Modulation of in vivo cardiac performance using hyper phosphomimetic cmybp-c

Abstract

A variant cMyBPC protein comprises at least one set of substitutions selected from four sets of substitutions. The first set of substitutions comprises the substitutions Thr272 to Asp272, Ser273 to Asp273 and Leu274 to Asp274. The second set of substitutions comprising the substitutions Thr281 to Asp281 and Ser282 to Asp 282. The third set of substitutions comprising the substitutions Ser302 to Asp 302 and Phe303 to Asp 303. The fourth set of substitutions comprising the substitutions Ser307 to Asp 307 and Lys308 to Asp308. The variant cMyBPC protein may be encoded by a polynucleotide and may further be carried by a vector adapted to express the variant cMyBPC protein. In a method of treating heart failure, the variant cMyBPC protein is administered to a patient. The route of administration may be via an adeno-associated virus vector such as AAV9.

Description

Modulation of in vivo Cardiac Performance Using Hyper Phosphomimetic cMyBP-CCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of pending U.S. Provisional Application No. 63 / 559,674 filed on February 29, 2024, the disclosure of which is incorporated by reference herein.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] The material contained in the Sequence Listing provided herewith in XML format in the file entitled “200512-00509. xml” created on February 28, 2025, and containing 40,555 bytes, is hereby incorporated by reference herein.BACKGROUND OF THE INVENTION

[0003] Heart failure (HF) is a complex clinical syndrome characterized by contractile dysfunction at the level of the myofilament that translates to impaired contraction and relaxation of cardiac muscle. Current therapies for HF are aimed at managing symptoms rather than targeting the pathophysiological mechanism of impaired contractile function itself. As a result, mortality for HF patients remains high. Previous attempts to address the underlying pathophysiology using a sarcomeric target for HF therapy resulted in the development of myosin activating drugs, but these failed clinical trials due to intolerable side effects caused by the difficulty in augmenting systolic function without causing diastolic impairment.

[0004] Cardiac myosin binding protein C (cMyBPC) is a better target for therapeutic intervention. cMyBPC is a sarcomere protein known to regulate contractile function. It is a long protein made up of 11 sequential domains numbered from the N-terminus as CO through CIO, separated by linking sequences. The C-terminal domains C8-C10 anchor cMyBPC to the thick filament. Expression is constrained to the C-zone of the sarcomere, with a stoichiometry of 3 cMyBPC molecules for every 9 myosins within this region. The lengths and flexibility of cMyBPC allow it to interact with multiple different myosins. Additionally, cMyBPC can span the distance between the thick and thin filaments in order to interact with both actin and myosin.

[0005] The N-terminal domains C0-C2 are the best studied, and are known to interact with both actin and myosin and allow cMyBPC to tunc contractility. Interactions with myosin have been shown to promote the “off’ state of myosin, likely by tethering myosin heads to the thick filamentbackbone and preventing them from participating in crossbridge cycling. This may serve as an energy-conserving mechanism and provides a recruitable pool of myosins when force must be augmented to meet physiologic demands. Interactions of cMyBPC N-terminal domains with the thin filament regulate the rate of crossbridge detachment, and thus the longevity of crossbridges. This corresponds with systolic ejection time in vivo.

[0006] The biological activity of cMyBPC is controlled via phosphorylation of key sites within the regulatory M-domain of cMyBPC. The M-domain comprises the linking sequence between domains Cl and C2 at the N-terminus of the molecule, and contains 4 primary serine residues whose phosphorylation is known to modulate cMyBPC function: S273, S282, S302, and S307. S273 and S282 are heavily phosphorylated at baseline, while S302 and S307 are primarily phosphorylated in response to beta-adrenergic signaling during stress. The M-domain is a highly dynamic and disordered region of the protein, which has made structural determination impossible thus far. It is hypothesized that the introduction of negative charges that occurs with phosphorylation alters N-terminal domain orientation and binding affinity, favoring or disfavoring interactions with different partners. Phosphorylation of M-domain sites has been shown to abolish myosin binding, thus relieving the inhibition on myosin heads and allowing them to participate in force generation. In HF, adrenergic signaling perturbations lead to dephosphorylation of cMyBPC. Dephosphorylation of cMyBPC is believed to contribute to the contractile dysfunction in HF. Overall, cMyBPC is a critical regulator of crossbridge cycling at the level of the sarcomere, which translates to regulation of force production and relaxation at the whole organ level.SUMMARY OF THE INVENTION

[0007] The present invention provides a form of cMyBPC that mimics the phosphorylated state of the native protein. When serine residues are phosphorylated, the uncharged OH group of the serine is replaced by a highly negatively charged phosphate group. In order to recapitulate the charge density accomplished by phosphorylation, the present invention provides full-length cMyBPC with one or more of the 4 regulatory serines and appropriate flanking amino acids replaced by negatively charged aspartate residues. A total of nine mutations arc possible: Thr272 to Asp272, Ser273 to Asp273, Leu274 to Asp274; Thr281 to Asp281 , Ser282 to Asp 282; Ser302 to Asp 302, Phc303 to Asp 303; Scr307 to Asp 307, Lys308 to Asp308. Full-length cMyBPC with the full set of these substitutions is designated herein as “4x3D-FL.”

[0008] The relevant portion of the native mouse amino acid sequence of cMyBPC is shown below with S273, S282, S302, and S307 in bold:273 282 302 307270 -RRTSLAGAGRRTSDSHEDAGTPDFSSLLKKRDSFRRDSKLE- 310 (SEQ ID NO: 1) Sites 282, 302, and 307 each have a native aspartate residue adjacent to the relevant serine such that only two mutations are necessary to produce the 3-Asp charge density. The present version of the phosphorylation-mimicking sequence is shown below with mutations in bold and underlined: 273 282 302 307

[0009] 270 - RRDDDAGAGRRDDDSHEDAGTPDFSSLLKKRDDDRRDDDLE - 310 (SEQID NO: 2)

[0010] Previous studies using transgenic mouse models with conventional approaches (serine substituted with a single D mutation) to mimic phosphorylation of cMyBPC have not shown significant differences in in vivo function compared to wild-type mice. While not wishing to condition patentability on any particular theory, it is proposed that this is because a single Asp substitution does not fully mimic the negative charge of biological phosphorylation added by a phosphate ion to a serine residue.

[0011] In general, the present invention provides a variant cMyBPC protein comprising at least one set of substitutions selected from the group consisting of: Thr272 to Asp272, Ser273 to Asp273, Leu274 to Asp274; Thr281 to Asp281, Ser282 to Asp 282; Ser302 to Asp 302, Phe303 to Asp 303; Ser307 to Asp 307, Lys308 to Asp3O8. The variant cMyBPC protein may comprise the amino acids of SEQ ID NO: 2. The protein may be expressed by a vector comprising a nucleotide sequence expressing the polypeptide of SEQ ID NO: 2.

[0012] In one particular embodiment, the variant cMyBPC protein comprises the substitutions Thr272 to Asp272, Ser273 to Asp273 and Leu274 to Asp274. In another embodiment, the variant cMyBPC protein comprises the substitutions , the variant cMyBPC protein comprises the substitutions Thr281 to Asp281 and Ser282 to Asp 282. In a further embodiment, the variant cMyBPC protein comprises the substitutions Ser302 to Asp 302 andPhe3O3 to Asp 303. In still another embodiment, the variant cMyBPC protein comprises the substitutions Ser307 to Asp 307 and Lys308 to Asp308. In yet another embodiment, the variant cMyBPC protein may comprise two or more of these sets of substitutions, such as substitutions Thr272 to Asp272, Scr273 to Asp273 and Leu274 to Asp274 together with Thr281 to Asp281 and Ser282 to Asp 282.

[0013] The present invention also provides a method of treatment for heart failure, the method comprising administering a vector to a patient, where the vector comprises a polynucleotide sequence encoding a variant cMyBPC polypeptide as described above where the vector is adapted to express the variant cMyBPC polypeptide. The vector may be an Adeno-associated virus (AAV) vector such as AAV9. An example of such a vector comprises SEQ ID NO: 5.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0014] Fig 1 A is a photograph of a Western blot showing expression of wild-type cMyBPC in wild-type mice and in cMyBPCmice expressing AAV9-delivered wild-type full length cMyBPC (WT-FL) or expressing AAV9-delivered full length 4x3D cMyBPC (4x3D-FL)

[0015] Fig. IB is a set of graphs showing the ejection fraction (EF, left panel), aortic ejections time (AAET, middle panel) and isovolumic relaxation time (IVRT, right panel) in cMyBPC - / - mice injected with AAV9-delivered wild-type full length cMyBPC (AAV9-WT-FL) or AAV9- delivered 4x3D-FL cMyBPC, or a Blank control.

[0016] Fig. 2A are a set of graphs showing isovolumic relaxation time (IVRT, left panel), ejection fraction (EF, middle panel), and global longitudinal strain (GLS, right panel) from wild type mice injected with AAV9-WT-FL (WT+WT-FL) or wild type mice injected with AAV9- delivered-4x3D (WT+4x3D).

[0017] Fig. 2B is a representative Western blot from WT mice injected with WT+WT-FL or WT+4x3D.

[0018] Fig. 3 is a set graphs showing ejection fraction (EF) and isovolumic relaxation time (IVRT) from cMyBPC- / - (KO) mice injected with AAV9-delivered 4x3D-FL cMyBPC (KO+4x3D), AAV9-delivered 273D cMyBPC (KO+273D), and AAV9-delivered 302D cMyBPC (KO+302D). AAV9-delivered 273D cMyBPC contains the substitutions T272D, S273D and L274D. AAV9-delivered 302D cMyBPC contains the substitutions S302D and F303D.DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is directed toward a variant cMyBPC protein comprising at least one set of substitutions at and surrounding cMyBPC amino acids 273, 282 302 and 307. The substitutions for locus 273 arc Thr272 to Asp272, Scr273 to Asp273 and Lcu274 to Asp274. The substitutions for locus 282 are Thr281 to Asp281 and Ser282 to Asp 282. The substitutions forlocus 302 are Ser302 to Asp 302 and Phe303 to Asp 303. The substitutions for locus 307 are Ser307 to Asp 307 and Lys308 to Asp3O8. The following examples should not be viewed as limiting the scope of the invention. The claims will serve to define the inventions.

[0020] In one embodiment, the variant cMyBPC protein comprises SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 9.

[0021] In another embodiment, the variant cMyBPC protein comprises SEQ ID NO: 4.

[0022] In another embodiment, the variant cMyBPC protein is encoded by the nucleotide sequence of SEQ ID NO: 3.

[0023] In another embodiment, the variant cMyBPC protein is expressed by a vector comprising the nucleotide sequence of SEQ ID NO: 5.

[0024] The present invention also provides a method of treatment for heart failure, the method comprising administering a vector to a patient, where the vector comprises a polynucelotide sequence encoding the variant cMyBPC polypeptide and the vector is adapted to express the variant cMyBPC polypeptide. The vector may be an Adeno-associated virus (AAV) vector such as AAV9.

[0025] We tested the ability of our mutant constructs to modulate cardiac function in vivo by delivering the constructs described below to cMyBPC- / - mice using AAV9 gene therapy vectors.

[0026] Fig. 1 A is a Western blot showing cMyBPC expression from wild type mice expressing native cMyBPC, and cMyBPC -I- mice expressing AAV9-delivered wild type full length cMyBPC (WT-FL) shows that these constructs demonstrate the ability of the 4x3D-FL mutations to induce a phosphorylated-like state of cMyBPC that produces functional changes similar to those caused by phosphorylation. Constructs are expressed at equivalent levels to non-mutated full-length cMyBPC (WT-FL), and both constructs express equivalent levels to natively produced cMyBPC in WT mice.

[0027] As shown in Fig. IB, expression of 4x3D-FL and WT-FL result in similar levels of overall functional improvement as indicated by ejection fraction (51.37+0.8% in WT-FL vs 49.52+1.2% in 4x3D-FL, p=0.46, n=13 per group), however, both ejection and relaxation are accelerated in the 4x3D-FL group. This is indicated by a faster aortic ejection time (40.21±0.4ms vs 49.87+0.5ms, p<0.0001) and faster isovolumic relaxation time (15.83+0.3ms vs 18.29+0.2ms, p<0.0001). These results demonstrate that triplc-D phosphomimetic mutations in cMyBPC can modulate its function in order to fine tune different aspects of cardiac contraction and relaxation.This work provides a novel mechanism of engineering cMyBPC to specifically manipulate cardiac function to achieve desired therapeutic outcomes.

[0028] Further data are provided Fig. 2A, which provides graphs of isovolumic relaxation time (IVRT), ejection fraction (EF), and global longitudinal strain (GLS) from WT mice injected with AAV9-WT-FL or -4x3D. n = 5 for WT-FL and 15 for -4x3D. Mice were anesthetized with 2% isoflurane and echoc ardiography images were collected using a Vevo 3100 ultrasonography 40MHz transducer (Visual Sonics). Vevo Lab 3.1.1 software (Visual Sonics) was used for image analysis. IVRT was measured from apical four-chamber pulsed wave Doppler images with the marquee placed over the region of peak inflow and outflow. EF was calculated over 3-4 consecutive cardiac cycles from midventricular short axis M-mode images. GLS was calculated using the VevoStrain module (Visual Sonics) over 3 consecutive cardiac cycles from parasternal long axis B-mode images. * p < 0.05, ** p < 0.01.

[0029] Fig. 2B is a representative Western blot from WT mice injected with AAV9-WT-FL or -4x3D. Commercially available primary antibodies were used to probe the N-terminal amino acids 1-120 of cMyBPC to assess total cMyBPC expression (cMyBPC, catalog sc-137237; Santa Cruz Biotechnology Inc.), the cMyc tag attached to vector-derived cMyBPC constructs to assess vector- driven cMyBPC expression as a proportion of total cMyBPC expression (cMyc, catalog sc-40; Santa Cruz Biotechnology Inc.), and the sarcomere protein alpha-actinin as a loading control (I±- actinin, catalog SAB4503474; MilliporeSigma). Secondary detection was performed using HorseRadish Peroxidase (HRP)-conjugated mouse IgG kappa binding protein for murine primaries (cMyBPC and cMyc, catalog sc-516102; Santa Cruz Biotechnology Inc.) and HRP-conjugated mouse anti-rabbit for rabbit primary (I±-actinin, catalog sc-2357, Santa Cruz Biotechnology Inc.). Blots were developed using the SuperSignal West Pico PLUS Chemiluminescent Substrate Kit (ref 34580, Thermo Scientific) and imaged using the Azure C600 (Azure Biosystems).

[0030] This mechanism can be applied to developing cMyBPC with single site-specific 3D mutations to further refine the specific therapeutic functions of cMyBPC gene therapy. Fig. 3 is a set graphs of ejection fraction (EF) and isovolumic relaxation time (IVRT) from cMyBPC- / - (KO) mice injected with AAV9-4x3D, -273D, and -302D demonstrating the graded effects of different sequence modifications on cardiac function, n = 13 for 4x3D, 6 for 273D, and 9 for 302D. AAV9- dclivcrcd 273D cMyBPC contains the substitutions T272D, S273D and L274D. AAV9-dclivcrcd 302D cMyBPC contains the substitutions S302D and F303D. 4x3D cMyBPC contains thesubstitutions Thr272 to Asp272, Ser273 to Asp273, Leu274 to Asp274; Thi‘281 to Asp281, Ser282 to Asp 282; Ser302 to Asp 302, Phe303 to Asp 303; Ser307 to Asp 307, Lys308 to Asp308. Mice were anesthetized with 2% isoflurane and echocardiography images were collected using a Vevo 3100 ultrasonography 40MHz transducer (Visual Sonics). Vevo Lab 3.1.1 software (Visual Sonics) was used for image analysis. EF was calculated over 3-4 consecutive cardiac cycles from midventricular short axis M-mode images. IVRT was measured from apical four-chamber pulsed wave Doppler images with the marquee placed over the region of peak inflow and outflow.ProceduresAAV9 VectorsAdeno- Associated Vims (AAV) 9 vectors were purchased from UPenn Vector core. AAV9 is known to be cardiotropic, with high baseline expression in cardiac tissue. Vectors are designed under the control of a truncated cardiac troponin T promoter to maximize expression in cardiac tissue and prevent expression in non-cardiac tissue. The MyBPC3 gene with appropriate mutations is fused with a c-myc tag at the C-terminus to allow for detection of virally derived protein. The nucleotide sequence of this cMyBPC / c-myc construct is provided as SEQ ID NO: 3. The corresponding amino acid sequence of the cMyBPC / c-myc fusion protein is provided as SEQ ID NO: 4. The nucleotide sequence of the entire vector is provided as SEQ ID NO: 5. For comparison, the nucleotide sequence of the wild type cMyBPC / c-myc construct is provided as SEQ ID NO: 6Gene deliveryTransgenic cMyBPC- / - mice on a 129SVE background are retro-orbitally injected with AAV9 vectors containing native full-length cMyBPC (WT-FL) or hyper phosphorylated cMyBPC (4x3D- FL) 2 weeks after birth. Injections consist of 2el4 genome copies (GC) of vector per kilogram of bodyweight. Briefly, pups are lightly anesthetized with 4% isoflurane and placed in the lateral recumbence position. A 31 -gauge insulin syringe is advanced through the medial canthus into the retro-orbital sinus and the injection is slowly delivered. Pups are recovered on warm soiled bedding and returned to their home cage.Echocardiography protocolConventional and speckle tracking echocardiography are used to non-invasively monitor systolic and diastolic function with B-mode, M-mode, and Doppler images and cine loops of the parasternal long and short axes and apical four-chamber views. Mice are anesthetized by 1-2% isoflurane during the procedure, and heart rate is managed using heat support. Echo is performed before and after intraperitoneal injection with dobutamine to characterize the response to acute stress.Echocardiography images are analyzed using Vevo Lab 3.1.1 (Visual Sonics). Left ventricular (LV) ejection fraction (EF), anterior and posterior wall thickness, chamber diameter, and LV mass are measured over 3-4 cardiac cycles from midvcntricular short axis M-mode images. IVRT and AET are measured from apical four chamber pulsed wave Doppler images with the marquee placed over the region of peak inflow and outflow. Speckle tracking echocardiography -based longitudinal strain parameters arc measured from parasternal long-axis B mode video using the semi-automated Vevo Strain module of Vevo Lab.Time course of experimentsMice are injected at 2 weeks of age, and echocardiography analysis is performed 6 weeks later. Mice are euthanized via cardiectomy after final echocardiography analysis, and hearts are immediately frozen in liquid nitrogen. Hearts are then used to prepare samples for Western Blotting, gel electrophoresis, fluorescence microscopy, and mechanical fiber experiments.Evaluation of protein expression and localizationMyocardial protein samples are prepared from hearts excised from injected mice. Western blot against the c-myc epitope, and against the N-terminal 120 amino acids of cMyBPC, allow for quantification of AAV9 replacement of endogenous protein. The c-Myc signal is compared with the total cMyBPC signal in injected mice to determine the extent of replacement. The localization of virally-delivered protein within the sarcomere is determined using immunohistochemistry and fluorescence microscopy. Slides are prepared using myofibrils stained with fluorescent antibodies against the c-Myc tag, the N-terminal 120 amino acids of cMyBPC, and alpha-actinin to allow visualization of virally-delivered cMyBPC, total cMyBPC, and sarcomere Z-discs, respectively.In the sequences described herein, nucleotides should be understood to be represented by their standard one letter abbreviations: A for adenine, G for guanine, C for cytosine, T for thymine and U for uracil. The presence of a mixture of nucleotides may also be indicated with an abbreviation as recognized in the art: N for any base (A,C,G or T / U), R for purine (G or A), Y for pyrimidine (T / U or C), M for amino (A or C), K for keto (G or T / U), S for G or C, W for A or T / U, V for nucleotides other than T (A, C, or G) , D for nucleotides other than C (A, G, or T), B for nucleotides other than A (C, G, or T), and H for nucleotides other than G (A, C, or T).The word “comprising” and forms of the word “comprising” as used in this description and in the claims does not limit the invention claimed to exclude any variants or additions.Based upon the foregoing disclosure, it should now be apparent that the present invention will carry out the objects set forth hereinabove. It is, therefore, to be understood that any variations evident fall within the scope of the claimed invention and thus, the selection of specific component elements can be determined without departing from the spirit of the invention herein disclosed and described.SEQUENCESSEQ ID NO:1RRTSLAGAGRRTSDSHEDAGTPDFSSLLKKRDSFRRDSKLESEQ ID NO: 2RRDDDAGAGRRDDDSHEDAGTPDFSSLLKKRDDDRRDDDLESEQ ID NO: 3 (Variations in the from wild type shown in capital letters) atgccggagccagggaagaaaccagtgtcagccttcaacaagaagccaaggtcagcggaggtgaccgctggcagtgctgc cgtgttcgaggctgagacggagcggtcaggcgtgaaggtgcggtggcagcgggatggcagcgacatcaccgccaatgaca agtatggtttggcagcagagggcaagcggcacacactgacagtgcgggatgcgagccctgatgaccagggttcctacgcg gtcattgcaggctcctcaaaggtcaagtttgacctcaaggtcacagagccagcccctccagagaaggcagaatctgaagt tgctccaggagcccccaaagaagtccctgctccagccactgagttggaagaaagtgtctcaagtcctgaagggtcagtctcggtaacccaggatggctcagctgcagagcatcagggagcccctgatgaccctattggcctctttctgatgcgaccacag gatggtgaggtgaccgtgggcggcagcattgtcttctcagcccgagtggctggggccagcctcctgaaaccgcctgtggt caagtggttcaagggcaagtgggtggacctgagcagcaaagtgggccagcacctgcagctgcatgacagctatgacagag ccagcaaggtctacttgtttgagttgcacatcacagatgctcagaccacttctgctgggggctaccgctgtgaggtgtct accaaggacaaatttgacagctgtaacttcaacctcactgtccatgaggccattggttctggagacctggacctcagatc agctttccgacgcGACGACGACgcgggagcaggtcggagaGACGACgacagccatgaagatgctgggactctggacttta gttccctgctgaagaagagagacGACGACcggagggacGACGACctggaggcacctgctgaagaagacgtgtgggagatc ctgagacaggcaccgccgtcagaatatgagcgcatcgccttccagcacggagtcacagaccttcgaggcatgctgaagag gctcaagggcatgaagcaggatgaaaagaagagcacagcctttcagaagaagctggagcctgcctaccaggtaaacaagg gccacaagattcggcttactgtggaactggctgatccggacgccgaagtcaagtggcttaagaatggacaggagatccag atgagtggcagcaagtacatcttcgagtccgtcggtgccaagcgcaccctgaccatcagccagtgctcactggctgacga cgcagcctaccagtgtgtggtggggggcgagaagtgcagcacggagctctttgtcaaagagcccccggtgctgatcactc ggtccctggaagaccagctggtgatggtgggtcagcgggtggagtttgagtgtgaggtctcagaagaaggggcccaagtc aaatggctgaaggatggggttgagctgacacgtgaggagaccttcaaataccggttcaagaaagatgggcggaaacacca cttgatcatcaatgaagcaaccctggaggatgcaggacactatgcagtacgcacaagtggaggccagtcactggctgagc tcattgtgcaagagaagaagttggaggtataccaaagcatcgcggacctggcagtgggagccaaggaccaggctgtgttt aagtgtgaggtttcagatgagaatgtacgcggcgtgtggctgaagaatgggaaggaactggtgcctgacaaccgcataaa ggtgtcccatataggccgggtccacaaactgaccattgacgatgtcacacctgctgatgaggctgactacagctttgtcc ctgaagggtttgcctgcaacctgtctgccaagctccacttcatggaggtcaagattgactttgtgcctaggcaggaacct cccaagatccacttggattgtcccggcagcacaccagacaccattgtggttgttgctgggaacaagttacgcctggatgt ccctatttctggagaccctgctcccactgtggtctggcagaagactgtaacacaggggaagaaggcctcaactgggccac accctgatgccccagaagatgctggtgctgatgaggagtgggtgtttgataagaagctgttgtgtgagactgagggccgg gtccgggtggagaccaccaaagaccgcagcgtctttacagtcgaaggggcagagaaggaagatgaaggtgtctacacagt cacagtaaagaaccccgtgggcgaggaccaggtcaacctcacagtcaaggtcatcgatgtcccagatgctcctgcggccc ctaagatcagcaacgtgggcgaggactcctgcactgtgcagtgggaaccgcctgcctatgatggcgggcagccggtcctg ggatacatcctggagcgcaagaagaaaaagagctacaggtggatgaggctcaactttgatctgctgcgggagctgagcca cgaggcgaggcgcatgatcgagggtgtagcctatgagatgcgagtctacgcagtcaatgccgtgggaatgtccaggccca gccctgcctctcagcccttcatgcctattgggccccctggcgaaccaacccacttggctgtggaggatgtgtcagacacc actgtctcactcaagtggcggcccccagagcgcgtgggggccggtggcctggacggatacagcgtggagtactgccagga gggatgctccgagtggacacctgctctgcaggggctgacagagcgcacatcgatgctggtgaaggacctacccactgggg cacggctgctgttccgagtacgggcacacaatgtggcaggtcctggaggccctatcgtcaccaaggagcctgtgacagtgcaggagatactgcaacgaccacggctccaactgcccagacacctgcgccagaccatccagaagaaagttggggagcctgt gaacctcctcatccctttccagggcaaaccccggcctcaggtgacctggaccaaagaggggcagcccctggcaggtgagg aggtgagcatccggaacagccccacagacacgatcttgttcatccgagctgcccgccgcacccactcgggcacctaccag gtgacagttcgcattgagaacatggaggacaaggcaacgctgatcctgcagattgtggacaagccaagtcctccccagga tatccggatcgttgagacttggggtttcaatgtggctctggagtggaagccaccccaagatgatggcaatacagagatct ggggttatactgtacagaaagctgacaagaagaccatggagtggttcacggttttggaacactaccgacgcactcactgt gtggtatcagagcttatcattggcaatggctactacttccgggtcttcagccataacatggtgggttccagtgacaaagc tgccgccaccaaggagccagtctttattccaagaccaggcatcacatatgagccacccaaatacaaggccctggacttct ctgaggccccaagcttcacccagcccttggcaaatcgctccatcattgcaggctataatgccatcctctgctgtgctgtc cgaggtagtcctaagcccaagatttcctggttcaagaatggcctggatctgggagaagatgctcgcttccgcatgttctg caagcagggagtattgaccctggagatcaggaaaccctgcccctatgatggtggtgtctatgtctgcagggccaccaact tgcagggcgaggcacagtgtgagtgccgcctggaggtgcgagttcctcaggaacagaagctcatctcggaagaggatctgSEQ ID NO: 4MPEPGKKPVSAFNKKPRSAEVTAGSAAVFEAETERSGVKVRWQRDGSD1TANDKYGLAAEGKRHTLTVRDASPDDQGSYAVIAGSSKVKFDLKVTEPAPPEKAESEVAPGAPKEVPAPATELEESVSSPEGSVSVTQDGSAAEHQGAPDDPIGLFLMRPQDGEVTVGGSIVFSARVAGASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELHITDAQTTSAGGYRCEVSTKDKFDSCNFNLTVHEAIGSGDLDLRSAFRRDDDAGAGRRDDDSHEDAGTLDFSSLLKKRDDDRRDDDLEAPAEEDVWEILRQAPPSEYERIAFQHGVTDLRGMLKRLKGMKQDEKKSTAFQKKLEPAYQVNKGHKIRLTVELADPDAEVKWLKNGQEIQMSGSKYIFESVGAKRTLTISQCSLADDAAYQCVVGGEKCSTELFVKEPPVLITRSLEDQLVMVGQRVEFECEVSEEGAQVKWLKDGVELTREETFKYRFKKDGRKHHLIINEATLEDAGHYAVRTSGGQSLAELIVQEKKLEVYQSIADLAVGAKDQAVFKCEVSDENVRGVWLKNGKELVPDNRIKVSHIGRVHKLTIDDVTPADEADYSFVPEGFACNLSAKLHFMEVKIDFVPRQEPPKIHLDCPGSTPDTIVVVAGNKLRLDVPISGDPAPTVVWQKTVTQGKKASTGPHPDAPEDAGADEEWVFDKKLLCETEGRVRVETTKDRSVFTVEGAEKEDEGVYTVTVKNPVGEDQVNLTVKVIDVPDAPAAPKISNVGEDSCTVQWEPPAYDGGQPVLGYILERKKKKSYRWMRLNFDLLRELSHEARRMIEGVAYEMRVYAVNAVGMSRPSPASQPFMPIGPPGEPTHLAVEDVSDTTVSLKWRPPERVGAGGLDGYSVEYCQEGCSEWTPALQGLTERTSMLVKDLPTGARLLFRVRAHNVAGPGGPIVTKEPVTVQEILQRPRLQLPRHLRQTIQKKVGEPVNLLIPFQGKPRPQVTWTKEGQPLAGEEVSIRNSPTDTILFIRAARRTHSGTYQVTVRIENMEDKATLILQIVDKPSPPQDIRIVETWGFNVALEWKPPQDDGNTEIWGYTVQKADKK TMEWFTVLEHYRRTHCVVSELIIGNGYYFRVFSHNMVGSSDKAAATKEPVFIPRPGITYEPPK YKALDFSEAPSFTQPLANRSIIAGYNAILCCAVRGSPKPKISWFKNGLDLGEDARFRMFCKQG VLTLEIRKPCPYDGGVYVCRATNLQGEAQCECRLEVRVPQEQKLISEEDLSEQ ID NO: 5 - AAV9 vector ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctact tatctaccagggtaatggggatcctctagaactatagctagaattcgcccttacgggccccccctcgaggtcgggataaa agcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtcccc agccctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatag caaaactctgaggccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggcc gctgctgccaaaatagcagctcacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgcccc ggggtgggcgccggggggaccttaaagcctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccg tgggacgatccccgaagctctagagctttattgcggtagtttatcacagttaaattgctaacgcagtcagtgcttctgac acaacagtctcgaacttaacgcgtatgccggagccagggaagaaaccagtgtcagccttcaacaagaagccaaggtcagc ggaggtgaccgctggcagtgctgccgtgttcgaggctgagacggagcggtcaggcgtgaaggtgcggtggcagcgggatg gcagcgacatcaccgccaatgacaagtatggtttggcagcagagggcaagcggcacacactgacagtgcgggatgcgagc cctgatgaccagggttcctacgcggtcattgcaggctcctcaaaggtcaagtttgacctcaaggtcacagagccagcccc tccagagaaggcagaatctgaagttgctccaggagcccccaaagaagtccctgctccagccactgagttggaagaaagtg tctcaagtcctgaagggtcagtctcggtaacccaggatggctcagctgcagagcatcagggagcccctgatgaccctatt ggcctctttctgatgcgaccacaggatggtgaggtgaccgtgggcggcagcattgtcttctcagcccgagtggctggggc cagcctcctgaaaccgcctgtggtcaagtggttcaagggcaagtgggtggacctgagcagcaaagtgggccagcacctgc agctgcatgacagctatgacagagccagcaaggtctacttgtttgagttgcacatcacagatgctcagaccacttctgct gggggctaccgctgtgaggtgtctaccaaggacaaatttgacagctgtaacttcaacctcactgtccatgaggccattgg ttctggagacctggacctcagatcagctttccgacgcGACGACGACgcgggagcaggtcggagaGACGACgacagccatg aagatgctgggactctggactttagttccctgctgaagaagagagacGACGACcggagggacGACGACctggaggcacct gctgaagaagacgtgtgggagatcctgagacaggcaccgccgtcagaatatgagcgcatcgccttccagcacggagtcac agaccttcgaggcatgctgaagaggctcaagggcatgaagcaggatgaaaagaagagcacagcctttcagaagaagctgg agcctgcctaccaggtaaacaagggccacaagattcggcttactgtggaactggctgatccggacgccgaagtcaagtgg cttaagaatggacaggagatccagatgagtggcagcaagtacatcttcgagtccgtcggtgccaagcgcaccctgaccatcagccagtgctcactggctgacgacgcagcctaccagtgtgtggtggggggcgagaagtgcagcacggagctctttgtca aagagcccccggtgctgatcactcggtccctggaagaccagctggtgatggtgggtcagcgggtggagtttgagtgtgag gtctcagaagaaggggcccaagtcaaatggctgaaggatggggttgagctgacacgtgaggagaccttcaaataccggtt caagaaagatgggcggaaacaccacttgatcatcaatgaagcaaccctggaggatgcaggacactatgcagtacgcacaa gtggaggccagtcactggctgagctcattgtgcaagagaagaagttggaggtataccaaagcatcgcggacctggcagtg ggagccaaggaccaggctgtgtttaagtgtgaggtttcagatgagaatgtacgcggcgtgtggctgaagaatgggaagga actggtgcctgacaaccgcataaaggtgtcccatataggccgggtccacaaactgaccattgacgatgtcacacctgctg atgaggctgactacagctttgtccctgaagggtttgcctgcaacctgtctgccaagctccacttcatggaggtcaagatt gactttgtgcctaggcaggaacctcccaagatccacttggattgtcccggcagcacaccagacaccattgtggttgttgc tgggaacaagttacgcctggatgtccctatttctggagaccctgctcccactgtggtctggcagaagactgtaacacagg ggaagaaggcctcaactgggccacaccctgatgccccagaagatgctggtgctgatgaggagtgggtgtttgataagaag ctgttgtgtgagactgagggccgggtccgggtggagaccaccaaagaccgcagcgtctttacagtcgaaggggcagagaa ggaagatgaaggtgtctacacagtcacagtaaagaaccccgtgggcgaggaccaggtcaacctcacagtcaaggtcatcg atgtcccagatgctcctgcggcccctaagatcagcaacgtgggcgaggactcctgcactgtgcagtgggaaccgcctgcc tatgatggcgggcagccggtcctgggatacatcctggagcgcaagaagaaaaagagctacaggtggatgaggctcaactt tgatctgctgcgggagctgagccacgaggcgaggcgcatgatcgagggtgtagcctatgagatgcgagtctacgcagtca atgccgtgggaatgtccaggcccagccctgcctctcagcccttcatgcctattgggccccctggcgaaccaacccacttg gctgtggaggatgtgtcagacaccactgtctcactcaagtggcggcccccagagcgcgtgggggccggtggcctggacgg atacagcgtggagtactgccaggagggatgctccgagtggacacctgctctgcaggggctgacagagcgcacatcgatgc tggtgaaggacctacccactggggcacggctgctgttccgagtacgggcacacaatgtggcaggtcctggaggccctatc gtcaccaaggagcctgtgacagtgcaggagatactgcaacgaccacggctccaactgcccagacacctgcgccagaccat ccagaagaaagttggggagcctgtgaacctcctcatccctttccagggcaaaccccggcctcaggtgacctggaccaaag aggggcagcccctggcaggtgaggaggtgagcatccggaacagccccacagacacgatcttgttcatccgagctgcccgc cgcacccactcgggcacctaccaggtgacagttcgcattgagaacatggaggacaaggcaacgctgatcctgcagattgt ggacaagccaagtcctccccaggatatccggatcgttgagacttggggtttcaatgtggctctggagtggaagccacccc aagatgatggcaatacagagatctggggttatactgtacagaaagctgacaagaagaccatggagtggttcacggttttg gaacactaccgacgcactcactgtgtggtatcagagcttatcattggcaatggctactacttccgggtcttcagccataa catggtgggttccagtgacaaagctgccgccaccaaggagccagtctttattccaagaccaggcatcacatatgagccac ccaaatacaaggccctggacttctctgaggccccaagcttcacccagcccttggcaaatcgctccatcattgcaggctat aatgccatcctctgctgtgctgtccgaggtagtcctaagcccaagatttcctggttcaagaatggcctggatctgggaga agatgctcgcttccgcatgttctgcaagcagggagtattgaccctggagatcaggaaaccctgcccctatgatggtggtgtctatgtctgcagggccaccaacttgcagggcgaggcacagtgtgagtgccgcctggaggtgcgagttcctcaggaacag aagctcatctcggaagaggatctgtgactcgacccgggcggcctcgaggacggggtgaactacgcctgaggatccgatct ttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattt tcattgcaatagtgtgttggaattttttgtgtctctcactcggaagcaattcgttgatctgaatttcgaccacccataat acccattaccctggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactc cctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca gtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcc cttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggtt acgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgtt cgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgacccca aaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtcc acgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagg gattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaa cgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaata tgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttc cgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaaga tgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgcc ccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacgga tggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacga tcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaac tggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgc gctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagca ctggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatag acagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattg atttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgt gagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaat ctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccga aggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttac cgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagct tggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatct ttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgga aaaacgccagcaacgcggcctttttacggttcctggccttttgctggccUttgctcacatgttctttcctgcgttatcc cctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcga gtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagct ggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccc caggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagcta tgaccatgattacgccagatttaattaaggccttaattaggSEQ ID NO: 6 atgccggagccagggaagaaaccagtgtcagccttcaacaagaagccaaggtcagcggaggtgaccgctggcagtgctgc cgtgttcgaggctgagacggagcggtcaggcgtgaaggtgcggtggcagcgggatggcagcgacatcaccgccaatgaca agtatggtttggcagcagagggcaagcggcacacactgacagtgcgggatgcgagccctgatgaccagggttcctacgcg gtcattgcaggctcctcaaaggtcaagtttgacctcaaggtcacagagccagcccctccagagaaggcagaatctgaagt tgctccaggagcccccaaagaagtccctgctccagccactgagttggaagaaagtgtctcaagtcctgaagggtcagtct cggtaacccaggatggctcagctgcagagcatcagggagcccctgatgaccctattggcctctttctgatgcgaccacag gatggtgaggtgaccgtgggcggcagcattgtcttctcagcccgagtggctggggccagcctcctgaaaccgcctgtggt caagtggttcaagggcaagtgggtggacctgagcagcaaagtgggccagcacctgcagctgcatgacagctatgacagag ccagcaaggtctacttgtttgagttgcacatcacagatgctcagaccacttctgctgggggctaccgctgtgaggtgtct accaaggacaaatttgacagctgtaacttcaacctcactgtccatgaggccattggttctggagacctggacctcagatc agctttccgacgcacgagcctggcgggagcaggtcggagaaccagtgacagccatgaagatgctgggactctggacttta gttccctgctgaagaagagagacagtttccggagggactcaaagctggaggcacctgctgaagaagacgtgtgggagatc ctgagacaggcaccgccgtcagaatatgagcgcatcgccttccagcacggagtcacagaccttcgaggcatgctgaagag gctcaagggcatgaagcaggatgaaaagaagagcacagcctttcagaagaagctggagcctgcctaccaggtaaacaagg gccacaagattcggcttactgtggaactggctgatccggacgccgaagtcaagtggcttaagaatggacaggagatccag atgagtggcagcaagtacatcttcgagtccgtcggtgccaagcgcaccctgaccatcagccagtgctcactggctgacga cgcagcctaccagtgtgtggtggggggcgagaagtgcagcacggagctctttgtcaaagagcccccggtgctgatcactc ggtccctggaagaccagctggtgatggtgggtcagcgggtggagtttgagtgtgaggtctcagaagaaggggcccaagtcaaatggctgaaggatggggttgagctgacacgtgaggagaccttcaaataccggttcaagaaagatgggcggaaacacca cttgatcatcaatgaagcaaccctggaggatgcaggacactatgcagtacgcacaagtggaggccagtcactggctgagc tcattgtgcaagagaagaagttggaggtataccaaagcatcgcggacctggcagtgggagccaaggaccaggctgtgttt aagtgtgaggtttcagatgagaatgtacgcggcgtgtggctgaagaatgggaaggaactggtgcctgacaaccgcataaa ggtgtcccatataggccgggtccacaaactgaccattgacgatgtcacacctgctgatgaggctgactacagctttgtcc ctgaagggtttgcctgcaacctgtctgccaagctccacttcatggaggtcaagattgactttgtgcctaggcaggaacct cccaagatccacttggattgtcccggcagcacaccagacaccattgtggttgttgctgggaacaagttacgcctggatgt ccctatttctggagaccctgctcccactgtggtctggcagaagactgtaacacaggggaagaaggcctcaactgggccac accctgatgccccagaagatgctggtgctgatgaggagtgggtgtttgataagaagctgttgtgtgagactgagggccgg gtccgggtggagaccaccaaagaccgcagcgtctttacagtcgaaggggcagagaaggaagatgaaggtgtctacacagt cacagtaaagaaccccgtgggcgaggaccaggtcaacctcacagtcaaggtcatcgatgtcccagatgctcctgcggccc ctaagatcagcaacgtgggcgaggactcctgcactgtgcagtgggaaccgcctgcctatgatggcgggcagccggtcctg ggatacatcctggagcgcaagaagaaaaagagctacaggtggatgaggctcaactttgatctgctgcgggagctgagcca cgaggcgaggcgcatgatcgagggtgtagcctatgagatgcgagtctacgcagtcaatgccgtgggaatgtccaggccca gccctgcctctcagcccttcatgcctattgggccccctggcgaaccaacccacttggctgtggaggatgtgtcagacacc actgtctcactcaagtggcggcccccagagcgcgtgggggccggtggcctggacggatacagcgtggagtactgccagga gggatgctccgagtggacacctgctctgcaggggctgacagagcgcacatcgatgctggtgaaggacctacccactgggg cacggctgctgttccgagtacgggcacacaatgtggcaggtcctggaggccctatcgtcaccaaggagcctgtgacagtg caggagatactgcaacgaccacggctccaactgcccagacacctgcgccagaccatccagaagaaagttggggagcctgt gaacctcctcatccctttccagggcaaaccccggcctcaggtgacctggaccaaagaggggcagcccctggcaggtgagg aggtgagcatccggaacagccccacagacacgatcttgttcatccgagctgcccgccgcacccactcgggcacctaccag gtgacagttcgcattgagaacatggaggacaaggcaacgctgatcctgcagattgtggacaagccaagtcctccccagga tatccggatcgttgagacttggggtttcaatgtggctctggagtggaagccaccccaagatgatggcaatacagagatct ggggttatactgtacagaaagctgacaagaagaccatggagtggttcacggttttggaacactaccgacgcactcactgt gtggtatcagagcttatcattggcaatggctactacttccgggtcttcagccataacatggtgggttccagtgacaaagc tgccgccaccaaggagccagtctttattccaagaccaggcatcacatatgagccacccaaatacaaggccctggacttct ctgaggccccaagcttcacccagcccttggcaaatcgctccatcattgcaggctataatgccatcctctgctgtgctgtc cgaggtagtcctaagcccaagatttcctggttcaagaatggcctggatctgggagaagatgctcgcttccgcatgttctg caagcagggagtattgaccctggagatcaggaaaccctgcccctatgatggtggtgtctatgtctgcagggccaccaact tgcagggcgaggcacagtgtgagtgccgcctggaggtgcgagttcctcaggaacagaagctcatctcggaagaggatctgSEQ ID NO: 7MPEPGKKPVSAFNKKPRSAEVTAGSAAVFEAETERSGVKVRWQRDGSDITANDKYGLAAEGKRHTLTVRDASPDDQGSYAVIAGSSKVKFDLKVTEPAPPEKAESEVAPGAPKEVPAPATELEESVSSPEGSVSVTQDGSAAEHQGAPDDPIGLFLMRPQDGEVTVGGSIVFSARVAGASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELHITDAQTTSAGGYRCEVSTKDKFDSCNFNLTVHEAIGSGDLDLRSAFRRTSLAGAGRRTSDSHEDAGTLDFSSLLKKRDSFRRDSKLEAPAEEDVWEILRQAPPSEYERIAFQHGVTDLRGMLKRLKGMKQDEKKSTAFQKKLEPAYQVNKGHKIRLTVELADPDAEVKWLKNGQEIQMSGSKYIFESVGAKRTLTISQCSLADDAAYQCVVGGEKCSTELFVKEPPVLITRSLEDQLVMVGQRVEFECEVSEEGAQVKWLKDGVELTREETFKYRFKKDGRKHHLIINEATLEDAGHYAVRTSGGQSLAELIVQEKKLEVYQSIADLAVGAKDQAVFKCEVSDENVRGVWLKNGKELVPDNRIKVSHIGRVHKLTIDDVTPADEADYSFVPEGFACNLSAKLHFMEVKIDFVPRQEPPKIHLDCPGSTPDTIVVVAGNKLRLDVPISGDPAPTVVWQKTVTQGKKASTGPHPDAPEDAGADEEWVFDKKLLCETEGRVRVETTKDRSVFTVEGAEKEDEGVYTVTVKNPVGEDQVNLTVKVIDVPDAPAAPKISNVGEDSCTVQWEPPAYDGGQPVLGY1LERKKKKSYRWMRLNFDLLRELSHEARRM1EGVAYEMRVYAVNAVGMSRPSPASQPFMPIGPPGEPTHLAVEDVSDTTVSLKWRPPERVGAGGLDGYSVEYCQEGCSEWTPALQGLTERTSMLVKDLPTGARLLFRVRAHNVAGPGGPIVTKEPVTVQEILQRPRLQLPRHLRQTIQKKVGEPVNLLIPFQGKPRPQVTWTKEGQPLAGEEVSIRNSPTDTILFIRAARRTHSGTYQVTVRIENMEDKATLILQIVDKPSPPQDIRIVETWGFNVALEWKPPQDDGNTEIWGYTVQKADKKTMEWFTVLEHYRRTHCVVSELIIGNGYYFRVFSHNMVGSSDKAAATKEPVFIPRPGITYEPPKYKALDFSEAPSFTQPLANRSIIAGYNAILCCAVRGSPKPKISWFKNGLDLGEDARFRMFCKQGVLTLEIRKPCPYDGGVYVCRATNLQGEAQCECRLEVRVPQEQKLISEEDLSEQ ID NO: 8RRDDDAGAGR RTSDSHEDAG TLDFSSLLKK RDSFRRDSKLSEQ ID NO: 9RRTSLAGAGR RTSDSHEDAG TLDFSSLLKK RDDDRRDSKL ESEQ ID NO: 10 - Modified CmyBPC / c-myc sequence [[273DDD]] atgccggagccagggaagaaaccagtgtcagccttcaacaagaagccaaggtcagcggaggtgaccgctggcagtgctgccgtgttcga ggctgagacggagcggtcaggcgtgaaggtgcggtggcagcgggatggcagcgacatcaccgccaatgacaagtatggtttggcagca gagggcaagcggcacacactgacagtgcgggatgcgagccctgatgaccagggttcctacgcggtcattgcaggctcctcaaaggtcaa gtttgacctcaaggtcacagagccagcccctccagagaaggcagaatctgaagttgctccaggagcccccaaagaagtccctgctccagc cactgagttggaagaaagtgtctcaagtcctgaagggtcagtctcggtaacccaggatggctcagctgcagagcatcagggagcccctgat gaccctattggcctctttctgatgcgaccacaggatggtgaggtgaccgtgggcggcagcattgtcttctcagcccgagtggctggggccag cctcctgaaaccgcctgtggtcaagtggttcaagggcaagtgggtggacctgagcagcaaagtgggccagcacctgcagctgcatgacag ctatgacagagccagcaaggtctacttgtttgagttgcacatcacagatgctcagaccacttctgctgggggctaccgctgtgaggtgtctacc aaggacaaatttgacagctgtaacttcaacctcactgtccatgaggccattggttctggagacctggacctcagatcagctttccgacgcGA CGACGACgcgggagcaggtcggagaaccagtgacagccatgaagatgctgggactctggactttagttccctgctgaagaagaga gacagtttccggagggactcaaagctggaggcacctgctgaagaagacgtgtgggagatcctgagacaggcaccgccgtcagaatatga gcgcatcgccttccagcacggagtcacagaccttcgaggcatgctgaagaggctcaagggcatgaagcaggatgaaaagaagagcaca gcctttcagaagaagctggagcctgcctaccaggtaaacaagggccacaagattcggcttactgtggaactggctgatccggacgccgaa gtcaagtggcttaagaatggacaggagatccagatgagtggcagcaagtacatcttcgagtccgtcggtgccaagcgcaccctgaccatca gccagtgctcactggctgacgacgcagcctaccagtgtgtggtggggggcgagaagtgcagcacggagctctttgtcaaagagcccccg gtgctgatcactcggtccctggaagaccagctggtgatggtgggtcagcgggtggagtttgagtgtgaggtctcagaagaaggggcccaa gtcaaatggctgaaggatggggttgagctgacacgtgaggagaccttcaaataccggttcaagaaagatgggcggaaacaccacttgatca tcaatgaagcaaccctggaggatgcaggacactatgcagtacgcacaagtggaggccagtcactggctgagctcattgtgcaagagaaga agttggaggtataccaaagcatcgcggacctggcagtgggagccaaggaccaggctgtgtttaagtgtgaggtttcagatgagaatgtacg cggcgtgtggctgaagaatgggaaggaactggtgcctgacaaccgcataaaggtgtcccatataggccgggtccacaaactgaccattga cgatgtcacacctgctgatgaggctgactacagctttgtccctgaagggtttgcctgcaacctgtctgccaagctccacttcatggaggtcaag attgactttgtgcctaggcaggaacctcccaagatccacttggattgtcccggcagcacaccagacaccattgtggttgttgctgggaacaag ttacgcctggatgtccctatttctggagaccctgctcccactgtggtctggcagaagactgtaacacaggggaagaaggcctcaactgggcc acaccctgatgccccagaagatgctggtgctgatgaggagtgggtgtttgataagaagctgttgtgtgagactgagggccgggtccgggtg gagaccaccaaagaccgcagcgtctttacagtcgaaggggcagagaaggaagatgaaggtgtctacacagtcacagtaaagaaccccgt gggcgaggaccaggtcaacctcacagtcaaggtcatcgatgtcccagatgctcctgcggcccctaagatcagcaacgtgggcgaggactc ctgcactgtgcagtgggaaccgcctgcctatgatggcgggcagccggtcctgggatacatcctggagcgcaagaagaaaaagagctaca ggtggatgaggctcaactttgatctgctgcgggagctgagccacgaggcgaggcgcatgatcgagggtgtagcctatgagatgcgagtctacgcagtcaatgccgtgggaatgtccaggcccagccctgcctctcagcccttcatgcctattgggccccctggcgaaccaacccacttggct gtggaggatgtgtcagacaccactgtctcactcaagtggcggcccccagagcgcgtgggggccggtggcctggacggatacagcgtgga gtactgccaggagggatgctccgagtggacacctgctctgcaggggctgacagagcgcacatcgatgctggtgaaggacctacccactgg ggcacggctgctgttccgagtacgggcacacaatgtggcaggtcctggaggccctatcgtcaccaaggagcctgtgacagtgcaggagat actgcaacgaccacggctccaactgcccagacacctgcgccagaccatccagaagaaagttggggagcctgtgaacctcctcatccctttc cagggcaaaccccggcctcaggtgacctggaccaaagaggggcagcccctggcaggtgaggaggtgagcatccggaacagccccaca gacacgatcttgttcatccgagctgcccgccgcacccactcgggcacctaccaggtgacagttcgcattgagaacatggaggacaaggca acgctgatcctgcagattgtggacaagccaagtcctccccaggatatccggatcgttgagacttggggtttcaatgtggctctggagtggaag ccaccccaagatgatggcaatacagagatctggggttatactgtacagaaagctgacaagaagaccatggagtggttcacggttttggaaca ctaccgacgcactcactgtgtggtatcagagcttatcattggcaatggctactacttccgggtcttcagccataacatggtgggttccagtgaca aagctgccgccaccaaggagccagtctttattccaagaccaggcatcacatatgagccacccaaatacaaggccctggacttctctgaggc cccaagcttcacccagcccttggcaaatcgctccatcattgcaggctataatgccatcctctgctgtgctgtccgaggtagtcctaagcccaag atttcctggttcaagaatggcctggatctgggagaagatgctcgcttccgcatgttctgcaagcagggagtattgaccctggagatcaggaaa ccctgcccctatgatggtggtgtctatgtctgcagggccaccaacttgcagggcgaggcacagtgtgagtgccgcctggaggtgcgagttc ctcaggaacagaagctcatctcggaagaggatctgSEQ ID NO: 11 - Modified CMyBPC / c-myc fusion protein sequence, 273 DDDMPEPGKKPVSAFNKKPRSAEVTAGSAAVFEAETERSGVKVRWQRDGSDITANDKYGLA AEGKRHTLTVRDASPDDQGSYAVIAGSSKVKFDLKVTEPAPPEKAESEVAPGAPKEVPA PATELEESVSSPEGSVSVTQDGSAAEHQGAPDDPIGLFLMRPQDGEVTVGGSIVFSARVA GASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELHITDAQTTSAGGY RCEVSTKDKFDSCNFNLTVHEAIGSGDLDLRSAFRRDDDAGAGRRTSDSHEDAGTLDFS SLLKKRDSFRRDSKLEAPAEEDVWEILRQAPPSEYERIAFQHGVTDLRGMLKRLKGMKQ DEKKSTAFQKKLEPAYQVNKGHKIRLTVELADPDAEVKWLKNGQEIQMSGSKYIFESVG AKRTLTISQCSLADDAAYQCVVGGEKCSTELFVKEPPVLITRSLEDQLVMVGQRVEFECE VSEEGAQVKWLKDGVELTREETFKYRFKKDGRKHHLIINEATLEDAGHYAVRTSGGQS LAELIVQEKKLEVYQSIADLAVGAKDQAVFKCEVSDENVRGVWLKNGKELVPDNRIKV SHIGRVHKLTIDDVTPADEADYSFVPEGFACNLSAKLHFMEVKIDFVPRQEPPKIHLDCPG STPDTIVVVAGNKLRLDVPISGDPAPTVVWQKTVTQGKKASTGPHPDAPEDAGADEEW VFDKKLLCETEGRVRVETTKDRSVFTVEGAEKEDEGVYTVTVKNPVGEDQVNLTVKVIDVPDAPAAPKISNVGEDSCTVQWEPPAYDGGQPVLGYILERKKKKSYRWMRLNFDLLR ELSHEARRMIEGVAYEMRVYAVNAVGMSRPSPASQPFMPIGPPGEPTHLAVEDVSDTTV SLKWRPPERVGAGGLDGYSVEYCQEGCSEWTPALQGLTERTSMLVKDLPTGARLLFRV RAHNVAGPGGPIVTKEPVTVQEILQRPRLQLPRHLRQTIQKKVGEPVNLLIPFQGKPRPQV TWTKEGQPLAGEEVSIRNSPTDTILFIRAARRTHSGTYQVTVRIENMEDKATLILQIVDKP SPPQDIRIVETWGFNVALEWKPPQDDGNTEIWGYTVQKADKKTMEWFTVLEHYRRTHC VVSELIIGNGYYFRVFSHNMVGSSDKAAATKEPVFIPRPGITYEPPKYKALDFSEAPSFTQ PLANRSIIAGYNAILCCAVRGSPKPKISWFKNGLDLGEDARFRMFCKQGVLTLEIRKPCPY DGGVYVCRATNLQGEAQCECRLEVRVPQEQKLISEEDLSEQ ID NO: 12 - Modified CmyBPC / c-myc sequence, 302D atgccggagccagggaagaaaccagtgtcagccttcaacaagaagccaaggtcagcggaggtgaccgctggcagtgctgccgtgttcga ggctgagacggagcggtcaggcgtgaaggtgcggtggcagcgggatggcagcgacatcaccgccaatgacaagtatggtttggcagca gagggcaagcggcacacactgacagtgcgggatgcgagccctgatgaccagggttcctacgcggtcattgcaggctcctcaaaggtcaa gtttgacctcaaggtcacagagccagcccctccagagaaggcagaatctgaagttgctccaggagcccccaaagaagtccctgctccagc cactgagttggaagaaagtgtctcaagtcctgaagggtcagtctcggtaacccaggatggctcagctgcagagcatcagggagcccctgat gaccctattggcctctttctgatgcgaccacaggatggtgaggtgaccgtgggcggcagcattgtcttctcagcccgagtggctggggccag cctcctgaaaccgcctgtggtcaagtggttcaagggcaagtgggtggacctgagcagcaaagtgggccagcacctgcagctgcatgacag ctatgacagagccagcaaggtctacttgtttgagttgcacatcacagatgctcagaccacttctgctgggggctaccgctgtgaggtgtctacc aaggacaaatttgacagctgtaacttcaacctcactgtccatgaggccattggttctggagacctggacctcagatcagctttccgacgcacg agcctggcgggagcaggtcggagaaccagtgacagccatgaagatgctgggactctggactttagttccctgctgaagaagagagacG ACGACcggagggactcaaagctggaggcacctgctgaagaagacgtgtgggagatcctgagacaggcaccgccgtcagaatatga gcgcatcgccttccagcacggagtcacagaccttcgaggcatgctgaagaggctcaagggcatgaagcaggatgaaaagaagagcaca gcctttcagaagaagctggagcctgcctaccaggtaaacaagggccacaagattcggcttactgtggaactggctgatccggacgccgaa gtcaagtggcttaagaatggacaggagatccagatgagtggcagcaagtacatcttcgagtccgtcggtgccaagcgcaccctgaccatca gccagtgctcactggctgacgacgcagcctaccagtgtgtggtggggggcgagaagtgcagcacggagctctttgtcaaagagcccccg gtgctgatcactcggtccctggaagaccagctggtgatggtgggtcagcgggtggagtttgagtgtgaggtctcagaagaaggggcccaa gtcaaatggctgaaggatggggttgagctgacacgtgaggagaccttcaaataccggttcaagaaagatgggcggaaacaccacttgatca tcaatgaagcaaccctggaggatgcaggacactatgcagtacgcacaagtggaggccagtcactggctgagctcattgtgcaagagaagaagttggaggtataccaaagcatcgcggacctggcagtgggagccaaggaccaggctgtgtttaagtgtgaggtttcagatgagaatgtacg cggcgtgtggctgaagaatgggaaggaactggtgcctgacaaccgcataaaggtgtcccatataggccgggtccacaaactgaccattga cgatgtcacacctgctgatgaggctgactacagctttgtccctgaagggtttgcctgcaacctgtctgccaagctccacttcatggaggtcaag attgactttgtgcctaggcaggaacctcccaagatccacttggattgtcccggcagcacaccagacaccattgtggttgttgctgggaacaag ttacgcctggatgtccctatttctggagaccctgctcccactgtggtctggcagaagactgtaacacaggggaagaaggcctcaactgggcc acaccctgatgccccagaagatgctggtgctgatgaggagtgggtgtttgataagaagctgttgtgtgagactgagggccgggtccgggtg gagaccaccaaagaccgcagcgtctttacagtcgaaggggcagagaaggaagatgaaggtgtctacacagtcacagtaaagaaccccgt gggcgaggaccaggtcaacctcacagtcaaggtcatcgatgtcccagatgctcctgcggcccctaagatcagcaacgtgggcgaggactc ctgcactgtgcagtgggaaccgcctgcctatgatggcgggcagccggtcctgggatacatcctggagcgcaagaagaaaaagagctaca ggtggatgaggctcaactttgatctgctgcgggagctgagccacgaggcgaggcgcatgatcgagggtgtagcctatgagatgcgagtct acgcagtcaatgccgtgggaatgtccaggcccagccctgcctctcagcccttcatgcctattgggccccctggcgaaccaacccacttggct gtggaggatgtgtcagacaccactgtctcactcaagtggcggcccccagagcgcgtgggggccggtggcctggacggatacagcgtgga gtactgccaggagggatgctccgagtggacacctgctctgcaggggctgacagagcgcacatcgatgctggtgaaggacctacccactgg ggcacggctgctgttccgagtacgggcacacaatgtggcaggtcctggaggccctatcgtcaccaaggagcctgtgacagtgcaggagat actgcaacgaccacggctccaactgcccagacacctgcgccagaccatccagaagaaagttggggagcctgtgaacctcctcatccctttc cagggcaaaccccggcctcaggtgacctggaccaaagaggggcagcccctggcaggtgaggaggtgagcatccggaacagccccaca gacacgatcttgttcatccgagctgcccgccgcacccactcgggcacctaccaggtgacagttcgcattgagaacatggaggacaaggca acgctgatcctgcagattgtggacaagccaagtcctccccaggatatccggatcgttgagacttggggtttcaatgtggctctggagtggaag ccaccccaagatgatggcaatacagagatctggggttatactgtacagaaagctgacaagaagaccatggagtggttcacggttttggaaca ctaccgacgcactcactgtgtggtatcagagcttatcattggcaatggctactacttccgggtcttcagccataacatggtgggttccagtgaca aagctgccgccaccaaggagccagtctttattccaagaccaggcatcacatatgagccacccaaatacaaggccctggacttctctgaggc cccaagcttcacccagcccttggcaaatcgctccatcattgcaggctataatgccatcctctgctgtgctgtccgaggtagtcctaagcccaag atttcctggttcaagaatggcctggatctgggagaagatgctcgcttccgcatgttctgcaagcagggagtattgaccctggagatcaggaaa ccctgcccctatgatggtggtgtctatgtctgcagggccaccaacttgcagggcgaggcacagtgtgagtgccgcctggaggtgcgagttc ctcaggaacagaagctcatctcggaagaggatctgSEQ ID NO: 13 Modified CMyBPC / c-myc fusion protein sequence, 302DMPEPGKKPVSAFNKKPRSAEVTAGSAAVFEAETERSGVKVRWQRDGSDITANDKYGLA AEGKRHTLTVRDASPDDQGSYAVIAGSSKVKFDLKVTEPAPPEKAESEVAPGAPKEVPA PATELEESVSSPEGSVSVTQDGSAAEHQGAPDDPIGLFLMRPQDGEVTVGGSIVFSARVAGASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELHITDAQTTSAGGYRCEVSTKDKFDSCNFNLTVHEAIGSGDLDLRSAFRRTSLAGAGRRTSDSHEDAGTLDFSSLLKKRDDDRRDSKLEAPAEEDVWEILRQAPPSEYERIAFQHGVTDLRGMLKRLKGMKQDEKKSTAFQKKLEPAYQVNKGHKIRLTVELADPDAEVKWLKNGQEIQMSGSKYIFESVGAKRTLTISQCSLADDAAYQCVVGGEKCSTELFVKEPPVLITRSLEDQLVMVGQRVEFECEVSEEGAQVKWLKDGVELTREETFKYRFKKDGRKHHLIINEATLEDAGHYAVRTSGGQSLAELIVQEKKLEVYQSIADLAVGAKDQAVFKCEVSDENVRGVWLKNGKELVPDNRIKVSHIGRVHKLTIDDVTPADEADYSFVPEGFACNLSAKLHFMEVKIDFVPRQEPPKIHLDCPGSTPDTIVVVAGNKLRLDVPISGDPAPTVVWQKTVTQGKKASTGPHPDAPEDAGADEEWVFDKKLLCETEGRVRVETTKDRSVFTVEGAEKEDEGVYTVTVKNPVGEDQVNLTVKVIDVPDAPAAPKISNVGEDSCTVQWEPPAYDGGQPVLGYILERKKKKSYRWMRLNFDLLRELSHEARRMIEGVAYEMRVYAVNAVGMSRPSPASQPFMPIGPPGEPTHLAVEDVSDTTVSLKWRPPERVGAGGLDGYSVEYCQEGCSEWTPALQGLTERTSMLVKDLPTGARLLFRVRAHNVAGPGGPIVTKEPVTVQEILQRPRLQLPRHLRQTIQKKVGEPVNLLIPFQGKPRPQVTWTKEGQPLAGEEVS1RNSPTDT1LF1RAARRTHSGTYQVTVR1ENMEDKATL1LQ1VDKPSPPQDIRIVETWGFNVALEWKPPQDDGNTEIWGYTVQKADKKTMEWFTVLEHYRRTHCVVSELIIGNGYYFRVFSHNMVGSSDKAAATKEPVFIPRPGITYEPPKYKALDFSEAPSFTQPLANRSIIAGYNAILCCAVRGSPKPKISWFKNGLDLGEDARFRMFCKQGVLTLEIRKPCPYDGGVYVCRATNLQGEAQCECRLEVRVPQEQKLISEEDL

Claims

CLAIMSWe claim:

1. A variant cMyBPC protein comprising at least one set of substitutions selected from the following sets of substitutions: the first set of substitutions comprising the substitutions Thr272 to Asp272, Ser273 to Asp273 and Leu274 to Asp274; the second set of substitutions comprising the substitutions Thr281 to Asp281 and Ser282 to Asp 282; the third set of substitutions comprising the substitutions Ser302 to Asp 302 and Phe303 toAsp 303; the fourth set of substitutions comprising the substitutions Ser307 to Asp 307 and Lys308 to Asp308.

2. The variant cMyBPC protein of claim 1, comprising all four sets of substitutions.

3. The variant cMyBPC protein of claim 2 comprising the amino acid sequence of SEQ ID NO: 2.

4. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 2.

5. The polynucleotide of claim 4, comprising SEQ ID NO: 3.

6. The variant cMyBPC protein according to claim 3, comprising the amino acid sequence of SEQ ID NO: 4.

7. The variant cMyBPC protein of claim 1, comprising the first set of substitutions.

8. The variant cMyBPC protein of claim 1, comprising the second set of substitutions.

9. The variant cMyBPC protein of claim 1 , comprising the third set of substitutions.

10. The variant cMyBPC protein of claim 1, comprising the fourth set of substitutions.

11. The variant cMyBPC protein of claim 1, comprising a plurality of sets of substitutions selected from the group consisting of the first set of substitutions, the second set of substitutions, the third set of substitutions and the fourth set of substitutions.

12. A vector adapted to express a polypeptide comprising the variant cMyPBC protein of claim 1.

13. The vector of claim 12, comprising an adeno associated virus vector.

14. The vector of claim 13, comprising Adeno associated virus 9.

15. The vector of claim 14, comprising SEQ ID NO: 3.

16. The vector of claim 15, additionally comprising SEQ ID NO: 5.

17. A method of treatment for heart failure, the method comprising administering a vector to a patient, where the vector comprises a polynucelotide sequence encoding the variant cMyBPC polypeptide and the vector is adapted to express the variant cMyBPC polypeptide, and wherein the variant cMyBPC protein comprises at least one set of substitutions selected from the following sets of substitutions: the first set of substitutions comprising the substitutions Thr272 to Asp272, Ser273 to Asp273 and Leu274 to Asp274; the second set of substitutions comprising the substitutions Thr281 to Asp281 and Ser282 to Asp 282; the third set of substitutions comprising the substitutions Ser302 to Asp 302 and Phe303 to Asp 303; the fourth set of substitutions comprising the substitutions Ser307 to Asp 307 and Lys308 to Asp308.

18. The method of claim 17, wherein the variant cMyBPC protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:8 and SEQ ID NO: 9.

19. The variant cMyBPC protein of claim 1 for the treatment of disease.

20. The variant cMyBPC protein of claim 1 for the manufacture of a medicament.

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