rep and Cap genes.

[0045] 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 does not integrate into the chromosomal DNA of the cell. Those skilled in the art will understand that the portion of the rAAV genome containing the transcriptional regulatory element operably linked to the coding sequence can be in the sense or antisense orientation relative to the transcriptional direction of the coding sequence.

[0046] As used herein, "percent identity" between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the aligned sequence pairs by 100 and dividing by the length of the aligned region including internal gaps. Identity scores count only perfect matches and do not consider the degree of similarity of the amino acids to each other. The length includes only internal gaps and not gaps at the ends of the sequences.

[0047] As used herein, the term "coding sequence" refers to the complementary DNA (cDNA) portion encoding a polypeptide, starting with a start codon and ending with a stop codon. Due to alternative splicing, alternative translation initiation, and variation within a population, a gene can have one or more coding sequences. The coding sequence can be wild-type or codon-optimized.

[0048] As used herein, the term "codon optimization" refers to altering the coding sequence of a gene (e.g., by nucleotide substitution) without changing the amino acid sequence of the polypeptide encoded by the coding sequence. Such codon changes are advantageous because they can increase the translational 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.

[0049] As used herein, the term "transcription regulatory element" or "TRE" refers to a cis-acting nucleotide sequence, such as a DNA sequence, that regulates (e.g., controls, increases, or decreases) the transcription of an operably linked nucleotide sequence by RNA polymerase to form an RNA molecule. A TRE relies on one or more trans-acting molecules, such as transcription factors, to regulate transcription. Thus, a TRE can regulate transcription in different ways when it contacts different trans-acting molecules, e.g., when it is in different types of cells. A TRE can comprise one or more promoter elements and / or enhancer sequences. One of ordinary skill in the art will understand that promoter and enhancer sequences in a gene can be in close proximity in position, and the term "promoter" can refer to a sequence that comprises a promoter element and an enhancer sequence. Thus, the term "promoter" does not exclude enhancer sequences in the sequence. Promoter and enhancer sequences need not be derived from the same gene or species, and the sequence of each promoter or enhancer sequence can be the same as or substantially the same as the corresponding endogenous sequence in the genome.

[0050] As used herein, the term "operably linked" is used to describe the connection between a TRE and a coding sequence to be transcribed. Generally, gene expression is placed under the control of a TRE that comprises one or more promoter and / or enhancer sequences. A coding sequence is "operably linked" to a TRE if the transcription of the coding sequence is controlled or affected by the TRE. The promoter and enhancer sequences of a 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.

[0051] As used herein, the term "polyadenylation signal" or "polyadenylation sequence" refers to a DNA sequence that constitutes a polyadenylation signal sequence when transcribed into RNA. The polyadenylation sequence can be native (e.g., relative to the coding sequence of a gene) or exogenous. An exogenous polyadenylation sequence can be a mammalian or viral polyadenylation sequence (e.g., the SV40 polyadenylation sequence).

[0052] As used herein, an "exogenous polyadenylation sequence" refers to a polyadenylation sequence that is not the same as or substantially the same as the endogenous polyadenylation sequence of the coding sequence of a gene. In certain embodiments, the exogenous polyadenylation sequence can be of the same species (e.g., human) or a different species (e.g., viral).

[0053] As used herein, in the context of administering a viral vector (e.g., recombinant AAV) to a subject, the term "effective amount" refers to the amount of the viral vector that achieves the desired prophylactic or therapeutic effect. In the context of administering a compound, the effective amount is the amount of the compound that achieves the desired prophylactic or therapeutic effect.

[0054] As used herein, the term "polynucleotide" in its broadest sense includes any compound and / or substance that comprises a polymer of nucleotides linked via phosphodiester bonds.

[0055] As used herein, the term "treatment" refers to the therapeutic or prophylactic measures described herein. A "treatment" method involves administering a polynucleotide to a subject having a disease or disorder or predisposed to such a disease or disorder in order to prevent, cure, delay the disease or disorder or a recurrent disease or disorder, reduce its severity, or improve one or more of its symptoms, or in order to extend the survival of the subject beyond that expected in the absence of such treatment.

[0056] As used herein, in the context of administering a therapy to a subject, the term "effective amount" refers to the amount of the therapy that achieves the desired prophylactic or therapeutic effect.

[0057] 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 canine. In certain embodiments, the canine subject is a canine breed such as a Cavalier King Charles Spaniel, Miniature Poodle, Shih Tzu, Maltese, Chihuahua, Cocker Spaniel, Miniature Schnauzer, Dachshund, Whippet, Pomeranian, or a mixed breed thereof.

[0058] As used herein, the term "about" when used in conjunction with a numerical value means a value that encompasses a range that is 5% less than the indicated value at the lower limit and 5% greater than the indicated value at the upper limit.

[0059] Polynucleotides, vectors, and compositions

[0060] In one aspect, the methods disclosed herein employ a first nucleic acid encoding soluble transforming growth factor β receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

[0061] In certain embodiments, the sTGFβR2 coding sequence encodes a polypeptide comprising all or substantially all of the extracellular portion of TGFβR2 and not encoding 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, murine, or canine TGFβR2.

[0062] 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 sequences shown 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 sequences shown in SEQ ID NO:1, 2, or 3.

[0063] 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 secretion signal sequence. In certain embodiments, the TGFβR2 secretion signal sequence is an endogenous secretion signal sequence. In certain embodiments, the TGFβR2 secretion signal sequence is a heterologous secretion signal sequence. For example, the heterologous secretion signal sequence can be obtained from the TGFβR2 secretion signal sequences of different species. Exemplary TGFβR2 secretion signal sequences include, but are not limited to, the secretion signal sequences from human, murine, and canine TGFβR2. Thus, the sTGFβR2 coding sequence can also encode a heterologous or endogenous secretion signal sequence. In certain embodiments, the heterologous or endogenous secretion 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 shown in SEQ ID NO:7 or 8. In certain embodiments, the heterologous or endogenous secretion 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 shown in SEQ ID NO:4, 5, or 6.

[0064] 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 shown 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 shown in SEQ ID NO:9, 10, 11, or 12.

[0065] In certain embodiments, the sTGFβR2 coding sequence also encodes a peptide to achieve an extended half-life. Such peptides include, but are not limited to, an IgG constant region or a fragment thereof (e.g., the Fc domain), human serum albumin (HSA), or an albumin-binding polypeptide. In certain embodiments, the sTGFβR2 coding sequence also encodes an Fc domain (referred to herein as the sTGFβR2-Fc coding sequence). Exemplary Fc domains include wild-type Fc domains from human, murine, 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 shown 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 shown in SEQ ID NO: 20 or 21.

[0066] In certain embodiments, the sTGFβR2-Fc 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 shown 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 shown in SEQ ID NO: 24 or 25.

[0067] 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, murine, or canine FGF21. In certain embodiments, the FGF21 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 shown 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 shown in SEQ ID NO: 28, 29, 30, or 31.

[0068] In certain embodiments, the first nucleic acid encoding soluble transforming growth factor β receptor 2 (sTGFβR2) is each operably linked to a first transcriptional regulatory element (TRE). In certain embodiments, the 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).

[0069] In certain embodiments, TRE is active in a wide range of mammalian cells. Such TREs can include constitutive promoters and / or enhancer sequences, including the cytomegalovirus (CMV) 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 shown in SEQ ID NO: 41); the CMV enhancer sequence, the CBA promoter, and the splice acceptor from exon 3 of the rabbit β-globin gene, collectively referred to as the CAG 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 shown in SEQ ID NO: 42); the 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 shown in SEQ ID NO: 43); the chicken β-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 shown in SEQ ID NO: 44); the 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 shown in SEQ ID NO: 45); the 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 shown in SEQ ID NO: 46);The human elongation factor 1α (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 sequences shown 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 β-actin promoter; the human neuron-specific enolase (ENO2) promoter; the human β-glucuronidase (GUSB) promoter; and / or the human methyl-CpG binding protein 2 (MeCP2) promoter. Any of these TREs or elements within these TREs can be combined in any order to drive efficient transcription.;

[0070] Alternatively, the TRE can be a tissue-specific TRE, i.e., it is active in a specific tissue and / or organ. Tissue-specific TREs include 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 tissues by methods well known in the art.

[0071] In certain embodiments, the TRE is liver-specific, i.e., it is active in liver cells. Liver-specific TREs include, but are not limited to, those provided in the Liver-Specific Promoter Database (LSPD, rulai.cshl.edu / LSPD / ); the human alpha-1-antitrypsin (hAAT) 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 shown in SEQ ID NO:38); the apolipoprotein E (ApoE) binding site (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 shown in SEQ ID NO:39 or 40); the human albumin (hAlb) or minimal promoter; the transthyretin (TTR) promoter or the TTR minimal promoter (TTRm); the apolipoprotein A1 (APOA1) promoter or minimal promoter; the complement factor B (CFB) promoter; the ketohexokinase (KHK) promoter; the hemopexin (HPX) promoter or minimal promoter; the nicotinamide N-methyltransferase (NNMT) promoter or minimal promoter; the (liver) carboxylesterase 1 (CES1) promoter or minimal promoter; the protein C (PROC) promoter or minimal promoter; the apolipoprotein C3 (APOC3) promoter or minimal promoter; the mannan-binding lectin serine protease 2 (MASP2) promoter or minimal promoter; the hepcidin antimicrobial peptide (HAMP) promoter or minimal promoter; and the serine protease inhibitor (serpin) peptidase inhibitor clade C (antithrombin) member 1 (SERPINC1) promoter or minimal promoter.

[0072] The TRE can be an inducible promoter. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when such expression is needed, or turn off the expression when it is not needed. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0073] In certain embodiments, the first nucleic acid and / or the second nucleic acid comprises two or more TREs, optionally comprising at least one TRE disclosed herein. Those skilled in the art will understand that any of these TREs can be combined in any order, and combinations of constitutive TREs and tissue-specific TREs can drive efficient and tissue-specific transcription.

[0074] In certain embodiments, the TRE may further comprise an intron sequence. Such introns can increase transgene expression, for example, by reducing transcriptional silencing and enhancing mRNA export from the nucleus to the cytoplasm. The intron may comprise the native intron sequence of sTGFβR2 or FGF21, intron sequences of the same gene from different species, intron sequences of different genes from the same species, and / or synthetic intron sequences. 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 (e.g., in Sibley et al. Nature Reviews Genetics. 2016, 17:407-21, which is incorporated herein by reference in its entirety). Exemplary intron sequences are provided in Lu et al., Molecular Therapy. 2013, 21(5):954-63, and Lu et al., Hum. Gene Ther. 2017, 28(1):125-34, which are incorporated herein by reference in their entirety. Suitable intron sequences include, but are not limited to, the minute virus of mice (MVM) intron (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 shown in SEQ ID NO:47); the β-globin intron 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 shown in SEQ ID NO:48); and the SV40 intron sequence.

[0075] In certain embodiments, the first nucleic acid and / or the second nucleic acid further comprises a post-transcriptional regulatory element. A post-transcriptional regulatory element can be any sequence that effectively terminates transcription, and those skilled in the art should understand that such sequences can be isolated from any gene expressed in the cell in which the transcription coding sequence is desired.

[0076] In certain embodiments, the post-transcriptional regulatory element comprises a polyadenylation signal sequence. In certain embodiments, the polyadenylation signal sequence is the same as or substantially the same as 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 the 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 shown in SEQ ID NO:51); the 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 shown in SEQ ID NO:52); the rabbit β-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 shown in SEQ ID NO:53); or the 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 shown in SEQ ID NO:54).

[0077] In certain embodiments, the post-transcriptional regulatory element includes the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE). In certain embodiments, the post-transcriptional regulatory element comprises a WPRE sequence (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 shown in SEQ ID NO:49); or a WPRE3 sequence (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 shown in SEQ ID NO:50 or 70).

[0078] The first nucleic acid and / or the 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 within a first vector, and the second nucleic acid is contained within a second vector. In certain embodiments, the first nucleic acid and the second nucleic acid are contained within a single vector. Where the first nucleic acid and the second nucleic acid are contained within a single vector, the first nucleic acid and the second nucleic acid can be separated by a polycistronic element.

[0079] In certain embodiments, the polycistronic element comprises a nucleotide sequence encoding an internal ribosome entry site (IRES). An IRES is an element that facilitates direct entry of internal ribosomes into the start codon (such as ATG) of the protein-coding region, resulting in cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to those obtainable 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 obtainable from, for example, encephalomyocarditis virus, rhinovirus, foot-and-mouth disease virus, 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 upon translation. Various 2A sequences are known to those skilled in the art, including but not limited to sequences found in members of the Picornaviridae virus 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 cardioviruses such as Theilovirus and encephalomyocarditis virus. The 2A sequences derived 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 sequences shown in SEQ ID NO:55, 56, or 57.

[0080] 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.

[0081] In certain embodiments, the vector is a viral vector. The viral vector can be replication-competent or replication-incompetent. The viral vector can be integrative or non-integrative. Many virus-based systems have been developed for gene transfer into mammalian cells, and one of ordinary skill in the art can select a suitable viral vector. Exemplary viral vectors include, but are not limited to, adenoviral vectors (e.g., adenovirus 5), adeno-associated virus (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, herpes viral vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flaviviruses (e.g., Kunjin virus, West Nile virus, dengue virus), rhabdoviral vectors (e.g., rabies virus, VSV), measles viral vectors, Newcastle disease virus vectors, poxviral vectors, and picornaviral vectors (e.g., coxsackievirus). In certain embodiments, the viral vector is selected from adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, and alphavirus.

[0082] In certain embodiments, the vector is an AAV vector. In certain embodiments, the vector is single-stranded AAV. In certain embodiments, the vector is self-complementary AAV.

[0083] In certain embodiments, the vector is an AAV vector contained in recombinant AAV (rAAV). In certain embodiments, rAAV comprises: an AAV capsid containing AAV capsid proteins, and an rAAV genome.

[0084] 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 proteins can be 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 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 shown 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 1-738 of SEQ ID NO:63; 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 138-738 of SEQ ID NO:63; 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 204-738 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; 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: 64; 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.

[0085] In certain embodiments, the rAAV comprises: an AAV capsid comprising an AAV capsid protein, and an rAAV genome comprising a nucleic acid encoding sTGFβR2. In certain embodiments, the rAAV comprises: an AAV capsid comprising an AAV capsid protein, and an rAAV genome comprising a nucleic acid encoding sTGFβR2-Fc. In certain embodiments, the rAAV comprises: an AAV capsid comprising an AAV capsid protein, 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 an AAV capsid protein, 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.

[0086] In certain embodiments, the rAAV comprises: an AAV capsid comprising an AAV capsid protein, and an rAAV genome comprising a nucleic acid encoding FGF21. In certain embodiments, the rAAV comprises: an AAV capsid comprising an AAV capsid protein, 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 shown in SEQ ID NO:33. In certain embodiments, the rAAV comprises: an AAV capsid comprising an AAV capsid protein, 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 shown in SEQ ID NO:67.

[0087] In certain embodiments, the rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence encoding the sequence and a 3' inverted terminal repeat (3' ITR) nucleotide sequence at the 3' of the coding sequence. In certain embodiments, the rAAV genome comprises a 5' ITR at the 5' of the TRE and a 3' ITR at the 3' of the coding sequence. ITR sequences from any AAV serotype or variants thereof can be used in the rAAV genomes disclosed herein. The 5' and 3' ITRs can be from the same serotype of AAV or from different serotypes of AAV. Exemplary ITRs used in the rAAV genomes disclosed herein are shown in SEQ ID NO:58, 59, 60 and 61.

[0088] In certain embodiments, the 5’ ITR or the 3’ ITR is from AAV2. In certain embodiments, both the 5’ ITR and the 3’ ITR are 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 shown 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 shown in SEQ ID NO:60 or 61. 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 shown 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 shown in SEQ ID NO:61.

[0089] 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 shown in SEQ ID NO:59); a transcriptional regulatory element operably linked to a nucleic acid encoding sTGFβR2 or sTGFβR2-Fc (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 shown in SEQ ID NO:2, 11, 20, 24, 25, 38, 39, 40, 47 or 48); a post-transcriptional regulatory element (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 shown in SEQ ID NO:50, 51 or 70); and a 3' 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 shown 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 shown in SEQ ID NO:67).

[0090] 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 shown in SEQ ID NO:59); a transcriptional regulatory element operably linked to a nucleic acid encoding FGF21 (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 shown in SEQ ID NO:30, 38, 39, 40, 47 or 48); a post-transcriptional regulatory element (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 shown in SEQ ID NO:50, 51 or 70); and a 3' 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 shown 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 shown in SEQ ID NO:69).

[0091] In certain embodiments, the rAAV comprises: (a) an rAAV capsid protein comprising the amino acid sequence of amino acids 1-738 of SEQ ID NO:63, the amino acid sequence of amino acids 138-738 of SEQ ID NO:63, and / or the amino acid sequence of amino acids 204-738 of SEQ ID NO:63; and (b) an rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO:66, 67, 68, or 69. In certain embodiments, the rAAV comprises: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 1-738 of SEQ ID NO:63, and an rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO:66, 67, 68, or 69; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-738 of SEQ ID NO:63, and an rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO:66, 67, 68, or 69; and / or (c) an AAV capsid protein comprising the amino acid sequence of amino acids 204-738 of SEQ ID NO:63, and an rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO:66, 67, 68, or 69;

[0092] In another aspect, the present 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.

[0093] The polynucleotide can include 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 contained within a viral vector. In certain embodiments, the polynucleotide is contained within a plasmid vector.

[0094] In another aspect, the present disclosure provides a pharmaceutical composition comprising an rAAV as disclosed herein and a pharmaceutically acceptable excipient, adjuvant, diluent, vehicle, or carrier or a combination thereof. A "pharmaceutically acceptable carrier" includes any substance that, when combined with the active ingredient of a composition, allows the ingredient to retain its biological activity and does not cause a destructive physiological response (such as an unexpected 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 edition, 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. Ansel et al., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., 3rd edition, Amer. Pharmaceutical Assoc.

[0095] Method of treatment

[0096] In another aspect, the present disclosure provides a method for treating a subject having mitral valve disease. The method generally comprises administering to the subject an effective amount of a gene therapy comprising a first nucleic acid encoding soluble transforming growth factor β receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

[0097] In certain embodiments, a method for 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 for 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 shown 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 shown in SEQ ID NO:33. In certain embodiments, a method for 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 nucleotide sequence shown 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 a nucleotide sequence shown in SEQ ID NO:67 or 69.

[0098] In certain embodiments, a method for treating MVD in a subject comprises administering an rAAV comprising: an AAV capsid comprising an AAV capsid protein and an rAAV genome, the 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 for treating MVD in a subject comprises administering an rAAV comprising: an AAV capsid comprising an AAV capsid protein and an rAAV genome, the rAAV genome comprising: a first nucleic acid encoding the amino acid sequence shown in SEQ ID NO:26 and a second nucleic acid encoding the amino acid sequence shown in SEQ ID NO:33, wherein the first nucleic acid and the second nucleic acid are separated by a polycistronic element.

[0099] In certain embodiments, a method for treating MVD in a subject further includes determining the LA / Ao of the subject before administering gene therapy and determining the LA / Ao of the subject during a duration after administering gene therapy (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). Methods for determining the LA / Ao of a subject are known in the art. For example, the LA / Ao can be measured from the 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 and right coronary aortic valve leaflets at the first frame after aortic valve closure. Then, the internal short-axis diameter of the LA is measured in the same frame along a line extending from the commissure between the non-coronary and left coronary aortic valve leaflets and parallel to that commissure to the far edge of the left atrium.

[0100] In addition to LA / Ao measurements, various methods for diagnosing MVD are known in the art and include, but are not limited to, echocardiography, electrocardiography, chest x-ray, cardiac magnetic resonance imaging, exercise testing, stress testing, and / or cardiac catheterization. The staging system for MVD generally refers to four basic groups: Stage A - at risk: risk factors for MVD are present; Stage B - progressive: mild or moderate MVD and no cardiac valve symptoms; Stage C - asymptomatic severe: severe MVD and no cardiac valve symptoms; and Stage D - symptomatic severe: severe MVD and causing symptoms. Stage B can be further subdivided 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 that is not severe enough to meet the current clinical trial treatment criteria; Stage B2 is diagnosed when a heart murmur is detected and there is radiographic or echocardiographic evidence of cardiac remodeling severe enough to meet the current clinical trial treatment criteria. It is known in the art that an LA / Ao greater than 1.6 indicates Stage B2 of MVD.

[0101] In certain embodiments, a method for treating MVD in a subject comprises administering gene therapy to the subject, wherein the subject has an LA / Ao of from about 1.6 to about 2.1, such as about 1.6, about 1.7, about 1.8, about 1.9, about 2, 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 less than about 2.1. It has been found that regardless of the dose, over time, subjects with an initial LA / Ao less than 2.1 respond to gene therapy, showing reversal of left atrial enlargement over time. Those skilled in the art will readily understand that LA / Ao measurement is the only method for diagnosing MVD. Thus, when using other methods for diagnosing MVD, those skilled in the art will be able to determine what measurement results are equivalent to an initial LA / Ao in the subject less than 2.1. The present disclosure also contemplates methods for treating MVD in subjects having diagnostic measurements that yield an equivalent of an initial LA / Ao in the subject less than 2.1 (e.g., a diagnostic equivalent of an initial LA / Ao less than 2.1).

[0102] In certain embodiments, a method for treating MVD in a subject further comprises administering to the subject an effective amount of one or more additional therapeutic agents for treating MVD. Such additional therapeutic agents for treating MVD can include, but are not limited to, diuretics, blood thinners (i.e., anticoagulants), and blood pressure medications. For example, in canines, pimobendan is used to manage heart failure caused by MMVD. Pimobendan is often used in combination with ACE inhibitors such as enalapril or benazepril. In certain embodiments, a 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.

[0103] In certain embodiments, a method for treating MVD in a subject comprises administering to the subject: (a) an effective amount of a gene therapy comprising a first nucleic acid encoding soluble transforming growth factor β 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 for 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 for treating MVD in a subject comprises administering to the subject: (a) an rAAV comprising: an AAV capsid comprising an AAV capsid protein and an rAAV genome, the 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.

[0104] In canines, pimobendan can be orally administered at a total daily dose of 0.23 mg / lb (0.5 mg / kg) body weight. In humans, pimobendan can be administered at a dose of 2.5 mg / day. The total daily dose is typically divided into two parts and the two parts are administered approximately 12 hours apart. In certain embodiments, the effective amount of pimobendan is between about 0.05 mg / kg and about 0.5 mg / kg. In some embodiments, the effective amount of pimobendan is between about 0.05 mg / kg and about 0.5 mg / kg, administered twice daily, and the total daily dose is between about 0.10 mg / kg and about 1.0 mg / kg. In some embodiments, the effective amount of pimobendan is about 0.25 mg / kg, administered twice daily, and the total daily dose is about 0.5 mg / kg. In some embodiments, the effective amount of pimobendan is 0.25 mg / kg, administered twice daily, and the total daily dose is 0.5 mg / kg.

[0105] In certain embodiments, an effective amount of pimobendan is between about 0.2 mg / kg and about 0.6 mg / kg body weight, once daily. In certain embodiments, an effective amount of pimobendan is between about 0.2 mg / kg and about 0.6 mg / kg body weight per day of administration. In certain embodiments, an effective amount of pimobendan is between about 0.2 mg / kg and about 0.5 mg / kg body weight per day of administration. In certain embodiments, the daily dose of pimobendan is administered in two doses between about 0.1 mg / kg and about 0.3 mg / kg body weight. In certain embodiments, the daily dose of pimobendan is administered in two doses between about 0.1 mg / kg and about 0.3 mg / kg body weight every 12 hours. In certain embodiments, the daily dose of pimobendan is administered in two doses of 0.25 mg / kg body weight every 12 hours.

[0106] In certain embodiments, an additional therapeutic agent for treating MVD (e.g., pimobendan) is administered at the same time as gene therapy. In certain embodiments, an additional therapeutic agent for treating MVD (e.g., pimobendan) is administered at a different time from gene therapy.

[0107] In certain embodiments, a method of treating MVD in a subject comprises administering to a subject that has received an effective amount of a non-gene therapy for treating MVD an effective amount of gene therapy comprising a first nucleic acid encoding soluble transforming growth factor β receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21). In certain embodiments, a method of treating MVD in a subject comprises administering to a subject that has received an effective amount of pimobendan an effective amount of gene therapy comprising a first nucleic acid encoding soluble transforming growth factor β receptor 2 (sTGFβR2) and a second nucleic acid encoding fibroblast growth factor 21 (FGF21).

[0108] In another aspect, the present disclosure provides a method for determining the likelihood of successful treatment of a subject with mitral valve disease using a gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, the method comprising determining the LA / Ao of the subject, wherein an LA / Ao greater than 2.1 indicates a reduced likelihood of successful treatment with the gene therapy and an LA / Ao of from about 1.6 to about 2.1 indicates an increased likelihood of successful treatment with the gene therapy. In certain embodiments, successful treatment of MVD comprises a decrease in the subject's LA / Ao compared to the LA / Ao measured prior to the gene therapy. In certain embodiments, successful treatment of MVD comprises a slow increase in the subject's LA / Ao over time compared to the level of increase in LA / Ao of subjects who have not received gene therapy. In certain embodiments, successful treatment of MVD comprises maintenance of the subject's LA / Ao over time compared to the change in LA / Ao of subjects who have not received gene therapy.

[0109] In another aspect, the present disclosure provides a method for identifying a subject having mitral valve disease suitable for treatment with a gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, the method comprising determining the LA / Ao of the subject, wherein the subject is suitable for treatment with the gene therapy if the subject's LA / Ao is from about 1.6 to about 2.1.

[0110] 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.

[0111] In certain embodiments, the gene therapy is administered to the subject intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally.

[0112] 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, canines, felines, ungulates (e.g., horses, cows, pigs (e.g., swine)), birds, rodents (e.g., rats, mice), and other subjects. In certain embodiments, the subject is a human. In certain embodiments, the subject is a canine. In certain embodiments, the subject is a canine 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.

[0113] Examples

[0114] The following examples are provided by way of illustration and not by way of limitation.

[0115] Example 1: Canine sTGFβR2 and FGF21 Recombinant AAV Vectors

[0116] This example provides canine sTGFβR2 and FGF21 recombinant adeno-associated virus (rAAV) vectors for expressing canine sTGFβR2 and FGF21 in cells (e.g., canine liver cells) into which these vectors are transduced.

[0117] rAAV-sTGFβR2 contains an rAAV genome that contains the following genetic elements from 5' to 3': 5' ITR element, apolipoprotein E (ApoE) binding site, human α-1 antitrypsin (hAAT) promoter, β-globin intron sequence, canine sTGFβR2-Fc coding sequence, WPRE3 sequence, SV40 polyadenylation signal, and 3' ITR element. The sequences of these elements are shown in Table 1. This vector is capable of expressing canine sTGFβR2-Fc fusion protein in cells (e.g., liver cells) into which the vector is transduced.

[0118] rAAV-FGF21 contains an rAAV genome that contains the following genetic elements from 5' to 3': 5' ITR element, apolipoprotein E (ApoE) binding site, human α-1 antitrypsin (hAAT) promoter, β-globin intron sequence, canine FGF21 coding sequence, WPRE3 sequence, SV40 polyadenylation signal, and 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., liver cells) into which the vector is transduced.

[0119] Table 1: Genetic Elements in rAAV-sTGFβR2 and rAAV-FGF21

[0120]

[0121] The rAAV vectors disclosed herein can be packaged in AAV capsids, such as but not limited to AAV8 capsid. Generally, standard triple transfection of HEK293T cells and iodixanol gradient purification, CsCl purification, or affinity and anion column purification are used to produce viral particles. See, e.g., 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. The packaged viral particles can be administered to wild-type animals or animals with mitral valve disease.

[0122] Example 2: sTGFβ2 and FGF21 Protein Expression in Canines with Mitral Valve Disease

[0123] The effects of sTGFβR2 and FGF21 gene therapies were investigated in canines with mitral valve disease. Canines with stage B2 myxomatous mitral valve disease (MMVD) were recruited. Stage B2 MMVD refers to canines with MMVD who have not yet developed signs of heart failure but have a moderate or large mitral valve murmur due to mitral valve leakage and have cardiac enlargement.

[0124] The left atrial to aortic root ratio (LA / Ao) is the most commonly used method to evaluate the size of the left atrium (LA) in canines. Standard M-mode, two-dimensional Doppler echocardiogram images and video loops were recorded, and continuous ECG monitoring was performed for all measurements. As previously described, LA / Ao was measured from the right parasternal short-axis view at the base of the heart. The internal short-axis diameter of the aorta was measured along the commissure between the non-coronary and right coronary aortic valve leaflets at 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 from the commissure between the non-coronary and left coronary aortic valve leaflets and parallel to this commissure to the distal margin of the left atrium. A normal LA / Ao is defined as <1.6. Stage B2 MMVD is defined as a canine having an LA / Ao of 1.7 - 3, requiring pimobendan treatment but not yet in heart failure.

[0125] To examine protein expression in the liver transduced with rAAV-sTGFβR2 and rAAV-FGF21 vectors, canines with stage B2 MMVD were intravenously administered rAAV-sTGFβR2 and rAAV-FGF21 (AAV8-sTGFβR2 and AAV8-FGF21, respectively; sequences are shown in Table 1) each packaged in an AAV8 capsid. AAV8-sTGFβR2 was administered at a dose of 1E13, 3E13, or 5E13 vg / kg, and AAV8-FGF21 was administered at a dose of 1E13 or 3E13 vg / kg. The viruses were titrated by ddPCR using gene-specific primers for the genes of interest (e.g., FGF21 or sTGFβR2).

[0126] sTGFβR2-Fc and FGF21 expression were measured by ELISA using antibodies specific for canine TGFβR2 and FGF21, respectively. Figure 1A The expression levels of sTGFβR2 expression in the treated canines are shown and Figure 1B The expression levels of FGF21 in the treated canines are shown. As Figure 1AAs shown, dogs were administered AAV8-sTGFβR2 at 1E13, 3E13, or 5E13 vg / kg, and stable long-term expression of sTGFβR2 was achieved for 16 months, 16 months, and over 32 months, respectively. In dogs administered AAV8-FGF21 at 1E13 or 3E13 vg / kg, stable long-term expression of FGF21 was also achieved for over 16 months and 32 months, respectively ( Figure 1B ). In Figure 1B , one dog in the 3E13 vg / kg dose cohort was non-responsive.

[0127] Example 3: Gene Therapy for Mitral Valve Disease in Dogs

[0128] Dogs with MVD typically experience enlargement of the left atrium of the heart due to damage caused by mitral valve dysfunction. It has been confirmed that LA / Ao is associated with the progression of MVD, and an increase in LA / Ao of 0.1 increases the change in MVD progression to the next stage by approximately 11%.

[0129] To investigate whether sTGFβR2 and FGF21 gene therapies could treat MVD in dogs, dogs with stage B2 MMVD were administered AAV8-sTGFβR2 and AAV8-FGF21, each at a dose of 1E13 vg / kg, or each at a dose of 3E13 vg / kg. Over time, LA / Ao was measured in the treated dogs. Figure 2 Shown are the measured values of LA / Ao in dogs at different indicated time points over time. As Figure 2 shown, regardless of the dose, over time, dogs with an initial LA / Ao less than 2.1 responded to gene therapy, showing reversal of left atrial enlargement over time. In Figure 2 , the dashed line indicates the entry criterion for LA / Ao of 1.6, and the dotted line indicates LA / Ao of 2.1, at which value all dogs responded to the therapy.

[0130] So far, 17 dogs have received AAV8-sTGFβR2 and AAV8-FGF21 and there have been no records of adverse safety events. Four of these dogs have been in the study for over two years.

[0131] Example 4: Combination Therapy for Canine Mitral Valve Disease

[0132] Pimobendan is currently the best-in-class drug, labeled for use in dogs to manage congestive heart failure (CHF) caused by dilated cardiomyopathy (DCM) or degenerative MVD. Because it is unethical to not adhere to the standard of care in investigational trial studies, dogs diagnosed with stage B2 MMVD and prescribed pimobendan were administered sTGFβR2 and FGF21 gene therapy. Echocardiograms were performed at baseline (0 months), 2 months after treatment, 4 months after treatment, and every 4 months after treatment up to 32 months after treatment. Echocardiograms were used to measure the size of the left atrium relative to the aorta and the percentage left ventricular fractional shortening (FS%). Although pimobendan has a marginal ability to reverse the pathological progression measured by echocardiogram, with published data demonstrating a 0.08 decrease in LA / Ao 1 month after administration of pimobendan (Boswood et al., JVet 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”), which infers a ~33% reduction in the chance of disease progression based on the same study ( Figure 3 ). The size of the left atrium was quantified using echocardiogram by measuring the size of the left atrium relative to the aorta. As Figure 3 shown, dogs treated with the combination of pimobendan with AAV8-sTGFβR2 and AAV8-FGF21 (“Pimo+GT”) showed a ~0.3 decrease in LA / Ao over 32 months. In this experiment, 12 dogs were treated at the early time point and 3 dogs were treated at the final time point due to the rolling enrollment of subjects. In contrast, based on published data reporting late LA / Ao values (dashed line), pimobendan alone was only able to minimize progressive left atrial dilation to 0.8 over 32 months (see, e.g., Nakamura et al J.Vet.Intern.Med. 2017, 31(2):316–325).

[0133] Dogs treated with the combination of pimobendan with AAV8-sTGFβR2 and AAV8-FGF21 (“Pimo+GT”) were able to maintain cardiac function. Using fractional shortening as a measure of myocardial contractility, based on published data, treatment of dogs with MVD with pimobendan limited the reduction in contractility to approximately 5% over 28 months ( Figure 4 dashed line; see, e.g., Nakamura et al J.Vet.Intern.Med. 2017, 31(2):316–325). Thus, even with treatment with the current best-in-class drug, dogs with MVD generally show a decrease in fractional shortening. As Figure 4As shown, dogs with MVD receiving Pimo+GT showed a 3% increase in fractional shortening within 28 months, indicating a reversal of disease progression.

[0134] In addition, dogs with MVD receiving Pimo+GT showed a delay in progression of more than 1.5 years compared to dogs with MVD receiving standard care. As Figure 5 shown, dogs with MVD receiving Pimo+GT showed an increase in time to progression of approximately 600 days compared to published data on dogs with MVD receiving pimobendan alone, and an increase in time to progression of approximately 800 days compared to published data on dogs with MVD receiving placebo. According to published data, dogs with MVD receiving pimobendan alone showed an increase in time to progression of approximately 200 days compared to dogs with MVD receiving placebo. See, e.g., Boswood et al., J Vet Intern Med, 2018, 32(1):72-85. Time to progression was measured based on the percentage of animals that had not reached the primary endpoint of onset of congestive heart failure, cardiac-related death, or euthanasia.

[0135] Importantly, no safety issues have been reported in three dogs with MVD that have received AAV8-sTGFβR2 and AAV8-FGF21 therapies for over three years.

[0136] ***

[0137] The scope of the present invention is not limited to the specific embodiments described herein. Indeed, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and drawings in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

[0138] All references cited herein (e.g., publications or patents or patent applications) are incorporated herein by reference in their entirety and for all purposes to the 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.

[0139] Other embodiments are within the scope of the claims.

Claims

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

1.

2. The method of claim 1, wherein the LA / Ao of the subject is about 1.

6.

3. The method of claim 1, wherein the LA / Ao of the subject is about 1.

8.

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

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

6. The method of any one of claims 1-5, wherein the subject is a canine breed selected from: 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 1-6, wherein the subject is a Cavalier King Charles Spaniel.

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

9. The method of any one of claims 1-8, wherein the method further comprises determining the LA / Ao of the subject before administering the gene therapy and determining the LA / Ao of the subject for a duration after administering the gene therapy.

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

11. The method of any one of claims 1-10, wherein the subject has been administered an effective amount of pimobendan.

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

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

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

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

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

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

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

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

20. A method for identifying a subject having mitral valve disease suitable for treatment with a gene therapy comprising a first nucleic acid encoding sTGFβR2 and a second nucleic acid encoding FGF21, the method comprising determining the LA / Ao of the subject, wherein if the LA / Ao of the subject is from about 1.6 to about 2.1, the subject is suitable for treatment with the gene therapy.

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

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

23. The method according to claim 22, wherein the canine 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 according to any one of claims 18 - 23, wherein the subject is a Cavalier King Charles Spaniel.

25. The method according to any one of claims 18 - 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 according to any one of claims 1 - 25, wherein the gene therapy is administered intravenously.

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

28. The method according to any one of claims 1 - 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. The method according to any one of claims 1-28, wherein the sTGFβR2 coding sequence further comprises a heterologous or native secretion 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. The method according to any one of claims 1-29, 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. The method according to any one of claims 1-27, 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. The method according to claim 31, wherein the sTGFβR2 coding sequence further encodes a secretion 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. The method according to 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. The method according to any one of claims 1-33, wherein the second nucleic acid comprises a second transcriptional regulatory element operably linked to the FGF21 coding sequence.

35. The method according to any one of claims 1-34, 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. The method according to any one of claims 1-35, 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 with SEQ ID NO: 32, 33 or 34.

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

38. The method according to any one of claims 34-37, wherein each of the first transcriptional regulatory element and the second 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 with SEQ ID NO: 38, 39, 40 and / or 48.

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

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

41. The method according to claim 40, wherein the polyadenylation signal is the SV40 polyadenylation signal.

42. The method according to claim 40, wherein the WPRE sequence is the WPRE3 sequence.

43. The method according to 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 with SEQ ID NO: 50, 51 or 70.

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

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

46. The method according to claim 44 or 45, wherein: the first vector is an AAV vector contained in a first recombinant AAV (rAAV), wherein the first rAAV comprises: an AAV capsid containing an AAV capsid protein; and a first rAAV genome; and / or The second vector is an AAV vector contained within a second rAAV, wherein the second rAAV comprises: an AAV capsid comprising an AAV capsid protein; and a second rAAV genome.

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

33.

48. The method according to 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 with SEQ ID NO:

66.

49. The method according to 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. The method according to any one of claims 46-49, 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 with 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 with SEQ ID NO: 60 or 61.

51. The method according to 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 with SEQ ID NO:

67.

52. The method according to 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 with SEQ ID NO:

68.

53. The method according to 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. The method according to 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 with 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 with SEQ ID NO: 60 or 61.

55. The method according to 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 with SEQ ID NO:

69.

56. The method according to any one of claims 46 - 55, wherein the AAV capsid protein is 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 protein or an engineered variant thereof.

57. The method according to 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 with SEQ ID NO: 63, 64 and / or 65.

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

59. The method according to claim 58, wherein the vector comprises: the first nucleic acid, which comprises a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 26; and the second nucleic acid, which comprises a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:

33.

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

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

62. The method according to 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. The method according to any one of claims 58 - 62, wherein the vector is an AAV vector contained within a recombinant AAV (rAAV), wherein the rAAV comprises: an AAV capsid containing an AAV capsid protein; and an rAAV genome.

64. The method according to claim 63, wherein the AAV capsid protein is 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 protein or an engineered variant thereof.

65. The method according to 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 NO: 63, 64 and / or 65.

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