026389-0044-WO01 CBIN1 THERAPY IMPROVES ATRIAL ELECTRICAL AND FUNCTIONAL REMODELING, LIMITING ATRIAL ARRHYTHMIAS IN FAILING HEARTS CROSS-REFERENCE TO RELATED APPLICATION(S) 5 This application claims priority to U.S. Provisional Patent Application No.63/648,025, filed on May 15, 2024, which is incorporated by reference herein in its entirety. FEDERALLY SPONSORED RESEARCH This invention was made with government support under R01 HL171686 awarded by the 10 National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING This application was filed with a Sequence Listing XML in ST.26 XML format in accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file submitted in the 15 USPTO Patent Center, “026389-0044-WO01_sequence_listing_xml_12-MAY-2025.xml,” was created on May 12, 2025, contains 17 sequences, has a file size of 44.0 kilobytes (45,056 bytes), and is incorporated by reference in its entirety into the specification. BACKGROUND 20 Heart failure (HF) results in significant morbidity and mortality and few effective therapeutic options are available for treating patients with HF. Atrial dysfunction and atrial arrhythmias have been shown to be strongly associated with HF progression. A recently developed gene therapy that increases exogenous cardiac bridging integrator 1 (cBIN1) expression has been shown to improve left ventricular function and results in fewer HF signs and mortality. 25 What is needed are compositions and methods for improving atrial function and reducing atrial arrhythmias. SUMMARY One embodiment described herein is a method of improving atrial function and reducing 30 atrial arrhythmias in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a cardiac bridging integrator 1 (cBIN1) gene therapy. In one aspect, the subject exhibits one or more improved atrial properties following administration of the cBIN1 gene therapy relative to before administration of the cBIN1 gene therapy. In another aspect, the one or more improved atrial properties comprise shortened P-wave duration, reduced 1
026389-0044-WO01 atrial fibrosis, reduced left atrial dyssynchrony, increased intracellular T-tubule area, reduced atrial arrhythmia, or combinations thereof. In another aspect, the reduced atrial arrhythmia is reduced atrial fibrillation. In another aspect, the cBIN1 gene therapy is administered to the subject via intravenous injection, intramyocardial injection, cardiac catheter infusion to myocardium, or 5 combinations thereof. In another aspect, the cBIN1 gene therapy is administered to the subject more than once. In another aspect, the cBIN1 gene therapy comprises a transgene encoding a cBIN1 polypeptide or functional variant or fragment thereof. In another aspect, the cBIN1 gene therapy comprises a cBIN1 expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof. In another aspect, the 10 cBIN1 expression vector comprises a non-viral vector, a viral vector, an adeno-associated virus (AAV) vector, a recombinant AAV (rAAV) vector, a single-stranded AAV vector, a double-stranded AAV vector, a self-complementary AAV (scAAV) vector, or combinations thereof. In another aspect, the cBIN1 expression vector comprises an AAV vector of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, a hybrid 15 serotype thereof, or a derivative thereof. In another aspect, the cBIN1 gene therapy comprises an adeno-associated virus 9 transducing cBIN1 (AAV9-cBIN1). In another aspect, the cBIN1 expression vector comprises a muscle-tropic expression vector. In another aspect, the cBIN1 gene therapy expresses cBIN1 in cardiac tissue of the subject. In another aspect, the cBIN1 gene therapy selectively expresses cBIN1 in cardiac tissue of the subject. In another aspect, the non- 20 viral vector comprises a lipid carrier, an exosome, a polymer-based carrier, a chemical-based carrier, a conjugated carrier, or combinations thereof. In another aspect, the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence comprising DNA, RNA, or a combination thereof. In another aspect, the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence having at least 90–99% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, or 11. In another aspect, 25 the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence of any one of SEQ ID NO: 1, 3, 5, 7, or 11. In another aspect, the cBIN1 gene therapy is administered to the subject using a dosing regimen based on cBIN1 gene expression vector genome (vg) per kg body weight of the subject. In another aspect, the cBIN1 gene therapy is administered to the subject at a dose of about 1 × 1010 vg/kg to about 1 × 1014 vg/kg cBIN1 gene expression vector. In another aspect, 30 the subject has pre-existing heart failure. In another aspect, the cBIN1 gene therapy is administered after the subject is diagnosed with heart failure. In another aspect, the subject is a mammal. In another aspect, the subject is a human, primate, dog, pig, or mouse. 2
026389-0044-WO01 Another embodiment described herein is the use of a cardiac bridging integrator 1 (cBIN1) gene therapy in a medicament for improving atrial function and reducing atrial arrhythmias in a subject in need thereof. Another embodiment described herein is the use of a transgene encoding cardiac bridging 5 integrator 1 (cBIN1) in the preparation of a medicament for use in a method of improving atrial function and reducing atrial arrhythmias in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the transgene encoding cBIN1. DESCRIPTION OF THE DRAWINGS 10 The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIG.1 shows that the P-wave duration was significantly shorter at the terminal study for the cBIN1 group as compared to the GFP group. There was also a significant decrease in P- 15 wave duration between the injection study and the terminal study for both the cBIN1 and GFP groups. FIG.2 shows that the left atrial (LA) dyssynchrony was similar at the baseline and injection time points between GFP and cBIN1 treatment groups. The dyssynchrony improved from injection to terminal studies in the cBIN1 group while it continued to worsen in the GFP group. 20 FIG.3 shows that the LA fibrosis was lower in the cBIN1 treated group compared to both the GFP and untreated HF (UHF) control groups. FIG.4 shows that cBIN1 therapy limited LA T-tubule dilation compared to the GFP treated group. FIG. 5A–D show mitochondrial analyses with various treatments. FIG. 5A shows that 25 matrix swelling resulted in collapse of intermembrane space of the crista. The mitochondrial area was calculated by marking the circumference of the mitochondria (shown with white squares). Mitochondrial images are binary segmented into black and white areas and the matrix area, shown in white, was calculated as a percentage of the total mitochondrial area. FIG.5B shows that the percentage of matrix area (matrix area divided by mitochondrial area) increased in HF 30 but was limited in the cBIN1 group compared to the GFP treated group. FIG.5C shows that the mitochondrial area was not significantly different between groups. FIG. 5D shows the percent matrix area. FIG. 6A–C show confocal imaging of cardiomyocytes with various treatments. FIG. 6A shows that confocal imaging of cardiomyocytes revealed high LA T-tubule density and regularity 3
026389-0044-WO01 in cBIN1 treated hearts. The box shows a magnified view of T-tubule organization, and an intensity analysis demonstrates the high intensity peaks and regularity that were observed in cBIN1 treated hearts. FIG. 6B shows confocal imaging of cardiomyocytes LA T-tubule in GFP treated hearts. T-tubule intensity peaks were low and irregular. FIG.6C shows confocal imaging 5 of LA cardiomyocytes showing T-tubule distribution in untreated HF (UHF) hearts. T-tubule intensity peaks were low and irregular. FIG.7 shows the percentage of LA whole cell intracellular area with T-tubules compared between groups. The area with T-tubules was significantly higher in the cBIN1 treated group compared to GFP and untreated HF control groups. 10 FIG.8 shows that the T-tubule intensity, as shown in FIG.6A–C, was higher in the cBIN1 treated group compared to GFP and untreated HF control groups. DETAILED DESCRIPTION Unless otherwise defined, all technical and scientific terms used herein have the same 15 meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are 20 described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein. As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and 25 standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein. As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements30 presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed 4
026389-0044-WO01 inventions. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim. As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular 5 and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified. As used herein, the term “or” can be conjunctive or disjunctive. As used herein, the term “and/or” refers to both the conjunctive and disjunctive. As used herein, the term “substantially” means to a great or significant extent, but not 10 completely. As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement 15 system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments 20 within 2-fold, of a value. As used herein, the symbol “~” means “about” or “approximately.” All ranges disclosed herein include both en
d points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1–2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard 25 deviations, including the end points, or as described above in the definition of “about.” As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15–30 30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15–30 °C; about 20–30 °C; about 22–30 °C; about 25–30 °C; about 27–30 °C; about 15–22 °C; about 15–25 °C; about 15–27 °C; about 20–22 °C; about 20–25 °C; about 20–27 °C; about 22–25 °C; about 22–27 °C; about 25–27 °C; about 15 °C 5
026389-0044-WO01 ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure. As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures 5 thereof, that provide a pharmacological, often beneficial, effect. As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells. 10 As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein. As used herein, the term “prophylaxis” refers to preventing or reducing the progression of 15 a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art. As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, therapy, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or 20 more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of 25 supplemental therapy used, ongoing disease process, and type of treatment desired. As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non- human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. 30 As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments. 6
026389-0044-WO01 As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process. As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, 5 repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves 10 administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or 15 compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifested. As used herein, “cBIN1” refers to cardiac bridging integrator 1 (cBIN1). In the cardiac system, the alternate splicing of the Bin1 gene produces 4–6 transcript variants and the corresponding protein isoforms. The two cardiac characteristic isoforms feature the inclusion of 20 exon 13 (deficient of exons 14–16) with or without the ubiquitously alternatively spliced exon 17 to form the protein isoforms of BIN1 + exon 13 and BIN1 + exon 13 + exon 17 (called cBIN1). Of these two cardiac isoforms, cBIN1 containing exons 13 and 17 is the isoform which localizes to cardiac T-tubules and acts as a critical membrane scaffolding protein. As used herein, “contacting” refers to contacting a target subject, cell, or tissue with a 25 therapeutic agent (e.g., a cBIN1 gene therapy, gene expression vector, transgene, or pharmaceutical composition) using any method that is suitable for placing the agent on, in, or adjacent to the target. For example, when cells are in vitro, contacting the cells with the agent can comprise adding the agent to culture medium containing the cells. For example, when cells are in vivo, contacting the cells with the agent can comprise administering the agent to a subject. 30 In one aspect described herein, contacting comprises administering a cBIN1 gene therapy (e.g., gene expression vector) to cardiac tissue of a subject in need thereof. As used herein, “P-wave duration” refers to the length of time for atria to depolarize before ventricles depolarize, as seen on, for example, an electrocardiogram. 7
026389-0044-WO01 As used herein, “left atrial dyssychrony” or “LA dyssynchrony” refers to the time difference to peak strain between the earliest septal and latest lateral activated segments (i.e., “septal-lateral delay”). As used herein, “variants” can include, but are not limited to, those that include 5 conservative amino acid (AA) substitution, SNP variants, degenerate variants, and biologically active portions of a gene. A “degenerate variant” as used herein refers to a variant that has a mutated nucleotide sequence, but still encodes the same polypeptide due to the redundancy of the genetic code. There are 20 naturally occurring amino acids; however, some of these share similar characteristics. For example, leucine and isoleucine are both aliphatic, branched, and 10 hydrophobic. Similarly, aspartic acid and glutamic acid are both small and negatively charged. Conservative substitutions in proteins often have a smaller effect on function than non- conservative mutations. Although there are many ways to classify amino acids, they are often sorted into six main groups on the basis of their structure and the general chemical characteristics of their R groups. A mutation among the same class of amino acids is considered a conservative 15 amino acid substitution. The term “functional” when used in conjunction with “variant” or “fragment” refers to an entity or molecule which possess a biological activity that is substantially similar to a biological activity of the entity or molecule of which it is a variant or fragment thereof. As described herein, a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment 20 thereof may be modified, for example, to facilitate or improve identification, expression, isolation, storage and/or administration, so long as such modifications do not reduce its function to an unacceptable level. In various embodiments, a cBIN1 polypeptide functional variant or fragment thereof has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the function of a full-length wildtype cBIN1 polypeptide. 25 As used herein, “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity compared to a reference sequence as determined using programs known in the art (e.g., Basic Local Alignment Search Tool; BLAST). In preferred embodiments, percent identity can be any integer from 25% to 100%. More preferred embodiments include polynucleotide sequences that have at least 30 about: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Accordingly, 8
026389-0044-WO01 polynucleotides of the present invention encoding a protein or polypeptide of the present invention include nucleic acid sequences that have substantial identity to the nucleic acid sequences that encode the proteins or polypeptides of the present invention. Polynucleotides encoding a polypeptide comprising an amino acid sequence that has at least about: 25%, 30%, 35%, 40%, 5 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference polypeptide sequence are also preferred. As used herein, “substantial identity” of amino acid sequences (and of polypeptides having these amino acid sequences) means that an amino acid sequence comprises a sequence that 10 has at least 25% sequence identity compared to a reference sequence as determined using programs known in the art (e.g., BLAST). In preferred embodiments, percent identity can be any integer from 25% to 100%. More preferred embodiments include amino acid or polypeptide sequences that have at least about: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 15 99% sequence identity compared to a reference sequence. Polypeptides that are “substantially identical” share amino acid sequences except that residue positions which are not identical may differ by one or more conservative amino acid changes, as described above. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, 20 and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. 25 Exemplary conservative amino acid substitution groups include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Accordingly, polypeptides or proteins, encoded by the polynucleotides of the present invention, include amino acid sequences that have substantial identity to the amino acid sequences of the reference polypeptide sequences. 30 The polynucleotides described herein include variants that have substitutions, deletions, and/or additions that can involve one or more nucleotides. The variants can be altered in coding regions, non-coding regions, or both. Alterations in the coding regions can produce conservative or non-conservative amino acid substitutions, deletions, or additions. Especially preferred among 9
026389-0044-WO01 these are silent substitutions, additions, and deletions, which do not alter the properties and activities of the binding. Further embodiments described herein include nucleic acid molecules comprising polynucleotides having nucleotide sequences about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 5 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and more preferably at least about 90–99% or 100% identical to (a) nucleotide sequences, or degenerate, homologous, or codon-optimized variants thereof, encoding polypeptides having the amino acid sequences in SEQ ID NO: 2, 4, 6, 8 or 12; (b) nucleotide sequences, or degenerate, homologous, or codon- optimized variants thereof, encoding polypeptides having the amino acid sequences in SEQ ID 10 NO: 2, 4, 6, 8 or 12; and (c) nucleotide sequences capable of hybridizing to the complement of any of the nucleotide sequences in (a) or (b) above and capable of expressing functional polypeptides of amino acid sequences in SEQ ID NO: 2, 4, 6, 8 or 12. By a polynucleotide having a nucleotide sequence at least, for example, 90–99% “identical” to a reference nucleotide sequence encoding a cBIN1 polypeptide is intended that the 15 nucleotide sequence of the polynucleotide be identical to the reference sequence except that the polynucleotide sequence can include up to about 10 to 1 point mutations, additions, or deletions per each 100 nucleotides of the reference nucleotide sequence encoding the cBIN1 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence about at least 90–99% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the 20 reference sequence can be deleted, added, or substituted, with another nucleotide, or a number of nucleotides up to 10% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5′- or 3′- terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one 25 or more contiguous groups within the reference sequence. The same is applicable to polypeptide sequences about at least 90–99% identical to a reference polypeptide sequence. As noted above, two or more polynucleotide sequences can be compared by determining their percent identity. Two or more amino acid sequences likewise can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or 30 peptide sequences, is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein 10
026389-0044-WO01 Sequences and Structure, Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.14(6): 6745- 6763 (1986). For example, due to the degeneracy of the genetic code, one having ordinary skill in the 5 art will recognize that a large number of the nucleic acid molecules having a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7, or 11 or degenerate, homologous, or codon-optimized variants thereof, will encode a functional cBIN1 polypeptide. 10 The polynucleotides described herein include those encoding mutations, variations, substitutions, additions, deletions, and particular examples of the polypeptides described herein. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247: 1306-1310 (1990), wherein the authors indicate that 15 proteins are surprisingly tolerant of amino acid substitutions. Thus, fragments, derivatives, or analogs of the polypeptides of SEQ ID NO: 2, 4, 6, 8 or 12 can be (i) ones in which one or more of the amino acid residues (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues, or even more) are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino 20 acid residue). Such substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) ones in which one or more of the amino acid residues includes a substituent group (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues or even more), or (iii) ones in which the mature polypeptide is fused with another polypeptide or compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 25 (iv) ones in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives, and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. In addition, fragments, derivatives, or analogs of the polypeptides of SEQ ID NO: 2, 4, 6, 30 8 or 12 can be substituted with one or more conserved or non-conserved amino acid residue (preferably a conserved amino acid residue). In some cases, these polypeptides, fragments, derivatives, or analogs thereof will have a polypeptide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide sequence shown in SEQ ID NO: 2, 4, 6, 8 or 12 and will comprise functional or 11
026389-0044-WO01 non-functional proteins. Similarly, additions or deletions to the polypeptides can be made either at the N- or C-termini or within non-conserved regions of the polypeptide (which are assumed to be non-critical because they have not been photogenically conserved). As described herein, in many cases the amino acid substitutions, mutations, additions, or 5 deletions are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein or additions or deletions to the N- or C- termini. Of course, the number of amino acid substitutions, additions, or deletions a skilled artisan would make depends on many factors, including those described herein. Generally, the number of substitutions, additions, or deletions for any given polypeptide will not be more than about 100, 10 90, 80, 70, 60, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 5, 6, 4, 3, 2, or 1. Another embodiment described herein is a polynucleotide vector comprising one or more nucleotide sequences described herein. Another embodiment described herein is a cell comprising one or more nucleotide sequences described herein or a polynucleotide vector described herein. 15 Another embodiment is a polypeptide encoded by a nucleotide sequence described herein. In one aspect, the polypeptide has at least 85% to 99% identity to SEQ ID NO: 2, 4, 6, 8 or 12. In another aspect, the polypeptide is selected from SEQ ID NO: 2, 4, 6, 8 or 12. Another embodiment described herein is a process for manufacturing one or more of the nucleotide sequence described herein or a polypeptide encoded by the nucleotide sequence 20 described herein, the process comprising: transforming or transfecting a cell with a nucleic acid comprising a nucleotide sequence described herein; growing the cells; optionally isolating additional quantities of a nucleotide sequence described herein; inducing expression of a polypeptide encoded by a nucleotide sequence of described herein; isolating the polypeptide encoded by a nucleotide described herein. 25 Another embodiment described herein is a means for manufacturing one or more of the nucleotide sequences described herein or a polypeptide encoded by a nucleotide sequence described herein, the process comprising: transforming or transfecting a cell with a nucleic acid comprising a nucleotide sequence described herein; growing the cells; optionally isolating additional quantities of a nucleotide sequence described herein; inducing expression of a 30 polypeptide encoded by a nucleotide sequence of described herein; isolating the polypeptide encoded by a nucleotide described herein. Another embodiment described herein is a nucleotide sequence or a polypeptide encoded by the nucleotide sequence produced by the method or the means described herein. 12
026389-0044-WO01 Another embodiment described herein is the use of an effective amount of a polypeptide encoded by one or more of the nucleotide sequences described herein. Another embodiment described herein is a research tool comprising a polypeptide encoded by a nucleotide sequence described herein. 5 Another embodiment described herein is a reagent comprising a polypeptide encoded by a nucleotide sequence described herein. Described herein are compositions and methods comprising a cBIN1 gene therapy for improving atrial function and reducing atrial arrhythmias in a subject in need thereof. In some embodiments, the disclosed cBIN1 gene therapy is capable of improving cardiac atrial electrical 10 and functional remodeling and reducing the incidence of or treating atrial fibrillation in a subject. In some aspects, the subjects being treated with cBIN1 gene therapy may have pre-existing heart failure. Various embodiments described herein provide pharmaceutical compositions and methods comprising a cBIN1 gene therapy for improving atrial function and reducing atrial 15 arrhythmias in a subject. In various embodiments, the cBIN1 gene therapy is of a mammal. In various embodiments, the cBIN1 gene therapy is of a primate, for example, a human, a chimpanzee, a gorilla, or a monkey. In various embodiments, the cBIN1 gene therapy is of a dog, horse, a goat, a donkey, a cow, a bull, or a pig. In various embodiments, the cBIN1 gene therapy is of a rodent, for example, a mouse, a rat, or a guinea pig. In various embodiments, the cBIN1 20 gene therapy is of a chicken, a duck, a frog, a dog, a cat, or a rabbit. Gene Therapy Compositions The present disclosure provides cBIN1 gene therapy pharmaceutical compositions. In some embodiments, the cBIN1 gene therapy may comprise a transgene encoding a cBIN1 25 polypeptide or functional variant or fragment thereof. For example, the cBIN1 gene therapy may comprise a cBIN1 expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof. In some embodiments, the disclosed compositions may further comprise one or more pharmaceutically acceptable carriers or excipients. 30 cBIN1 Polynucleotide Sequences and Gene Expression Vectors The disclosed pharmaceutical compositions may comprise a cBIN1 gene expression vector comprising a cBIN1 polynucleotide sequence (i.e., transgene) encoding a cBIN1 polypeptide or functional variant or fragment thereof. In some embodiments, the cBIN1 13
026389-0044-WO01 polynucleotide sequence or transgene may comprise DNA, RNA (e.g., mRNA), or a combination thereof encoding a cBIN1 polypeptide or functional variant or fragment thereof. In some embodiments, various gene expression vectors as described herein are used to produce various cBIN1 polypeptides or functional variants or fragments thereof. For example, 5 various gene expression vectors may be introduced into bacteria or yeast to produce various cBIN1 polypeptides or functional variants thereof, which are later isolated. In various embodiments, the gene expression vector is a plasmid. In a nonlimiting exemplary embodiment, the cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof is the mouse cBIN1 (BIN1 + exon 13 10 + exon 17) coding sequence (SEQ ID NO: 1–2): SEQ ID NO: 1 – Mouse (Mus musculus) cBIN1 Polynucleotide Sequence ATGGCAGAGATGGGGAGCAAGGGGGTGACGGCGGGGAAGATCGCCAGCAACGTACAGAAGAAGCTGACC CGAGCGCAGGAGAAGGTCCTGCAGAAACTGGGGAAGGCGGACGAGACGAAGGACGAGCAGTTTGAGCAG TGTGTCCAGAACTTCAATAAGCAGCTGACAGAGGGTACCCGGCTGCAGAAGGATCTTCGGACCTATCTG GCTTCTGTTAAAGCGATGCACGAAGCCTCCAAGAAGCTGAGTGAGTGTCTTCAGGAGGTGTATGAGCCC GAGTGGCCTGGCAGGGATGAAGCAAACAAGATTGCAGAGAACAATGACCTACTCTGGATGGACTACCAC CAGAAGCTGGTGGACCAGGCTCTGCTGACCATGGACACCTACCTAGGCCAGTTCCCTGATATCAAGTCG CGCATTGCCAAGCGGGGGCGGAAGCTGGTGGACTATGACAGTGCCCGGCACCACTATGAGTCTCTTCAA ACCGCCAAAAAGAAGGATGAAGCCAAAATTGCCAAGGCAGAAGAGGAGCTCATCAAAGCCCAGAAGGTG TTCGAGGAGATGAACGTGGATCTGCAGGAGGAGCTGCCATCCCTGTGGAACAGCCGTGTAGGTTTCTAT GTCAACACGTTCCAGAGCATCGCGGGTCTGGAGGAAAACTTCCATAAAGAGATGAGTAAGCTCAATCAG AACCTCAATGATGTCCTGGTCAGCCTAGAGAAGCAGCACGGGAGCAACACCTTCACAGTCAAGGCCCAA CCCAGTGACAATGCCCCTGAGAAAGGGAACAAGAGCCCGTCACCTCCTCCAGATGGCTCCCCTGCTGCT ACCCCTGAGATCAGAGTGAACCATGAGCCAGAGCCGGCCAGTGGGGCCTCACCCGGGGCTACCATCCCC AAGTCCCCATCTCAGCTCCGGAAAGGCCCACCTGTCCCTCCGCCTCCCAAACACACCCCATCCAAGGAG ATGAAGCAGGAGCAGATTCTCAGCCTTTTTGATGACGCATTTGTCCCTGAGATCAGCGTGACCACCCCC TCCCAGCCAGCAGAGGCCTCCGAGGTGGTGGGTGGAGCCCAGGAGCCAGGGGAGACAGCAGCCAGTGAA GCAACCTCCAGCTCTCTTCCGGCTGTGGTGGTGGAGACCTTCTCCGCAACTGTGAATGGGGCGGTGGAG GGCAGCGCTGGGACTGGACGCTTGGACCTGCCCCCGGGATTCATGTTCAAGGTTCAAGCCCAGCATGAT TACACGGCCACTGACACTGATGAGCTGCAACTCAAAGCTGGCGATGTGGTGTTGGTGATTCCTTTCCAG AACCCAGAGGAGCAGGATGAAGGCTGGCTCATGGGTGTGAAGGAGAGCGACTGGAATCAGCACAAGGAA CTGGAGAAATGCCGCGGCGTCTTCCCGGAGAATTTTACAGAGCGGGTGCAGTGA Underlined – Exon 13; Bolded – Exon 17 SEQ ID NO: 2 – Mouse cBIN1 Polypeptide Sequence MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTRLQKDLRTYL ASVKAMHEASKKLSECLQEVYEPEWPGRDEANKIAENNDLLWMDYHQKLVDQALLTMDTYLGQFPDIKS RIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKAEEELIKAQKVFEEMNVDLQEELPSLWNSRVGFY VNTFQSIAGLEENFHKEMSKLNQNLNDVLVSLEKQHGSNTFTVKAQPSDNAPEKGNKSPSPPPDGSPAA TPEIRVNHEPEPASGASPGATIPKSPSQLRKGPPVPPPPKHTPSKEMKQEQILSLFDDAFVPEISVTTP SQPAEASEVVGGAQEPGETAASEATSSSLPAVVVETFSATVNGAVEGSAGTGRLDLPPGFMFKVQAQHD YTATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFPENFTERVQ 14
026389-0044-WO01 In another nonlimiting exemplary embodiment, the cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof is the rat cBIN1 coding sequence (SEQ ID NO: 3–4): SEQ ID NO: 3 – Rat (Rattus norvegicus) cBIN1 Polynucleotide Sequence ATGGCAGAGATGGGGAGCAAGGGGGTGACGGCGGGGAAGATCGCAAGCAATGTTCAGAAGAAGCTGACTCGAGCTC AGGAGAAGGTCCTGCAGAAACTGGGGAAGGCGGATGAAACGAAGGATGAGCAGTTCGAACAGTGCGTCCAGAATTT CAATAAGCAGCTGACAGAGGGCACCCGGCTGCAGAAGGATCTTCGGACCTACCTGGCTTCTGTTAAAGCCATGCAC GAAGCCTCCAAGAAGCTGAGTGAGTGTCTCCAGGAGGTGTATGAGCCTGAGTGGCCTGGCAGGGATGAAGCGAACA AGATAGCAGAGAACAATGACCTGCTATGGATGGACTATCACCAGAAGCTGGTGGACCAGGCTCTGCTGACCATGGA TACCTACCTGGGCCAGTTCCCTGATATCAAGTCACGCATTGCCAAGCGGGGGCGGAAGCTGGTGGACTACGACAGC GCCCGGCACCACTATGAGTCTCTTCAAACCGCCAAAAAGAAGGATGAAGCCAAAATTGCCAAGGCAGAAGAGGAGC TCATCAAAGCCCAGAAGGTGTTCGAGGAGATGAATGTGGACCTGCAGGAGGAGCTGCCATCCCTGTGGAACAGCCG TGTGGGTTTCTATGTCAACACGTTCCAGAGCATCGCGGGTCTGGAGGAAAACTTCCATAAAGAGATGAGTAAGCTC AATCAGAACCTCAATGATGTCCTGGTCAGCCTAGAGAAGCAACACGGGAGCAACACCTTCACAGTCAAGGCCCAGC CCAGAAAGAAAACTAAACTGTTCTCACGGCTGCGCAGAAAGAAGAACAGTGACAGCGCCCCTGAAAAAGGGAACAA GAGCCCTTCACCTCCTCCAGATGGTTCCCCTGCTGCTACCCCTGAGATCAGAGTGAACCATGAGCCAGAGCCGGCC AGTGGGGCATCGCCTGGGGCTACCATCCCCAAGTCCCCATCTCAGAGCTCTCTCCCGGCTGTGGTGGTGGAGACCT TCTCAGCAACTGTGAATGGCGCCGTGGAGGGCAGCACTACGACTGGACGCTTGGATCTGCCCCCGGGATTCATGTT CAAGGTGCAAGCCCAGCATGATTACACGGCCACTGACACTGACGAGCTGCAACTCAAAGCTGGCGATGTGGTACTG GTGATCCCCTTCCAGAACCCAGAGGAGCAGGATGAAGGCTGGCTCATGGGTGTGAAGGAGAGCGACTGGAATCAGC ACAAGGAACTGGAGAAATGCCGCGGCGTCTTCCCGGAGAATTTCACAGAGCGGGTGCAGTGA Underlined – Exon 13; Bolded – Exon 17 SEQ ID NO: 4 – Rat cBIN1 Polypeptide Sequence MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTRLQKDLRTYLASVKAMH EASKKLSECLQEVYEPEWPGRDEANKIAENNDLLWMDYHQKLVDQALLTMDTYLGQFPDIKSRIAKRGRKLVDYDS ARHHYESLQTAKKKDEAKIAKAEEELIKAQKVFEEMNVDLQEELPSLWNSRVGFYVNTFQSIAGLEENFHKEMSKL NQNLNDVLVSLEKQHGSNTFTVKAQPSDSAPEKGNKSPSPPPDGSPAATPEIRVNHEPEPASGASPGATIPKSPSQ LRKGPPVPPPPKHTPSKEMKQEQILSLFDDAFVPEISVTTPSQPAEASEVVGGTQEPGETAASEATSSSLPAVVVE TFSATVNGAVEGSTTTGRLDLPPGFMFKVQAQHDYTATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWN QHKELEKCRGVFPENFTERVQ 5 In another nonlimiting exemplary embodiment, the cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof is the dingo cBIN1 (SEQ ID NO: 5–6): SEQ ID NO: 5 – Dingo (Canis lupus dingo) cBIN1 Polynucleotide Sequence ATGGCCGAGATGGGCAGCAAGGGGGTGACGGCGGGGAAGATCGCCAGCAACGTGCAGAAGAAGCTCACGCGCGCGC AGGAGAAGGTCCTCCAGAAACTGGGGAAGGCGGATGAAACAAAGGATGAGCAGTTTGAACAGTGTGTCCAGAATTT CAACAAGCAGCTGACTGAGGGCACTCGACTACAGAAGGACCTCCGGACCTACCTGGCCTCAGTCAAGGCCATGCAC GAAGCCTCCAAGAAACTGAATGAATGTCTGCAGGAGGTGTACGAGCCCGACTGGCCTGGCAGGGATGAAGCAAACA AGATAGCTGAGAACAACGACCTCCTCTGGCTGGATTACCACCAGAAGCTGGTGGACCAGGCGCTGTTGACTATGGA CACATACCTGGGCCAGTTCCCTGACATCAAGTCACGCATTGCCAAGCGAGGGCGGAAGCTGGTGGACTATGACAGT GCCCGGCACCATTATGAGTCCCTCCAAACCGCCAAAAAGAAGGATGAAGCCAAGATCGCCAAGGCAGAGGAGGAGC TCATCAAAGCCCAGAAAGTGTTTGAGGAGATGAACGTGGATCTGCAGGAAGAGCTGCCATCCCTGTGGAACAGCCG GGTAGGTTTCTATGTCAACACGTTTCAGAGCATTGCAGGCCTGGAGGAGAACTTCCACAAGGAGATGAGTAAGCTC AACCAGAACCTCAACGATGTGCTGGTCAGCCTGGAGAAGCAGCATGGGAGCAACACCTTCACAGTCAAGGCCCAGC CCAGTGACAACGCCCCTGCAAAAGGGAACAAGAGCCCTTCGCCTCCGCCAGACGGCTCCCCAGCCGCCACCCCGGA GATCAGAGTCAACCACGAGCCTGAGCCCTCGGCAGCAGCCGGGGCAGCCCTCCCCAAGTCCCCATCTCAGCTCCGG 15
026389-0044-WO01 AAAGGGCCACCAGTCCCTCCGCCTCCCAAACACACCCCGTCCAAGGAGGTCAAGCAGGAGCAGATCCTCAGCCTGT TTGATGACACGTTTGTCCCTGAGATCAGCGTGACCACCCCCTCCCAGCCAGCGGAGGCCTCGGAGGTGGCGGGTGG GACCCCACCTGCGGCTGGAGCCCAGGAGCCCGGGGAAACAGCAGCAAGTGAAGCGGCCTCCAGCTCTCTCCCTGCG GTGGTGGTAGAGACCTTCTCAGCAACTGTGAATGGCACCGTGGAGGGTGGCGGTGGGGCGGGACGCTTGGACCTGC CCCCAGGCTTCATGTTCAAGGTGCAGGCCCAGCACGACTACGTGGCCACCGACACCGATGAGCTGCAGCTCAAGGC GGGTGACGTGGTGCTGGTGATCCCCTTCCAGAACCCGGAGGAGCAGGATGAAGGCTGGCTCATGGGCGTGAAGGAG AGTGACTGGAACCAGCACAAGGAGCTGGAGAAATGCCGGGGCGTCTTCCCTGAGAACTTCACAGAGCGGGTTCAGT GA Underlined – Exon 13; Bolded – Exon 17 SEQ ID NO: 6 – Dingo cBIN1 Polypeptide Sequence MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTRLQKDLRTYLASVKAMH EASKKLNECLQEVYEPDWPGRDEANKIAENNDLLWLDYHQKLVDQALLTMDTYLGQFPDIKSRIAKRGRKLVDYDS ARHHYESLQTAKKKDEAKIAKAEEELIKAQKVFEEMNVDLQEELPSLWNSRVGFYVNTFQSIAGLEENFHKEMSKL NQNLNDVLVSLEKQHGSNTFTVKAQPSDNAPAKGNKSPSPPPDGSPAATPEIRVNHEPEPSAAAGAALPKSPSQLR KGPPVPPPPKHTPSKEVKQEQILSLFDDTFVPEISVTTPSQPAEASEVAGGTPPAAGAQEPGETAASEAASSSLPA VVVETFSATVNGTVEGGGGAGRLDLPPGFMFKVQAQHDYVATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKE SDWNQHKELEKCRGVFPENFTERVQ In another nonlimiting exemplary embodiment, the cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof is the human cBIN1 (BIN1 + exon 13 + exon 17) coding sequence (SEQ ID NO: 7–8): 5 SEQ ID NO: 7 – Human (Homo sapiens) cBIN1 Polynucleotide Sequence ATGGCAGAGATGGGCAGTAAAGGGGTGACGGCGGGAAAGATCGCCAGCAACGTGCAGAAGAAGCTCACC CGCGCGCAGGAGAAGGTTCTCCAGAAGCTGGGGAAGGCAGATGAGACCAAGGATGAGCAGTTTGAGCAG TGCGTCCAGAATTTCAACAAGCAGCTGACGGAGGGCACCCGGCTGCAGAAGGATCTCCGGACCTACCTG GCCTCCGTCAAAGCCATGCACGAGGCTTCCAAGAAGCTGAATGAGTGTCTGCAGGAGGTGTATGAGCCC GATTGGCCCGGCAGGGATGAGGCAAACAAGATCGCAGAGAACAACGACCTGCTGTGGATGGATTACCAC CAGAAGCTGGTGGACCAGGCGCTGCTGACCATGGACACGTACCTGGGCCAGTTCCCCGACATCAAGTCA CGCATTGCCAAGCGGGGGCGCAAGCTGGTGGACTACGACAGTGCCCGGCACCACTACGAGTCCCTTCAA ACTGCCAAAAAGAAGGATGAAGCCAAAATTGCCAAGGCCGAGGAGGAGCTCATCAAAGCCCAGAAGGTG TTTGAGGAGATGAATGTGGATCTGCAGGAGGAGCTGCCGTCCCTGTGGAACAGCCGCGTAGGTTTCTAC GTCAACACGTTCCAGAGCATCGCGGGCCTGGAGGAAAACTTCCACAAGGAGATGAGCAAGCTCAACCAG AACCTCAATGATGTGCTGGTCGGCCTGGAGAAGCAACACGGGAGCAACACCTTCACGGTCAAGGCCCAG CCCAGTGACAACGCGCCTGCAAAAGGGAACAAGAGCCCTTCGCCTCCAGATGGCTCCCCTGCCGCCACC CCCGAGATCAGAGTCAACCACGAGCCAGAGCCGGCCGGCGGGGCCACGCCCGGGGCCACCCTCCCCAAG TCCCCATCTCAGCTCCGGAAAGGCCCACCAGTCCCTCCGCCTCCCAAACACACCCCGTCCAAGGAAGTC AAGCAGGAGCAGATCCTCAGCCTGTTTGAGGACACGTTTGTCCCTGAGATCAGCGTGACCACCCCCTCC CAGCCAGCAGAGGCCTCGGAGGTGGCGGGTGGGACCCAACCTGCGGCTGGAGCCCAGGAGCCAGGGGAG ACGGCGGCAAGTGAAGCAGCCTCCAGCTCTCTTCCTGCTGTCGTGGTGGAGACCTTCCCAGCAACTGTG AATGGCACCGTGGAGGGCGGCAGTGGGGCCGGGCGCTTGGACCTGCCCCCAGGTTTCATGTTCAAGGTA CAGGCCCAGCACGACTACACGGCCACTGACACAGACGAGCTGCAGCTCAAGGCTGGTGATGTGGTGCTG GTGATCCCCTTCCAGAACCCTGAAGAGCAGGATGAAGGCTGGCTCATGGGCGTGAAGGAGAGCGACTGG AACCAGCACAAGGAGCTGGAGAAGTGCCGTGGCGTCTTCCCCGAGAACTTCACTGAGAGGGTCCCATGA Underlined – Exon 13; Bolded – Exon 17 SEQ ID NO: 8 – Human cBIN1 Polypeptide Sequence MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTRLQKDLRTYL ASVKAMHEASKKLNECLQEVYEPDWPGRDEANKIAENNDLLWMDYHQKLVDQALLTMDTYLGQFPDIKS RIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKAEEELIKAQKVFEEMNVDLQEELPSLWNSRVGFY 16
026389-0044-WO01 VNTFQSIAGLEENFHKEMSKLNQNLNDVLVGLEKQHGSNTFTVKAQPSDNAPAKGNKSPSPPDGSPAAT PEIRVNHEPEPAGGATPGATLPKSPSQLRKGPPVPPPPKHTPSKEVKQEQILSLFEDTFVPEISVTTPS QPAEASEVAGGTQPAAGAQEPGETAASEAASSSLPAVVVETFPATVNGTVEGGSGAGRLDLPPGFMFKV QAQHDYTATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFPENFTERVP As shown in the Clustal Omega (v 1.2.4) alignment the cBIN1 polypeptide sequence is highly conserved among mammals such as mouse, rat, dingo and humans. CLUSTAL Omega Multiple Sequence Alignment Mouse MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTR 60 Rat MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTR 60 Dingo MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTR 60 Human MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTR 60 ************************************************************ Mouse LQKDLRTYLASVKAMHEASKKLSECLQEVYEPEWPGRDEANKIAENNDLLWMDYHQKLVD 120 Rat LQKDLRTYLASVKAMHEASKKLSECLQEVYEPEWPGRDEANKIAENNDLLWMDYHQKLVD 120 Dingo LQKDLRTYLASVKAMHEASKKLNECLQEVYEPDWPGRDEANKIAENNDLLWLDYHQKLVD 120 Human LQKDLRTYLASVKAMHEASKKLNECLQEVYEPDWPGRDEANKIAENNDLLWMDYHQKLVD 120 **********************.*********:******************:******** Mouse QALLTMDTYLGQFPDIKSRIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKAEEELIK 180 Rat QALLTMDTYLGQFPDIKSRIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKAEEELIK 180 Dingo QALLTMDTYLGQFPDIKSRIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKAEEELIK 180 Human QALLTMDTYLGQFPDIKSRIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKAEEELIK 180 ************************************************************ Mouse AQKVFEEMNVDLQEELPSLWNSRVGFYVNTFQSIAGLEENFHKEMSKLNQNLNDVLVSLE 240 Rat AQKVFEEMNVDLQEELPSLWNSRVGFYVNTFQSIAGLEENFHKEMSKLNQNLNDVLVSLE 240 Dingo AQKVFEEMNVDLQEELPSLWNSRVGFYVNTFQSIAGLEENFHKEMSKLNQNLNDVLVSLE 240 Human AQKVFEEMNVDLQEELPSLWNSRVGFYVNTFQSIAGLEENFHKEMSKLNQNLNDVLVGLE 240 *********************************************************.** Mouse KQHGSNTFTVKAQPSDNAPEKGNKSPSPPPDGSPAATPEIRVNHEPEPASGASPGATIPK 300 Rat KQHGSNTFTVKAQPSDSAPEKGNKSPSPPPDGSPAATPEIRVNHEPEPASGASPGATIPK 300 Dingo KQHGSNTFTVKAQPSDNAPAKGNKSPSPPPDGSPAATPEIRVNHEPEPSA--AAGAALPK 298 Human KQHGSNTFTVKAQPSDNAPAKGNKSPSP-PDGSPAATPEIRVNHEPEPAGGATPGATLPK 299 ****************.** ******** *******************:. : **::** Mouse SPSQLRKGPPVPPPPKHTPSKEMKQEQILSLFDDAFVPEISVTTPSQPAEASEVVGG--- 357 Rat SPSQLRKGPPVPPPPKHTPSKEMKQEQILSLFDDAFVPEISVTTPSQPAEASEVVGG--- 357 Dingo SPSQLRKGPPVPPPPKHTPSKEVKQEQILSLFDDTFVPEISVTTPSQPAEASEVAGGTPP 358 Human SPSQLRKGPPVPPPPKHTPSKEVKQEQILSLFEDTFVPEISVTTPSQPAEASEVAGGTQP 359 **********************:*********:*:*******************.** Mouse ---AQEPGETAASEATSSSLPAVVVETFSATVNGAVEGSAGTGRLDLPPGFMFKVQAQHD 414 Rat ---TQEPGETAASEATSSSLPAVVVETFSATVNGAVEGSTTTGRLDLPPGFMFKVQAQHD 414 Dingo AAGAQEPGETAASEAASSSLPAVVVETFSATVNGTVEGGGGAGRLDLPPGFMFKVQAQHD 418 Human AAGAQEPGETAASEAASSSLPAVVVETFPATVNGTVEGGSGAGRLDLPPGFMFKVQAQHD 419 :***********:************ *****:***. :****************** Mouse YTATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFPENFTE 474 Rat YTATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFPENFTE 474 17
026389-0044-WO01 Dingo YVATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFPENFTE 478 Human YTATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFPENFTE 479 *.********************************************************** Mouse RVQ 477 Rat RVQ 477 Dingo RVQ 481 Human RVP 482 ** Mouse – SEQ ID NO: 2; Rat – SEQ ID NO: 4; Dingo – SEQ ID NO: 6; Human – SEQ ID NO: 8 In various embodiments, the cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof may comprise one or more site directed or random mutations while maintaining activity (e.g., a function variant or mutant). For example, in 5 various embodiments, the cBIN1 polynucleotide sequence encodes cBIN1 polypeptide functional variants having one or more conservative amino acid substitutions, where the variant retains a substantial amount of biological activity. Exemplary conservative amino acid substitution groups include, but are not limited to, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. 10 In some embodiments, the cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof comprises one or more mutations and has at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to the wildtype cBIN1 gene while still retaining a substantial amount of biological activity. For example, the cBIN1 15 polynucleotide sequence may have greater than about 90%, greater than about 95%, or greater than about 99% sequence homology to the wildtype cBIN1 gene while still retaining a substantial amount of biological activity. In various embodiments, the cBIN1 gene expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof 20 can be modified for better expression, production, storage, administration, detection, delivery efficiency, etc. In various embodiments, the cBIN1 gene expression vector may comprise one or more molecular tags and/or linkers. In one embodiment, the cBIN1 gene expression vector may comprise a cBIN1 polynucleotide sequence encoding one or more fluorescent tags (e.g., GFP). 25 In another embodiment, the cBIN1 gene expression vector may comprise a cBIN1 polynucleotide sequence encoding a C-terminal or N-terminal V5 epitope tag sequence (SEQ ID NO: 9–10): SEQ ID NO: 9 – V5 Epitope Tag Polynucleotide Sequence 18
026389-0044-WO01 TACCCAGCTTTCTTGTACAAAGTGGTTGATCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAAC CCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGA SEQ ID NO: 10 – V5 Epitope Tag Polypeptide Sequence YPAFLYKVVDLEGPRFEGKPIPNPLLGLDSTRTG In one nonlimiting exemplary embodiment, the cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof is the mouse cBIN1 coding sequence having a C-terminal V5 epitope tag (SEQ ID NO: 11–12): 5 SEQ ID NO: 11 – Mouse cBIN1 with C-terminal V5 Epitope Tag Polynucleotide ATGGCAGAGATGGGGAGCAAGGGGGTGACGGCGGGGAAGATCGCCAGCAACGTACAGAAGAAGCTGACC CGAGCGCAGGAGAAGGTCCTGCAGAAACTGGGGAAGGCGGACGAGACGAAGGACGAGCAGTTTGAGCAG TGTGTCCAGAACTTCAATAAGCAGCTGACAGAGGGTACCCGGCTGCAGAAGGATCTTCGGACCTATCTG GCTTCTGTTAAAGCGATGCACGAAGCCTCCAAGAAGCTGAGTGAGTGTCTTCAGGAGGTGTATGAGCCC GAGTGGCCTGGCAGGGATGAAGCAAACAAGATTGCAGAGAACAATGACCTACTCTGGATGGACTACCAC CAGAAGCTGGTGGACCAGGCTCTGCTGACCATGGACACCTACCTAGGCCAGTTCCCTGATATCAAGTCG CGCATTGCCAAGCGGGGGCGGAAGCTGGTGGACTATGACAGTGCCCGGCACCACTATGAGTCTCTTCAA ACCGCCAAAAAGAAGGATGAAGCCAAAATTGCCAAGGCAGAAGAGGAGCTCATCAAAGCCCAGAAGGTG TTCGAGGAGATGAACGTGGATCTGCAGGAGGAGCTGCCATCCCTGTGGAACAGCCGTGTAGGTTTCTAT GTCAACACGTTCCAGAGCATCGCGGGTCTGGAGGAAAACTTCCATAAAGAGATGAGTAAGCTCAATCAG AACCTCAATGATGTCCTGGTCAGCCTAGAGAAGCAGCACGGGAGCAACACCTTCACAGTCAAGGCCCAA CCCAGTGACAATGCCCCTGAGAAAGGGAACAAGAGCCCGTCACCTCCTCCAGATGGCTCCCCTGCTGCT ACCCCTGAGATCAGAGTGAACCATGAGCCAGAGCCGGCCAGTGGGGCCTCACCCGGGGCTACCATCCCC AAGTCCCCATCTCAGCTCCGGAAAGGCCCACCTGTCCCTCCGCCTCCCAAACACACCCCATCCAAGGAG ATGAAGCAGGAGCAGATTCTCAGCCTTTTTGATGACGCATTTGTCCCTGAGATCAGCGTGACCACCCCC TCCCAGCCAGCAGAGGCCTCCGAGGTGGTGGGTGGAGCCCAGGAGCCAGGGGAGACAGCAGCCAGTGAA GCAACCTCCAGCTCTCTTCCGGCTGTGGTGGTGGAGACCTTCTCCGCAACTGTGAATGGGGCGGTGGAG GGCAGCGCTGGGACTGGACGCTTGGACCTGCCCCCGGGATTCATGTTCAAGGTTCAAGCCCAGCATGAT TACACGGCCACTGACACTGATGAGCTGCAACTCAAAGCTGGCGATGTGGTGTTGGTGATTCCTTTCCAG AACCCAGAGGAGCAGGATGAAGGCTGGCTCATGGGTGTGAAGGAGAGCGACTGGAATCAGCACAAGGAA CTGGAGAAATGCCGCGGCGTCTTCCCGGAGAATTTTACAGAGCGGGTGCAGTACCCAGCTTTCTTGTAC AAAGTGGTTGATCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGAT TCTACGCGTACCGGTTAGTAATGA SEQ ID NO: 12 – Mouse cBIN1 with C-terminal V5 Epitope Tag Polypeptide MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFEQCVQNFNKQLTEGTRLQKDLRTYL ASVKAMHEASKKLSECLQEVYEPEWPGRDEANKIAENNDLLWMDYHQKLVDQALLTMDTYLGQFPDIKS RIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKAEEELIKAQKVFEEMNVDLQEELPSLWNSRVGFY VNTFQSIAGLEENFHKEMSKLNQNLNDVLVSLEKQHGSNTFTVKAQPSDNAPEKGNKSPSPPPDGSPAA TPEIRVNHEPEPASGASPGATIPKSPSQLRKGPPVPPPPKHTPSKEMKQEQILSLFDDAFVPEISVTTP SQPAEASEVVGGAQEPGETAASEATSSSLPAVVVETFSATVNGAVEGSAGTGRLDLPPGFMFKVQAQHD YTATDTDELQLKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFPENFTERVQYPAFLY KVVDLEGPRFEGKPIPNPLLGLDSTRTG In various embodiments, the gene expression vector is a non-viral vector, a viral vector, an adeno-associated virus (AAV) vector, a recombinant AAV (rAAV) vector, a single-stranded AAV vector, a double-stranded AAV vector, a self-complementary AAV (scAAV) vector, or a 19
026389-0044-WO01 combination thereof. In various embodiments, non-viral vector delivery of the disclosed cBIN1 polynucleotide sequences may comprise the use of non-viral vector carriers including, but not limited to, lipid carriers (e.g., lipid nanoparticles), exosomes, polymer-based carriers, chemical- based carriers, conjugated carriers (e.g., transferrin-conjugated approaches), and the like. In 5 some embodiments, the gene expression vector is a non-viral vector comprising a lipid carrier such as a lipid nanoparticle (LNP). Non-viral vector delivery approaches of the disclosed cBIN1 polynucleotide sequences may also comprise administration of naked polynucleotide sequences (i.e., not protected and/or devoid of a carrier), including, for example, naked plasmid administration with reversible electroporation. 10 In various embodiments, the gene expression vector is a polynucleotide or a virus particle. In various embodiments, the gene expression vector is a virus particle having a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, a hybrid serotype thereof, or a derivative of one of these capsids. In various embodiments, the gene expression vector is a muscle-tropic AAV-based capsid that enables potent and specific muscle- 15 directed targeted delivery of cBIN1 polynucleotide sequences. These muscle-tropic AAV-based capsids may be either derivatives of existing capsids (e.g., AAV9, AAVrh74), or may be newly discovered or custom designed capsids. In various embodiments, the cBIN1 gene expression vector may be a vector containing a ubiquitous and/or constitutive promoter (e.g., CMV promoter) for ubiquitous and/or constitutive 20 expression of cBIN1. In various embodiments, the cBIN1 gene expression vector may be a vector containing a tissue-specific promoter (e.g., cardiac-specific promoter) for targeted and tissue- specific expression of cBIN1, such as specific expression of cBIN1 in the heart. In one nonlimiting exemplary embodiment, the cBIN1 gene expression vector is an AAV9 vector containing a CMV promoter for ubiquitous expression, wherein a cBIN1 polynucleotide 25 sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof is cloned into the AAV9 vector (i.e., AAV9-cBIN1). In one non-limiting embodiment, gene expression vector is a polynucleotide encoding the mouse cBIN1 polypeptide comprising a C-terminal V5 epitope tag (SEQ ID NO: 13). 30 SEQ ID NO: 13 – Mouse cBIN1 AAV Construct with C-terminal V5 tag (pAAV-CMV-mBin1) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTCTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA 20
026389-0044-WO01 CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTG TTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGA TTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTT TGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATT CTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTG ATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATT ATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTC TGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACATCGATTGAATTCAACAAGTTTGTACAAAAAAGCAGGCTTA ATGGCAGAGATGGGGAGCAAGGGGGTGACGGCGGGGAAGATCGCCAGCAACGTACAGAAGAAGCTGACCCGAGCGCAGGAGAAG GTCCTGCAGAAACTGGGGAAGGCGGACGAGACGAAGGACGAGCAGTTTGAGCAGTGTGTCCAGAACTTCAATAAGCAGCTGACA GAGGGTACCCGGCTGCAGAAGGATCTTCGGACCTATCTGGCTTCTGTTAAAGCGATGCACGAAGCCTCCAAGAAGCTGAGTGAG TGTCTTCAGGAGGTGTATGAGCCCGAGTGGCCTGGCAGGGATGAAGCAAACAAGATTGCAGAGAACAATGACCTACTCTGGATG GACTACCACCAGAAGCTGGTGGACCAGGCTCTGCTGACCATGGACACCTACCTAGGCCAGTTCCCTGATATCAAGTCGCGCATT GCCAAGCGGGGGCGGAAGCTGGTGGACTATGACAGTGCCCGGCACCACTATGAGTCTCTTCAAACCGCCAAAAAGAAGGATGAA GCCAAAATTGCCAAGGCAGAAGAGGAGCTCATCAAAGCCCAGAAGGTGTTCGAGGAGATGAACGTGGATCTGCAGGAGGAGCTG CCATCCCTGTGGAACAGCCGTGTAGGTTTCTATGTCAACACGTTCCAGAGCATCGCGGGTCTGGAGGAAAACTTCCATAAAGAG ATGAGTAAGCTCAATCAGAACCTCAATGATGTCCTGGTCAGCCTAGAGAAGCAGCACGGGAGCAACACCTTCACAGTCAAGGCC CAACCCAGTGACAATGCCCCTGAGAAAGGGAACAAGAGCCCGTCACCTCCTCCAGATGGCTCCCCTGCTGCTACCCCTGAGATC AGAGTGAACCATGAGCCAGAGCCGGCCAGTGGGGCCTCACCCGGGGCTACCATCCCCAAGTCCCCATCTCAGCTCCGGAAAGGC CCACCTGTCCCTCCGCCTCCCAAACACACCCCATCCAAGGAGATGAAGCAGGAGCAGATTCTCAGCCTTTTTGATGACGCATTT GTCCCTGAGATCAGCGTGACCACCCCCTCCCAGCCAGCAGAGGCCTCCGAGGTGGTGGGTGGAGCCCAGGAGCCAGGGGAGACA GCAGCCAGTGAAGCAACCTCCAGCTCTCTTCCGGCTGTGGTGGTGGAGACCTTCTCCGCAACTGTGAATGGGGCGGTGGAGGGC AGCGCTGGGACTGGACGCTTGGACCTGCCCCCGGGATTCATGTTCAAGGTTCAAGCCCAGCATGATTACACGGCCACTGACACT GATGAGCTGCAACTCAAAGCTGGCGATGTGGTGTTGGTGATTCCTTTCCAGAACCCAGAGGAGCAGGATGAAGGCTGGCTCATG GGTGTGAAGGAGAGCGACTGGAATCAGCACAAGGAACTGGAGAAATGCCGCGGCGTCTTCCCGGAGAATTTTACAGAGCGGGTG CAGTACCCAGCTTTCTTGTACAAAGTGGTTGATCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGT CTCGATTCTACGCGTACCGGTTAGAAGCTTGCCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCT GGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTT CTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTG GGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCC TCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATT GGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCAC TGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCC ACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCA GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC ATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTA CACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCAC GTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAA CTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATG AGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCT CTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCG CTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGG GCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATG TGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTC AATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTG TTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATC TCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCG CGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCAC CAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGG CCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTG ATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGC GCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATA GACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATT TAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCA GCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACAT ACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGT 21
026389-0044-WO01 TACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCG GAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCT TTTGCTGGCCTTTTGCTCACATGT In another non-limiting embodiment, gene expression vector is a polynucleotide encoding the human cBIN1 polypeptide (SEQ ID NO: 14). SEQ ID NO: 14 – Human cBIN1 AAV Construct (pAAV-CMV-hBin1) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTCTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTG TTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGA TTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTT TGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATT CTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTG ATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATT ATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTC TGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACATCGATTGAATTCCCCGGGGATCCACCATGGCAGAGATGGG CAGTAAAGGGGTGACGGCGGGAAAGATCGCCAGCAACGTGCAGAAGAAGCTCACCCGCGCGCAGGAGAAGGTTCTCCAGAAGCT GGGGAAGGCAGATGAGACCAAGGATGAGCAGTTTGAGCAGTGCGTCCAGAATTTCAACAAGCAGCTGACGGAGGGCACCCGGCT GCAGAAGGATCTCCGGACCTACCTGGCCTCCGTCAAAGCCATGCACGAGGCTTCCAAGAAGCTGAATGAGTGTCTGCAGGAGGT GTATGAGCCCGATTGGCCCGGCAGGGATGAGGCAAACAAGATCGCAGAGAACAACGACCTGCTGTGGATGGATTACCACCAGAA GCTGGTGGACCAGGCGCTGCTGACCATGGACACGTACCTGGGCCAGTTCCCCGACATCAAGTCACGCATTGCCAAGCGGGGGCG CAAGCTGGTGGACTACGACAGTGCCCGGCACCACTACGAGTCCCTTCAAACTGCCAAAAAGAAGGATGAAGCCAAAATTGCCAA GGCCGAGGAGGAGCTCATCAAAGCCCAGAAGGTGTTTGAGGAGATGAATGTGGATCTGCAGGAGGAGCTGCCGTCCCTGTGGAA CAGCCGCGTAGGTTTCTACGTCAACACGTTCCAGAGCATCGCGGGCCTGGAGGAAAACTTCCACAAGGAGATGAGCAAGCTCAA CCAGAACCTCAATGATGTGCTGGTCGGCCTGGAGAAGCAACACGGGAGCAACACCTTCACGGTCAAGGCCCAGCCCAGTGACAA CGCGCCTGCAAAAGGGAACAAGAGCCCTTCGCCTCCAGATGGCTCCCCTGCCGCCACCCCCGAGATCAGAGTCAACCACGAGCC AGAGCCGGCCGGCGGGGCCACGCCCGGGGCCACCCTCCCCAAGTCCCCATCTCAGCTCCGGAAAGGCCCACCAGTCCCTCCGCC TCCCAAACACACCCCGTCCAAGGAAGTCAAGCAGGAGCAGATCCTCAGCCTGTTTGAGGACACGTTTGTCCCTGAGATCAGCGT GACCACCCCCTCCCAGCCAGCAGAGGCCTCGGAGGTGGCGGGTGGGACCCAACCTGCGGCTGGAGCCCAGGAGCCAGGGGAGAC GGCGGCAAGTGAAGCAGCCTCCAGCTCTCTTCCTGCTGTCGTGGTGGAGACCTTCCCAGCAACTGTGAATGGCACCGTGGAGGG CGGCAGTGGGGCCGGGCGCTTGGACCTGCCCCCAGGTTTCATGTTCAAGGTACAGGCCCAGCACGACTACACGGCCACTGACAC AGACGAGCTGCAGCTCAAGGCTGGTGATGTGGTGCTGGTGATCCCCTTCCAGAACCCTGAAGAGCAGGATGAAGGCTGGCTCAT GGGCGTGAAGGAGAGCGACTGGAACCAGCACAAGGAGCTGGAGAAGTGCCGTGGCGTCTTCCCCGAGAACTTCACTGAGAGGGT CCCATGATAATAGAAGCTTCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTT GCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATG GGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGG AGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTG GGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTC TCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCT GTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGC GCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG CGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCA ACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGC CCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCT CCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATC GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT CAACCCTATCTCGGGCTATTCTTTTGATTTAATCCCCTATAGTGAGTCGTATTACATGGTCATAGCTGTTTCCTGGCAGCTCTG GCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAA CAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATA 22
026389-0044-WO01 TGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTT GTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCC TCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCA GGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGT TTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTC AGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGT CGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTT TCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGT TAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGCAAGCTCATGACCAAAATCCCTTAACGTGAGTTA CGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGG CTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT GGCCTTTTGCTGGCCTTTTGCTCACATGTT Controls One embodiment described herein is a control gene expression vector. In one aspect, the control may comprise an enhanced Green Fluorescent Protein (eGFP) polynucleotide sequence 5 encoding an eGFP protein (SEQ ID NO: 15–16): SEQ ID NO: 15 – eGFP Polynucleotide Sequence ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCC GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCA AGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTAT ATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGC AGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG CACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCC GGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA SEQ ID NO: 16 – eGFP Polypeptide Sequence MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYP DHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVY IMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA GITLGMDELYK Another embodiment described herein is control gene expression vector comprising polynucleotide encoding the enhanced Green Fluorescent Protein (eGFP) polynucleotide 10 sequence (SEQ ID NO: 17). SEQ ID NO: 17 – eGFP AAV Construct (pAAV-CMV-eGFP) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTCTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG 23
026389-0044-WO01 ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTG TTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGA TTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTT TGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATT CTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTG ATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATT ATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTC TGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACATCGATTGAATTCAACAAGTTTGTACAAAAAAGCAGGCTTA ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTC AGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAG GTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATC CGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG CCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAAGCTTGCCTCGAGAGATCTACGGGTGGCATCCCTG TGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCAT CATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAAC CTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGT TCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTG GTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATT GCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAG GAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGC CCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACG CATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGT TCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAA AACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCT TTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGA TTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTAT GGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCAT CACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGT CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA GACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCT TTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCAC GAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCA CTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTC AGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCA TAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACA TGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGC CTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGA TGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTG AGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTC AGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTT ACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGA CCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTT TTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTC TTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGG AGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACA GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG CGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT 24
026389-0044-WO01 Methods of Treatment The present disclosure also provides methods of improving atrial function and reducing atrial arrhythmias in a subject in need thereof by administering to the subject a therapeutically effective amount of a cBIN1 gene therapy. For example, described herein are methods for 5 treating, preventing, reducing the likelihood of having, reducing the severity of, and/or slowing the progression of atrial fibrillation in a subject using cBIN1 gene therapy. In some aspects, a subject may exhibit one or more improved atrial properties following administration of the cBIN1 gene therapy relative to before administration of the cBIN1 gene therapy. In certain aspects, the one or more improved atrial properties may comprise shortened P-wave duration, reduced atrial 10 fibrosis, reduced left atrial dyssynchrony, increased intracellular T-tubule area, reduced atrial arrhythmia (e.g., reduced atrial fibrillation), or combinations thereof. In other aspects, the improved atrial properties may further comprise one or more of reduced left atrial volume (LAV), increased left atrial (LA) reservoir function, or atrial mitochondrial improvements. In some embodiments, the cBIN1 gene therapy is administered to the subject via 15 intravenous injection, intramyocardial injection, cardiac catheter infusion to myocardium, or combinations thereof. The cBIN1 gene therapy being administered to the subject may comprise a cBIN1 expression vector comprising a cBIN1 polynucleotide sequence transgene encoding a cBIN1 polypeptide or functional variant or fragment thereof, as described herein. In some embodiments, the subject may have atrial fibrillation or may be at risk of developing atrial 20 fibrillation. In some embodiments, the subject may have pre-existing heart failure or may be at risk of developing heart failure. The heart failure may comprise ischemic or non-ischemic heart failure. In various embodiments, the pharmaceutical composition comprising the cBIN1 gene expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or 25 functional variant or fragment thereof may be administered as a therapeutic agent (e.g., cBIN1 gene therapy) to a subject in need thereof to improve atrial function and reduce atrial arrhythmias. In various embodiments, the pharmaceutical composition may be administered as a therapeutic agent (e.g., cBIN1 gene therapy) to a subject to treat, prevent, reduce the likelihood of having, reduce the severity of, and/or slow the progression of atrial fibrillation in the subject. 30 In various embodiments, the subject is a mammal. In various embodiments, the subject is a human. In various embodiments, the subject is a mammalian subject including but not limited to human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse, and rat. In various embodiments, the pharmaceutical composition comprising a cBIN1 gene expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or 25
026389-0044-WO01 functional variant or fragment thereof is administered to a subject by intravenous (i.v.) injection. Pharmaceutical compositions as described herein may also be administered using alternative routes, including but not limited to intravascular, intraarterial, intramuscular, subcutaneous, intraperitoneal, aerosol, nasal, via inhalation, oral, transmucosal, transdermal, parenteral, 5 implantable pump or reservoir, continuous infusion, enteral application, topical application, local application, capsules, and/or injections. For example, in one embodiment, the pharmaceutical composition comprising a cBIN1 gene expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof is administered to a subject by cardiac catheter infusion to myocardium or intramyocardial injection. 10 In some embodiments, the subject is administered a single dose of the disclosed pharmaceutical compositions (e.g., cBIN1 gene therapy). In other embodiments, the subject is administered a plurality of doses of the disclosed pharmaceutical compositions over a period of time (i.e., administered more than once). For example, in various nonlimiting embodiments, a pharmaceutical composition as described herein may be administered to a subject once a day 15 (SID/QD), twice a day (BID), three times a day (TID), four times a day (QID), or more, so as to administer a therapeutically effective amount of the pharmaceutical composition to the subject, where the therapeutically effective amount is any one or more of the doses described herein. In some embodiments, a pharmaceutical composition as described herein is administered to a subject 1–3 times per day, 1–7 times per week, 1–9 times per month, 1–12 times per year, or 20 more. In other embodiments, a pharmaceutical composition as described herein is administered for about 1–10 days, 10–20 days, 20–30 days, 30–40 days, 40–50 days, 50–60 days, 60–70 days, 70–80 days, 80–90 days, 90–100 days, 1–6 months, 6–12 months, 1–5 years, or more. In various embodiments, a pharmaceutical composition as described herein is administered at about 0.001–0.01, 0.01–0.1, 0.1–0.5, 0.5–5, 5–10, 10–20, 20–50, 50–100, 100–200, 200–300, 300– 25 400, 400–500, 500–600, 600–700, 700–800, 800–900, 900–1000 mg/kg, or a combination thereof. The actual dosing regimen can depend upon many factors, including but not limited to the judgment of a trained physician, the overall condition of the subject, and the specific type of atrial fibrillation. The actual dosage can also depend on the determined experimental effectiveness of 30 the specific pharmaceutical composition (e.g., a cBIN1 gene expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof) that is administered. For example, the dosage may be determined based on in vitro responsiveness of relevant cultured cells, or in vivo responses observed in appropriate animal models or human studies for the cBIN1 gene therapy. 26
026389-0044-WO01 In some embodiments, disclosed pharmaceutical compositions may be administered to a subject using a dosing regimen that is based on cBIN1 gene expression vector genome (vg) per kg body weight of the subject (vg/kg). In one embodiment, a cBIN1 gene therapy vg/kg dosing regimen is used to improve atrial function and reduce/treat atrial arrhythmias (e.g., atrial 5 fibrillation) in a subject in need thereof. In one nonlimiting exemplary embodiment, a pharmaceutical composition comprising a cBIN1 gene therapy is administered to a subject at a dose ranging from about 5 × 1011 vg/kg to about 5 × 1013 vg/kg cBIN1 gene expression vector. In other embodiments, a cBIN1 gene therapy is administered to a subject at a dose of about 1 × 1010 vg/kg to about 5 × 1013 vg/kg cBIN1 gene expression vector. 10 In some embodiments, the disclosed methods and pharmaceutical compositions are able to sufficiently deliver a cBIN1 transgene to a large mammalian (e.g., human) heart by i.v. administration of a very low dose of cBIN1 gene therapy (e.g., about 5 × 1011 vg/kg to about 5 × 1013 vg/kg cBIN1 gene expression vector). In some embodiments, the disclosed methods and pharmaceutical compositions are able to achieve robust and durable cBIN1 transgene 15 expression in a large mammalian heart, specifically in the apex, anterior wall, posterior wall, base, and/or septum regions of the heart. In some embodiments, the disclosed methods and pharmaceutical compositions are able to increase the selective expression of a cBIN1 transgene in a large mammalian heart by at least about 20% relative to an endogenous cBIN1 expression level in the heart. In some embodiments, the disclosed methods and pharmaceutical 20 compositions are able to increase the selective expression of a cBIN1 transgene in a large mammalian heart for at least 1 month, 3 months, 6 months, 12 months, 18 months, 2 years, or greater. Another embodiment described herein is the use of echocardiography or other imaging methods to measure cardiac and functional improvements of a subject in response to treatment 25 with the disclosed cBIN1 gene therapies. Another embodiment described herein is the use of a plasma or blood cardiac bridging integrator 1 (cBIN1) score (CS) to identify a subject having atrial fibrillation for cBIN1 gene therapy treatment. In one aspect, the cBIN1 gene therapy treatment comprises a therapeutically effective amount of a pharmaceutical composition comprising: a cBIN1 gene expression vector comprising 30 a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof. In another aspect, the plasma CS is a non-invasive measure of target engagement and therapeutic response of the subject to the cBIN1 gene therapy treatment. In another aspect, the plasma CS is the natural log of the ratio of a median plasma cBIN1 concentration in a normal human population to a measured cBIN1 concentration in the subject having atrial fibrillation. 27
026389-0044-WO01 In some embodiments, the disclosed methods and pharmaceutical compositions may reduce atrial fibrillation in a subject. In some embodiments, the disclosed methods and pharmaceutical compositions may stabilize (i.e., normalize or restore) the intracellular distribution of calcium handling machinery in myocardium of a subject, wherein the calcium handling 5 machinery may comprise one or more of SERCA2a, Cav1.2, or RyR2. In some embodiments, the disclosed methods and pharmaceutical compositions may rehabilitate or increase transverse- tubule microfolds or microdomains in myocardium of a subject. In some embodiments, the disclosed methods and pharmaceutical compositions may correct T-tubule muscle pathology present in an atrial fibrillation heart and/or improve, restore, or enhance T-tubule size, shape, 10 and/or structure, as measured by a tissue biopsy or other means for imaging cardiac T-tubules. In some embodiments, the disclosed methods and pharmaceutical compositions may express or selectively express cBIN1 in the heart (cardiac tissue) of a subject. In some embodiments, the disclosed methods and pharmaceutical compositions may express or selectively express cBIN1 in one or more of the apex, anterior wall, posterior wall, base, or septum 15 of the heart of a subject. In some embodiments, the disclosed methods and pharmaceutical compositions may increase the selective expression of cBIN1 in the heart of a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater relative to an endogenous cBIN1 expression level in the heart of the subject. In some embodiments, the disclosed methods and pharmaceutical compositions may 20 normalize cardiac expression of cBIN1 in a subject having one or more atrial arrhythmias (e.g., atrial fibrillation). In some embodiments, the disclosed methods and pharmaceutical compositions may express cBIN1 in a subject for at least 6 months. In some embodiments, the disclosed methods and pharmaceutical compositions may enhance and/or normalize the cardiac expression of cBIN1 in a subject to a sufficient level to improve atrial function and reduce atrial 25 arrhythmias in the subject. One embodiment described herein is a method of improving atrial function and reducing atrial arrhythmias in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a cardiac bridging integrator 1 (cBIN1) gene therapy. In one aspect, the subject exhibits one or more improved atrial properties following administration of the 30 cBIN1 gene therapy relative to before administration of the cBIN1 gene therapy. In another aspect, the one or more improved atrial properties comprise shortened P-wave duration, reduced atrial fibrosis, reduced left atrial dyssynchrony, increased intracellular T-tubule area, reduced atrial arrhythmia, or combinations thereof. In another aspect, the reduced atrial arrhythmia is reduced atrial fibrillation. In another aspect, the cBIN1 gene therapy is administered to the subject via 28
026389-0044-WO01 intravenous injection, intramyocardial injection, cardiac catheter infusion to myocardium, or combinations thereof. In another aspect, the cBIN1 gene therapy is administered to the subject more than once. In another aspect, the cBIN1 gene therapy comprises a transgene encoding a cBIN1 polypeptide or functional variant or fragment thereof. In another aspect, the cBIN1 gene 5 therapy comprises a cBIN1 expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof. In another aspect, the cBIN1 expression vector comprises a non-viral vector, a viral vector, an adeno-associated virus (AAV) vector, a recombinant AAV (rAAV) vector, a single-stranded AAV vector, a double-stranded AAV vector, a self-complementary AAV (scAAV) vector, or combinations thereof. In another 10 aspect, the cBIN1 expression vector comprises an AAV vector of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, a hybrid serotype thereof, or a derivative thereof. In another aspect, the cBIN1 gene therapy comprises an adeno-associated virus 9 transducing cBIN1 (AAV9-cBIN1). In another aspect, the cBIN1 expression vector comprises a muscle-tropic expression vector. In another aspect, the cBIN1 15 gene therapy expresses cBIN1 in cardiac tissue of the subject. In another aspect, the cBIN1 gene therapy selectively expresses cBIN1 in cardiac tissue of the subject. In another aspect, the non- viral vector comprises a lipid carrier, an exosome, a polymer-based carrier, a chemical-based carrier, a conjugated carrier, or combinations thereof. In another aspect, the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence comprising DNA, RNA, or a combination thereof. In 20 another aspect, the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence having at least 90–99% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, or 11. In another aspect, the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence of any one of SEQ ID NO: 1, 3, 5, 7, or 11. In another aspect, the cBIN1 gene therapy is administered to the subject using a dosing regimen based on cBIN1 gene expression vector genome (vg) per kg body weight of the 25 subject. In another aspect, the cBIN1 gene therapy is administered to the subject at a dose of about 1 × 1010 vg/kg to about 1 × 1014 vg/kg cBIN1 gene expression vector. In another aspect, the subject has pre-existing heart failure. In another aspect, the cBIN1 gene therapy is administered after the subject is diagnosed with heart failure. In another aspect, the subject is a mammal. In another aspect, the subject is a human, primate, dog, pig, or mouse. 30 Another embodiment described herein is the use of a cardiac bridging integrator 1 (cBIN1) gene therapy in a medicament for improving atrial function and reducing atrial arrhythmias in a subject in need thereof. Another embodiment described herein is the use of a transgene encoding cardiac bridging integrator 1 (cBIN1) in the preparation of a medicament for use in a method of improving atrial 29
026389-0044-WO01 function and reducing atrial arrhythmias in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the transgene encoding cBIN1. It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described 5 herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential 10 combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the 15 other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are 20 incorporated by reference herein for the specific teachings thereof. Various embodiments and aspects of the inventions described herein are summarized by the following clauses: Clause 1. A method of improving atrial function and reducing atrial arrhythmias in a subject in need thereof, the method comprising: 25 administering to the subject a therapeutically effective amount of a cardiac bridging integrator 1 (cBIN1) gene therapy. Clause 2. The method of clause 1, wherein the subject exhibits one or more improved atrial properties following administration of the cBIN1 gene therapy relative to before administration of the cBIN1 gene therapy. 30 Clause 3. The method of clause 1 or 2, wherein the one or more improved atrial properties comprise shortened P-wave duration, reduced atrial fibrosis, reduced left atrial dyssynchrony, increased intracellular T-tubule area, reduced atrial arrhythmia, or combinations thereof. 30
026389-0044-WO01 Clause 4. The method of any one of clauses 1–3, wherein the reduced atrial arrhythmia is reduced atrial fibrillation. Clause 5. The method of any one of clauses 1–4, wherein the cBIN1 gene therapy is administered to the subject via intravenous injection, intramyocardial injection, cardiac 5 catheter infusion to myocardium, or combinations thereof. Clause 6. The method of any one of clauses 1–5, wherein the cBIN1 gene therapy is administered to the subject more than once. Clause 7. The method of any one of clauses 1–6, wherein the cBIN1 gene therapy comprises a transgene encoding a cBIN1 polypeptide or functional variant or fragment thereof. 10 Clause 8. The method of any one of clauses 1–7, wherein the cBIN1 gene therapy comprises a cBIN1 expression vector comprising a cBIN1 polynucleotide sequence encoding a cBIN1 polypeptide or functional variant or fragment thereof. Clause 9. The method of any one of clauses 1–8, wherein the cBIN1 expression vector comprises a non-viral vector, a viral vector, an adeno-associated virus (AAV) vector, a 15 recombinant AAV (rAAV) vector, a single-stranded AAV vector, a double-stranded AAV vector, a self-complementary AAV (scAAV) vector, or combinations thereof. Clause 10. The method of any one of clauses 1–9, wherein the cBIN1 expression vector comprises an AAV vector of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, a hybrid serotype thereof, or 20 a derivative thereof. Clause 11. The method of any one of clauses 1–10, wherein the cBIN1 gene therapy comprises an adeno-associated virus 9 transducing cBIN1 (AAV9-cBIN1). Clause 12. The method of any one of clauses 1–11, wherein the cBIN1 expression vector comprises a muscle-tropic expression vector. 25 Clause 13. The method of any one of clauses 1–12, wherein the cBIN1 gene therapy expresses cBIN1 in cardiac tissue of the subject. Clause 14. The method of any one of clauses 1–13, wherein the cBIN1 gene therapy selectively expresses cBIN1 in cardiac tissue of the subject. Clause 15. The method of any one of clauses 1–14, wherein the non-viral vector comprises a 30 lipid carrier, an exosome, a polymer-based carrier, a chemical-based carrier, a conjugated carrier, or combinations thereof. Clause 16. The method of any one of clauses 1–15, wherein the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence comprising DNA, RNA, or a combination thereof. 31
026389-0044-WO01 Clause 17. The method of any one of clauses 1–16, wherein the cBIN1 gene therapy comprises a cBIN1 polynucleotide sequence having at least 90–99% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, or 11. Clause 18. The method of any one of clauses 1–17, wherein the cBIN1 gene therapy 5 comprises a cBIN1 polynucleotide sequence of any one of SEQ ID NO: 1, 3, 5, 7, or 11. Clause 19. The method of any one of clauses 1–18, wherein the cBIN1 gene therapy is administered to the subject using a dosing regimen based on cBIN1 gene expression vector genome (vg) per kg body weight of the subject. Clause 20. The method of any one of clauses 1–19, wherein the cBIN1 gene therapy is 10 administered to the subject at a dose of about 1 × 1010 vg/kg to about 1 × 1014 vg/kg cBIN1 gene expression vector. Clause 21. The method of any one of clauses 1–20, wherein the subject has pre-existing heart failure. Clause 22. The method of any one of clauses 1–21, wherein the cBIN1 gene therapy is 15 administered after the subject is diagnosed with heart failure. Clause 23. The method of any one of clauses 1–22, wherein the subject is a mammal. Clause 24. The method of any one of clauses 1–23, wherein the subject is a human, primate, dog, pig, or mouse. Clause 25. Use of a cardiac bridging integrator 1 (cBIN1) gene therapy in a medicament for 20 improving atrial function and reducing atrial arrhythmias in a subject in need thereof. Clause 26. Use of a transgene encoding cardiac bridging integrator 1 (cBIN1) in the preparation of a medicament for use in a method of improving atrial function and reducing atrial arrhythmias in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the transgene encoding cBIN1. 25 EXAMPLES An established canine model of ischemic dilated cardiomyopathy and subsequent HF was used to demonstrate the effects of cBIN1 gene therapy on atrial remodeling in HF. Canines (n = 18, 25–35 kg, 12 months old) underwent a thoracotomy and permanent ligation of the left anterior 30 descending (LAD) artery below the first diagonal artery. Potential collateral circulation to the infarction zone were also ligated, including the distal LAD and any epicardial branches towards the left and right ventricles (typically an additional 4–6 branches). The thoracotomies were closed, and HF progress was monitored for 2–4 months. Weekly to biweekly echocardiograms and blood work were conducted until left ventricular ejection fraction (LVEF) < 40% and NT-proBNP > 900 32
026389-0044-WO01 pg/mL. Once these criteria for HF were observed, the dogs were divided into one of 3 groups: (1) AAV9-cBIN1 therapy (n = 9); (2) AAV9-GFP therapy (n = 6); or (3) untreated HF (UHF, n = 3). Additionally, tissue samples were collected from 3 control canines that did not undergo LAD ligation (control). Gene therapy was delivered to the myocardium with a NOGA-XP system 5 (Johnson and Johnson) using a Myostar injection catheter to the left ventricle (LV) through myocardial injections delivered to 20 locations surrounding and away from the low voltage region denoting scar formation. The AAV9-mouse cBIN1 (SEQ ID NO: 13) and AAV9-GFP (SEQ ID NO: 17) constructs were delivered 250 µL per injection and 2.5 × 1011 vg for a total dose of 5 × 1012 vg per animal. The untreated HF group did not receive myocardial injections. 10 Following establishment of HF criteria, canine subjects were survived for 5–8 weeks and underwent echocardiograms, ECG analysis, and tissue sampling of the atria. P-wave duration was measured from the ECG using limb leads. Left atrial (LA) dyssynchrony was measured, defined as the time difference to peak strain between the earliest septal and latest lateral activated segments. LA reservoir strain was measured at end systole. LA volume was measured at end 15 diastole. Atrial arrhythmia incidence during terminal studies were defined as sustained atrial arrhythmias lasting for at least 2 minutes. After euthanasia, hearts were removed and left atrial tissue samples were preserved as follows: (1) in frozen in optimal cutting temperature (OCT) compound, (2) in formalin fixative, and (3) in fixative for transmission electron microscopy (TEM) by submersion in 2.5% glutaraldehyde, 20 1% paraformaldehyde, 0.1 M cacodylate buffer, pH 7.4. OCT preserved tissues were sliced into 10 µm sections and stained with wheat germ agglutinin (WGA) and imaged with confocal microscopy to visualize T-tubule distribution and regularity. Using QuPath software, the magnification of the images was standardized, and the regions of interest were extracted. T- tubule characteristics were quantified using ImageJ (or FIJI), including the T-tubule fraction per 25 cell and the T-tubule density within arbitrarily selected rectangular areas, which calculated the average density of each T-tubule peak au (ImageJ, plugin-orientation J). These parameters were compared among the cBIN1, GFP, and UHF treated groups. Formalin fixed tissues were cut in 4 µm sections and stained with Masson trichrome. The percentage of collagen (blue stained) over myocardium (red stained) was quantified with nine randomly selected areas from each sample,30 which increased statistical confidence of the measured parameters, using ImageJ software. TEM- fixed tissues were sliced into ultrathin 70 nm sections and imaged with TEM techniques to image T-tubule sections and mitochondrial characteristics. T-tubule cross-sectional areas and mitochondrial area matrix areas and percentage matrix were quantified using ImageJ software. For mitochondrial analysis, the images were converted to binary color data, which allowed for the 33
026389-0044-WO01 identification of the white area corresponding to the matrix portion, resulting in the calculation of the percentage of matrix (matrix/cristae × 100). During terminal studies, the incidence of spontaneous sustained atrial arrhythmias lasting at least 2 minutes were recorded. 5 Statistics Continuous variables were presented as mean ± standard error (SE) for normally distributed data and as median with interquartile range [IQR] for non-normally distributed data. Normality was assessed prior to statistical testing. For comparisons involving more than two 10 groups, analysis of variance (ANOVA) was performed, followed by pairwise post-hoc analysis when appropriate using the Tukey HSD test for normally distributed parameters or the Steel– Dwass test for non-normally distributed parameters. An unpaired t-test was used for between- group comparisons of normally distributed variables, while paired t-tests were used for within- subject (time-point) comparisons, such as ultrasound measurements. Non-parametric data were 15 compared using the Wilcoxon signed-rank test. A p-value of <0.05 was considered statistically significant. Ultrasound Findings A total of 15 canines (9 cBIN1-treated, 6 GFP-treated) underwent echocardiographic 20 (ECG) assessments at baseline, pre-injection, and terminal time points. ECG was captured simultaneously with echocardiograms. P-wave duration significantly increased after ligation in both groups. Following gene therapy treatment, the cBIN1 group demonstrated a significant shortening of P-wave duration (from 99 ± 1 ms to 88 ± 1 ms, p = 0.0001; FIG.1), whereas the GFP group exhibited further prolongation (from 89 ± 4 ms to 104 ± 25 4 ms, p = 0.0001). Septal-lateral delay in left atrial (LA) strain increased post-ligation in both groups. At terminal study, this delay significantly decreased in the cBIN1 group (from 57 ± 6 ms to 13 ± 6 ms, p = 0.001; FIG.2), while LA dyssynchrony continued to increase in the GFP group. Left atrial volume (LAV) increased significantly after ligation in both groups (cBIN1: 22 to 30 57 mL, p < 0.0001; GFP: 24 to 68 mL, p < 0.0001). The cBIN1 group showed a trend toward smaller LAV at the terminal point (57 to 48 mL, p = 0.09), though not statistically significant. LA reservoir strain worsened after ligation in both groups and showed a non-significant trend toward improvement after therapy in the cBIN1 group (from 14 ± 3% to 19 ± 3%, p = 0.28), while this was not seen in the GFP group (from 15 ± 3% to 14 ± 3%, p = 0.75). 34
026389-0044-WO01 Fibrosis Burden Fibrotic area was quantified in three atrial tissue samples per treatment group (total n = 9). 5 The cBIN1 group had significantly less fibrosis compared to both the GFP and UHF control groups (8.6 ± 0.8% vs.12.4 ± 0.9%, p = 0.01; vs.13.1 ± 1.0%, p = 0.002; FIG.3). No significant difference was observed between the GFP and UHF control groups (p = 0.86). 10 Transmission Electron Microscopy (TEM) TEM was performed on samples from 3 cBIN1-treated, 6 GFP-treated, and 3 nonfailing control animals. T-tubule area was significantly smaller in the cBIN1 group compared to the GFP group (0.012 [0.006–0.022] μm2 vs. 0.017 [0.012–0.027] μm2, p = 0.01; FIG. 4), but both were larger 15 than in unfailing controls (0.003 [0.001–0.01] μm2). Using binary images of mitochondria, the percentage of matrix area, total mitochondrial area, and matrix area were quantified and compared across the groups (FIG.5A). Mitochondrial matrix area (%) was significantly smaller in the cBIN1 group (49.6 ± 0.5%) than in the GFP group (54.4 ± 0.5%, p < 0.0001), both of which were larger than in the non-failing 20 control group (42.3 ± 0.7%; FIG.5B). Total mitochondrial area did not differ significantly across the groups (FIG.5C); however, matrix area was significantly greater in the cBIN1 and GFP groups compared to the non-failing control (0.29 ± 0.01 μm2 and 0.31 ± 0.01 μm2 vs. 0.24 ± 0.02 μm2, p = 0.048 and p = 0.01, respectively; FIG.5D), with no difference between cBIN1 and GFP (p = 0.62). 25 Confocal Imaging Analysis Confocal images were obtained from 4 cBIN1, 3 GFP, and 3 UHF samples (see examples in FIG.6A–C). Per-cell T-tubule fraction was significantly greater in the cBIN1 group (12 ± 1%) compared 30 to GFP (7 ± 1%) and UHF (6 ± 1%) groups (p = 0.002; FIG.7). Peak intensity (per-area analysis) was higher in the cBIN1 group than in GFP or UHF (74 ± 12 au vs.45 ± 4 au vs.40 ± 6 au, p = 0.01; FIG.8). Atrial Arrhythmia Incidence 35
026389-0044-WO01 Atrial fibrillation spontaneously occurred in 0 of 8 animals treated with cBIN1 and 2 of 6 animals treated with GFP. This trend did not reach statistical significance (p = 0.165). The data presented herein demonstrate improvement in atrial substrate remodeling during 5 HF progression with cBIN1 gene therapy. Improvements in P-wave duration, LA dyssynchrony, and sub-cellular, cellular, and tissue level remodeling are all indicators of improved atrial function and a reduced arrhythmogenic substrate. Increased atrial fibrosis leads to slower atrial conduction velocity, consistent with the decrease in LA synchrony and increased P-wave duration. Dysregulation of T-tubule structure and mitochondrial compromise results in decreased atrial 10 contractility. Sustained atrial arrhythmias require both a trigger for initiation of arrhythmias and atrial substrate remodeling. T-tubule dysregulation leads to calcium mishandling and triggered activity through calcium overload and calcium sparks. As disclosed herein, cBIN1 gene therapy delivered to the LV was found to reduce T-tubule remodeling in the atria, which reduces the risk of triggered activity. The atrial substrate that 15 sustains atrial fibrillation, including atrial fibrosis and slowed conduction velocity, were reduced in cBIN1-treated animals. Patients with HF are at increased risk for atrial arrhythmias and dysfunction. The data presented herein demonstrate that cBIN1 gene therapy provides a potent effect and therapeutic approach to reduce atrial dysfunction and arrhythmogenic substrate. 20 36