GENE THERAPY FOR DUCHENNE MUSCULAR DYSTROPHY

Abstract
The disclosure provides compositions and methods for producing micro-dystrophin proteins, and the use thereof (e.g., in gene therapy). The disclosure also provides skeletal muscle-specific promoters and the use thereof (e.g., in gene therapy). The disclosure further provides catheter-based methods of delivering gene therapy vectors to skeletal muscle and/or cardiac muscle.
Description
BACKGROUND

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness. DMD is caused by mutations in the dystrophin gene. The multifaceted pathologies associated with DMD represent major challenges to the development and clinical implementation of effective therapeutics for the disease. Adeno-associated virus (AAV) gene therapy, delivering a micro-dystrophin construct to all striated muscles, is now poised to bring about transformative alterations in DMD disease course.


However, a number of significant problems remain. Accordingly, there is a need for alternative gene therapies for treating DMD.


SUMMARY

In one aspect, the present disclosure provides a recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein comprising: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, and spectrin-like repeat 17, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin. In some embodiments, the micro-dystrophin protein further comprises spectrin-like repeat 18 and/or spectrin-like repeat 19.


In one aspect, the present disclosure provides a recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein comprising: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 17, spectrin-like repeat 18, and spectrin-like repeat 19, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin.


In some embodiments, the micro-dystrophin protein further comprises one or more of: spectrin-like repeat 16, hinge domain 3, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin. In some embodiments, the micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin. In some embodiments, the micro-dystrophin protein further comprises one or more coiled coil domains of dystrophin.


In some embodiments, the spectrin-like repeat 1 is directly coupled to the spectrin-like repeat 17.


In some embodiments, the spectrin-like repeat 19 is directly coupled to spectrin-like repeat 24. In some embodiments, the spectrin-like repeat 19 is directly coupled to hinge domain 4.


In some embodiments, the spectrin-like repeat 17 is directly coupled to hinge domain 3 or spectrin-like repeat 22.


In one aspect, the present disclosure provides a recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein comprising: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 2, and spectrin-like repeat 17, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 3 of dystrophin. In some embodiments, the micro-dystrophin protein further comprises spectrin-like repeat 16.


In one aspect, the present disclosure provides a recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein comprising: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 2, spectrin-like repeat 16, and spectrin-like repeat 17, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 3 of dystrophin.


In some embodiments, the micro-dystrophin protein further comprises one or more of: hinge domain 3, spectrin-like repeat 22, spectrin-like repeat 23, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin. In some embodiments, the micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin. In some embodiments, the micro-dystrophin protein further comprises one or more coiled coil domains of dystrophin.


In some embodiments, the spectrin-like repeat 2 is directly coupled to spectrin-like repeat 16. In some embodiments, the spectrin-like repeat 2 is directly coupled to spectrin-like repeat 17.


In some embodiments, the spectrin-like repeat 17 is directly coupled to hinge domain 3.


In some embodiments, the spectrin-like repeat 17 is directly coupled to spectrin-like repeat 24. In some embodiments, the spectrin-like repeat 17 is directly coupled to hinge domain 4.


In some embodiments, the micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the C-terminus. In some embodiments, the micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the end of the proline rich region following the first coiled coil domain. In some embodiments, the micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the C-terminus.


In some embodiments, the micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the proline rich region following the first coiled coil domain.


In some embodiments, the micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the second syntrophin binding domain.


In some embodiments, the recombinant nucleic acid is less than 5 kb in length.


In some embodiments, a promoter is operably linked to the nucleotide sequence encoding the micro-dystrophin protein. In some embodiments, the promoter is a cardiac-specific promoter.


In some embodiments, the cardiac-specific promoter is a cardiac troponin T (cTnT) promoter. In some embodiments, the promoter is a skeletal muscle-specific promoter. In some embodiments, the skeletal muscle-specific promoter is a skeletal muscle alpha-actin promoter. In some embodiments, the skeletal-muscle specific promoter comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 78 or SEQ ID NO: 80.


In some embodiments, the present disclosure provides a recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein, wherein the nucleotide sequence encoding the micro-dystrophin protein comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of any one of SEQ ID Nos. 7-12, 22-39, 92-97, 109-114, and 116-118.


In some embodiments, the present disclosure provides a recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein that comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6, 13-21, 86-91, 98, 106-108, and 115.


In some embodiments, the present disclosure provides a micro-dystrophin protein encoded by a recombinant nucleic acid of the disclosure.


In some embodiments, the present disclosure provides a micro-dystrophin protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6, 13-21, 86-91, 98, 106-108, and 115.


In one aspect, the present disclosure provides a skeletal muscle-specific promoter. In one aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80.


In some embodiments, the nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80 is operably linked to a nucleotide sequence that is at least 80% identical to the nucleotide sequence of any one of SEQ ID Nos. 7-12, 22-39, 92-97, 109-114, and 116-118.


In some embodiments, the nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80 is operably linked to a nucleotide sequence that encodes a micro-dystrophin protein that comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6,13-21, 86-91, 98, 106-108, and 115.


In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a recombinant nucleic acid or isolated nucleic acid of the disclosure.


In one aspect, the present disclosure provides an rAAV particle comprising a rAAV vector of the disclosure encapsidated in an AAV capsid. In some embodiments, the AAV capsid comprises a capsid protein derived from AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAVrh.74, AAVrh.10, AAV2/6 or AAV9 serotypes.


In one aspect, the present disclosure provides a composition comprising an rAAV particle.


In one aspect, the present disclosure provides a method of treating a skeletal muscle disorder in a subject in need thereof, comprising administering to the subject a recombinant or isolated nucleic acid of the disclosure, a micro-dystrophin protein of the disclosure, an rAAV particle of the disclosure, or a composition of the disclosure.


In one aspect, the present disclosure provides a method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, comprising administering to the subject a recombinant or isolated nucleic acid of the disclosure, a micro-dystrophin protein of the disclosure, an rAAV particle of the disclosure, or a composition of the disclosure.


In one aspect, the present disclosure provides a method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising delivering to the subject a first recombinant adeno-associated virus (rAAV) particle comprising a nucleotide sequence encoding a first micro-dystrophin protein to cardiac muscle and a second rAAV particle comprising a nucleotide sequence encoding a second micro-dystrophin to skeletal muscle.


In some embodiments, the first rAAV particle comprises a cardiac-specific promoter operably linked to the nucleotide sequence encoding the first micro-dystrophin protein. In some embodiments, the second rAAV particle comprises a skeletal muscle-specific promoter operably linked to the nucleotide sequence encoding the second micro-dystrophin protein.


In some embodiments, the first and second rAAV particles are of the same serotype. In some embodiments, the first and second rAAV particles are of different serotypes. In some embodiments, the first rAAV particle comprises a capsid protein derived from AAV9, AAVrh.74, or AAVrh.10. In some embodiments, the second rAAV particle comprises a capsid protein derived from AAV8.


In some embodiments, the first and second rAAV particles are delivered by the same method. In some embodiments, the first and second rAAV particles are delivered by different methods.


In some embodiments, the first and second rAAV particles are delivered via one or more catheters. In some embodiments, the method comprises: (i) introducing a catheter into the femoral artery and advancing to the heart; (ii) delivering the first rAAV particle into the left and right coronary arteries; (iii) retracting the catheter to the aortic arch; and (iv) delivering the second rAAV particle to the subclavian and/or carotid arteries. In some embodiments, the method further comprises (v) retracting the catheter into the descending aorta and delivering the second rAAV particle to skeletal muscle via descending aortic branches.


In some embodiments, the method further comprises administering a vasodilator to the subject prior to the delivery of the first and second rAAV particles.


In some embodiments, the vasodilator is a PDE5 inhibitor. In some embodiments, the PDE5 inhibitor is sildenafil or tadalafil.


In some embodiments, the vasodilator is administered 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour prior to the delivery of first rAAV and/or second rAAV.


In some embodiments, a blood pressure cuff is inflated on each limb at the time of delivery of the first rAAV particle and/or the second rAAV particle.


In some embodiments, the skeletal muscle is fast-twitch or slow-twitch.


In some embodiments, the first micro-dystrophin protein comprises: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, and spectrin-like repeat 17, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin. In some embodiments, the first micro-dystrophin protein comprises: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 17, spectrin-like repeat 18, and spectrin-like repeat 19, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin. In some embodiments, the first micro-dystrophin protein further comprises one or more of: spectrin-like repeat 16, hinge domain 3, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin. In some embodiments, the first micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin. In some embodiments, the first micro-dystrophin protein further comprises one or more coiled coil domains of dystrophin. In some embodiments, the spectrin-like repeat 1 of the first micro-dystrophin protein is directly coupled to the spectrin-like repeat 17. In some embodiments, the spectrin-like repeat 19 of the first micro-dystrophin protein is directly coupled to spectrin-like repeat 24. In some embodiments, the spectrin-like repeat 19 of the first micro-dystrophin protein is directly coupled to hinge domain 4. In some embodiments, the spectrin-like repeat 17 is directly coupled to hinge domain 3. In some embodiments, the spectrin-like repeat 17 is directly coupled to spectrin-like repeat 22.


In some embodiments, the second micro-dystrophin protein comprises: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 2, and spectrin-like repeat 17, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 3 of dystrophin. In some embodiments, the second micro-dystrophin protein comprises: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 2, spectrin-like repeat 16, and spectrin-like repeat 17, and wherein the micro-dystrophin protein does not contain spectrin-like repeat 3 of dystrophin. In some embodiments, the second micro-dystrophin protein further comprises one or more of: hinge domain 3, spectrin-like repeat 22, spectrin-like repeat 23, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin. In some embodiments, the second micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin. In some embodiments, the second micro-dystrophin protein further comprises one or more coiled coil domains of dystrophin. In some embodiments, the spectrin-like repeat 2 of the second micro-dystrophin protein is directly coupled to spectrin-like repeat 16. In some embodiments, the spectrin-like repeat 2 of the second micro-dystrophin protein is directly coupled to spectrin-like repeat 17. In some embodiments, the spectrin-like repeat 17 of the second micro-dystrophin protein is directly coupled to hinge domain 3. In some embodiments, the spectrin-like repeat 17 of the second micro-dystrophin protein is directly coupled to spectrin-like repeat 24. In some embodiments, the spectrin-like repeat 17 of the second micro-dystrophin protein is directly coupled to hinge domain 4.


In some embodiments, the first or second micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the C-terminus. In some embodiments, the first or second micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the end of the proline rich region following the first coiled coil domain. In some embodiments, the first or second micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the C-terminus. In some embodiments, the first or second micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the proline rich region following the first coiled coil domain. In some embodiments, the first or second micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the second syntrophin binding domain.


In one aspect, the present disclosure provides a method of delivering an rAAV particle to a skeletal muscle in a subject, the method comprising delivering the rAAV particle via a catheter.


In some embodiments, the method comprises delivering the rAAV particle to the subclavian and/or carotid arteries. In some embodiments, the catheter is first introduced into the femoral artery and advanced to the subclavian and/or carotid arteries. In some embodiments, the method further comprises retracting the catheter into the descending aorta and delivering the rAAV particle to skeletal muscle via descending aortic branches.


In some embodiments, the method comprises administering a vasodilator. In some embodiments, the vasodilator is a PDE5 inhibitor. In some embodiments, the PDE5 inhibitor is sildenafil or tadalafil. In some embodiments, the vasodilator is administered 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour prior to the delivery of the rAAV particle.


In some embodiments, the method further comprises inflating a blood pressure cuff on each limb at the time of delivery of the rAAV particle.


In some embodiments, the skeletal muscle is fast-twitch or slow-twitch.





BRIEF DESCRIPTION OF DRAWINGS


FIG. 1 shows a diagram of dystrophin and its interactions, showing known syntrophin binding sites.



FIGS. 2A and 2B show a diagram of full-length dystrophin showing order of Repeats (R) and hinges (H), as well as the cysteine-rich portion of the C-terminus (CR), which is commonly included in micro-dystrophins and referred to as the truncated C-terminus, which is then followed by the rest of the C-terminus (CT).



FIGS. 3A and 3B show skeletal muscle function at one month following injection of AAV micro-dystrophins. FIG. 3A shows the force per unit area values (specific tension) for the diaphragm and EDL muscles that received delivery of either one of three micro-dystrophins in clinical trials (PF-06939926, SRP-9001-10 or SGT-001) or one of the exemplary micro-dystrophins of the disclosure (Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin region, Nterm-R2_R17_H3_R22_H4 to end of syntrophin region, Nterm-R1_R17_R22_H4 to 1st coiled coil, or Nterm-R1_R16-R17_R21-R22_H4 to end of syntrophin region). “H4 to end of syntrophin region” is shown as “H4-SynBS.” FIG. 3B shows the ability of each of these micro-dystrophins to protect the skeletal muscles from contraction-induced injury.



FIG. 4 shows cardiac function at one year of age following injection of AAV micro-dystrophins at 5 weeks of age. Shown in the two graphs are cardiac output values for separate groups of mice that received the indicated AAV-micro-dystrophins. Cardiac outputs (mis/minute) were determined by measuring stroke volume using a Vevo3100 echocardiographic instrument and multiplying by heart rate.



FIG. 5 shows ventricular end diastolic volumes (EDV) at one year of age following injection of AAV micro-dystrophins at 5 weeks of age. Shown in the two graphs are cardiac EDV values (determined using a Vevo3100 echocardiographic instrument) for separate groups of mice that received the indicated AAV-micro-dystrophins.





DETAILED DESCRIPTION

The following detailed description is made by way of illustration of certain aspects of the disclosure. It is to be understood that other aspects are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense. Scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.


Overview

The present disclosure relates to compositions and methods of cardiac muscle and/or skeletal muscle gene therapy, e.g., for treating DMD.


Gene therapy for DMD has moved into the clinic, with four ongoing clinical trials and more planned. The ongoing trials deliver a truncated version of the deficient protein, dystrophin, to heart and skeletal muscle using AAV. The reason for delivery of a truncated protein, known as micro-dystrophin is that the full length protein will not fit within the packaging limits of AAV.


However, a number of significant problems remain. There is a need to prevent and resolve fibrosis in order to improve the efficiency of delivery and enable the use of lower viral doses. Additional therapeutics may be needed to slow the skeletal muscle disease progression that remains after micro-dystrophin therapy. Perhaps the most critical issue facing AAV gene therapy for DMD is that current micro-dystrophin designs and vector doses are optimized for skeletal muscle without sufficient evaluation of the potential impact on cardiac muscle function.


The micro-dystrophins in trials have resulted from more than a decade of investigation of constructs in the dystrophin-deficient mdx mouse. The inventors have recently analyzed these constructs in clinical trials in a newer, more severe mouse model (D2.mdx mouse) that is much more indicative of the human disease. Without wishing to be bound by theory, based on the results of these mouse studies, it is predicted that the long-term consequences of existing gene therapies will be a form of DMD with severe cardiomyopathy and slowly failing skeletal muscle function. Accordingly, the inventors have identified the need for a different approach to micro-dystrophin delivery to skeletal muscle versus to the heart.


Designing different micro-dystrophin constructs that are optimal for each tissue will significantly improve the therapeutic effects of gene therapy with micro-dystrophin. In some embodiments, this involves simultaneous delivery of two separate vectors, one delivered systemically to skeletal muscles and one delivered directly into the coronary arteries of the heart, using different micro-dystrophin constructs and doses. In some embodiments, a small, skeletal muscle (fast and slow)-specific promoter is used for skeletal muscle. In some embodiments, a cardiac-specific promoter is used for the heart. The disclosure provides micro-dystrophin constructs with improved cardiac-corrective properties, likely to be safer and more beneficial than those in current trials. By combining additional gene therapy approaches with micro-dystrophin, long-lasting benefits to both striated muscle types can be achieved.


Accordingly, in one aspect, the present disclosure provides compositions and methods for producing micro-dystrophin proteins, and the use thereof. The present disclosure provides nucleic acids encoding micro-dystrophin proteins optimized for either skeletal muscle or cardiac muscle. In some embodiments, the nucleic acids encoding micro-dystrophin proteins are optimized for cardiac muscle. In some embodiments, the nucleic acids encoding micro-dystrophin proteins are optimized for skeletal muscle.


In another aspect, the present disclosure relates to skeletal muscle-specific promoters and the use thereof.


In yet another aspect, the present disclosure relates to localized delivery (e.g., catheter-based delivery) of gene therapy vectors (e.g., rAAV vectors) to skeletal muscle and/or cardiac muscle. The inventors have found that catheter-based delivery of gene therapy vectors to skeletal muscle and/or cardiac muscle is more efficient and allows for a better distribution of the gene therapy vectors, enabling the use of lower doses of the gene therapy vectors. This in turn mitigates any unfavorable immune responses to the gene therapy vectors. In some embodiments, the present disclosure provides a catheter-based method of delivering a gene therapy vector (e.g., an rAAV vector) to skeletal muscle. In some embodiments, the present disclosure provides a catheter-based method of simultaneously delivering one or more gene therapy vectors (e.g., one or more rAAV vectors) to skeletal muscle and cardiac muscle. In some embodiments, the gene therapy vector targeted to skeletal muscle comprises a nucleic acid encoding a micro-dystrophin protein that is optimized for skeletal muscle and the gene therapy vector targeted to cardiac muscle comprises a nucleic acid encoding a micro-dystrophin protein that is optimized for cardiac muscle.


Micro-Dystrophins

A micro-dystrophin gene as used herein refers to a truncated dystrophin gene that is generally less than 5 kb in length. A micro-dystrophin gene is generally small enough to fit into an AAV vector. In some embodiments, a micro-dystrophin gene is less than about 4900 bp in length. In some embodiments, a micro-dystrophin gene is less than about 4800 bp in length. In some embodiments, a micro-dystrophin gene is less than about 4700, 4600, or 4500 bp in length.


In some embodiments, a micro-dystrophin gene is between about 3600-5000 bp in length. In some embodiments, a micro-dystrophin gene is between about 3600-4800 bp in length, about 3600-4500 bp in length, about 3300-5000 bp in length, about 3300 to 4800 bp in length, about 3300 to 4500 bp in length, about 3000-5000 bp in length, about 3000-4800 bp in length, or about 3000-4500 bp in length. A micro-dystrophin protein as used herein is less than about 1700 aa in length. In some embodiments, a micro-dystrophin protein is less than about 1666 aa in length. In some embodiments, a micro-dystrophin protein is less than about 1600 aa in length. In some embodiments, a micro-dystrophin protein is between about 1200-1700 aa in length. In some embodiments, a micro-dystrophin protein is between about 1200-1666 aa in length, about 1200-1600 aa in length, about 1100-1700 aa in length, about 1100-1666 aa in length, about 1100 to 1600 aa in length, about 1100-1700 aa in length, about 1000-1666 aa in length, or about 1000-1600 aa in length. A micro-dystrophin gene may include naturally occurring dystrophin sequences from any species and variants derived from such genes by mutagenesis, or other modifications.


Cardiac Muscle Micro-Dystrophins

The present disclosure provides micro-dystrophin genes and proteins optimized for cardiac muscle. The inventors have found that certain combinations of domains from full-length dystrophin are beneficial for cardiac muscle.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle does not comprise spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin. In some embodiments, a micro-dystrophin protein does not comprise spectrin-like repeat 2. In some embodiments, a micro-dystrophin protein does not comprise spectrin-like repeat 3. In some embodiments, a micro-dystrophin protein does not comprise spectrin-like repeat 2 and spectrin-like repeat 3. The inventors have found that including spectrin-like repeat 1 but not spectrin-like repeat 2 and/or spectrin-like repeat 3 is beneficial to the heart as such micro-dystrophin proteins outcompete utrophin for its membrane binding in the heart to a lesser extent, thus mitigating the harmful effects of displacing utrophin. Harmful effects of displacing utrophin include cardiomyopathy and heart failure.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, and spectrin-like repeat 17. In some embodiments, the micro-dystrophin protein further comprises spectrin-like repeat 18 of dystrophin. In some embodiments, the micro-dystrophin protein further comprises spectrin-like repeat 19 of dystrophin. In some embodiments, the micro-dystrophin protein further comprises spectrin-like repeat 18 and spectrin-like repeat 19 of dystrophin. In some embodiments, the micro-dystrophin protein does not contain spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin. In some embodiments, the micro-dystrophin protein does not comprise spectrin-like repeat 2. In some embodiments, the micro-dystrophin protein does not comprise spectrin-like repeat 3. In some embodiments, the micro-dystrophin protein does not comprise spectrin-like repeat 2 and spectrin-like repeat 3.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises one or more of: spectrin-like repeat 16, hinge domain 3, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 24 and hinge domain 4. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 24 and the dystroglycan binding site of dystrophin. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises hinge domain 4 and the dystroglycan binding site of dystrophin. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises hinge domain 3, spectrin-like repeat 21, spectrin-like repeat 22, hinge domain 4 and the dystroglycan binding site of dystrophin. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 22, hinge domain 4 and the dystroglycan binding site of dystrophin. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 16, spectrin-like repeat 21, spectrin-like repeat 22, hinge domain 4 and the dystroglycan binding site of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises one or more syntrophin binding domains of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises one or more coiled coil domains of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 1 directly coupled to spectrin-like repeat 17.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 19 directly coupled to spectrin-like repeat 24. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 19 directly coupled to hinge domain 4.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 17 directly coupled to hinge domain 3. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises spectrin-like repeat 17 directly coupled to spectrin-like repeat 22.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises the region of dystrophin that is C-terminal to the cysteine-rich portion of the C-terminal region (FIGS. 1 and 2). In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises the region of dystrophin that is C-terminal to the dystroglycan binding site of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises the region of dystrophin from spectrin-like repeat 24 to the C-terminus. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises the region of dystrophin from spectrin-like repeat 24 to the end of the proline rich region following the first coiled coil domain.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises the region of dystrophin from hinge domain 4 to the C-terminus. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises the region of dystrophin from hinge domain 4 to the end of the proline rich region following the first coiled coil domain. In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises the region of dystrophin from hinge domain 4 to the end of the second syntrophin binding domain.


Domains of the dystrophin protein are known in the art. Exemplary sequences of domains of the dystrophin protein or combinations thereof are provided in Example 1. In some embodiments, a micro-dystrophin protein of the disclosure comprises an amino-terminal actin-binding domain and hinge domain 1 region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 1 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 2 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 3 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 16 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 16 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44 without the linker sequence. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 17 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, a micro-dystrophin protein of the disclosure comprises spectrin-like repeats 17-19 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, a micro-dystrophin protein of the disclosure comprises spectrin-like repeats 17-19 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, a micro-dystrophin protein of the disclosure comprises a hinge domain 3 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 21 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 22 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 22 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121 without the linker sequence. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 23 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 23 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 24 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, a micro-dystrophin protein of the disclosure comprises a hinge domain 4 and dystroglycan binding site (also referred to herein as “trCterm”) comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49. In some embodiments, a micro-dystrophin protein of the disclosure comprises a syntrophin binding region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a micro-dystrophin protein of the disclosure comprises a 1st coiled coil region and proline rich region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, a micro-dystrophin protein of the disclosure comprises a 1st coiled coil region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51 without the proline rich region. In some embodiments, a micro-dystrophin protein of the disclosure comprises a 2nd coiled coil region to end of dystrophin comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, a micro-dystrophin protein of the disclosure comprises a an amino acid sequence that has up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions or deletions relative to a sequence provided herein (e.g., any one of SEQ ID NOs. 40-52, 81, 82, and 119-121).


Non-limiting examples of human micro-dystrophin proteins optimized for cardiac muscle are described below.










Nterm-R1_R17-R19_R24 to trCterm:



(SEQ ID NO: 1)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDREDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPL





RQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNH





LLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRL





LQELRAKQWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVS





HVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHELSTSVQGPWERAISPNKV





PYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI





LQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVAS





STGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRV





AAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKN





KFRTKRYFAKHPRMGYLPVQTVLEGDNMETP





Nterm-R1_R17-R19_R24 to trCterm:


(SEQ ID NO: 86)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRSVEKWRRFHY





DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL





NLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQG





ILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLL





WLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQE





LRAKQWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVN





DLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHELSTSVQGPWERAISPNKVPYY





INHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI





INCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTG





FCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAA





ETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFR





TKRYFAKHPRMGYLPVQTVLEGDNMETP





Nterm-R1_R17-R19_H4 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 2)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRFDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPL





RQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNH





LLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRL





LQELRAKQGPASQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRR





LQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRT





GRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASEGGSNIEPSVRSCFQ





FANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGR





VAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAP





ASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLE





SEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSP





Nterm-R1_R17-R19_H4 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 87)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRSVEKWRRFHY





DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL





NLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQG





ILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLL





WLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQE





LRAKQGPASQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRESAYRTAMKLRRLQK





ALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRI





RVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFAN





NKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAK





GHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASS





PQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEE





RGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSP





Nterm-R1_R17-R19_H4 to end of syntrophin region:


(SEQ ID NO: 3)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRFDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPL





RQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNH





LLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRL





LQELRAKQGPASQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRR





LQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRT





GRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASEGGSNIEPSVRSCFQ





FANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHENYDICQSCFFSGR





VAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAP





ASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRS





Nterm-R1_R17-R19_H4 to end of syntrophin region:


(SEQ ID NO: 88)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLILGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRSVEKWRRFHY





DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL





NLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQG





ILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLL





WLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQE





LRAKQGPASQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQK





ALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRI





RVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASEGGSNIEPSVRSCFQFAN





NKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAK





GHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASS





PQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCOSLNODSPLSQPRS





N-terminus through R1_R17-R19_R24 to 1st coiled coil and proline rich region that


follows:


(SEQ ID NO: 4)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDREDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPL





RQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNH





LLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRL





LQELRAKQWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVS





HVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKV





PYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI





LQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVAS





STGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRV





AAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKN





KFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYL





NDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHE





HKGLSPLPSPPEMMPTSPQSP





N-terminus through R1_R17-R19_R24 to 1st coiled coil and proline rich region that


follows:


(SEQ ID NO: 89)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRSVEKWRRFHY





DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL





NLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQG





ILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLL





WLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQE





LRAKQWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVN





DLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHELSTSVQGPWERAISPNKVPYY





INHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI





INCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTG





FCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAA





ETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFR





TKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDS





ISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKG





LSPLPSPPEMMPTSPQSP





N-terminus through R1_R17-R19_H4 to end of dystrophin:


(SEQ ID NO: 5)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRFDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPL





RQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNH





LLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRL





LQELRAKQGPASQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRESAYRTAMKLRR





LQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRT





GRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASEGGSNIEPSVRSCFQ





FANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGR





VAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAP





ASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLE





SEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARM





QILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTS





TGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM





N-terminus through R1_R17-R19_H4 to end of dystrophin:


(SEQ ID NO: 90)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRSVEKWRRFHY





DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL





NLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQG





ILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLL





WLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQE





LRAKQGPASQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQK





ALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRI





RVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFAN





NKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAK





GHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASS





PQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEE





RGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQIL





EDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGL





EEVMEQLNNSFPSSRGRNTPGKPMREDTM





N-terminus through R1_R17-R19_R24 to end of dystrophin:


(SEQ ID NO: 6)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDREDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPL





RQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNH





LLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRL





LQELRAKQWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVS





HVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHELSTSVQGPWERAISPNKV





PYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI





LQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVAS





STGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRV





AAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKN





KFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYL





NDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHE





HKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGT





TVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM





N-terminus through R1_R17-R19_R24 to end of dystrophin:


(SEQ ID NO: 91)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRSVEKWRRFHY





DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL





NLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQG





ILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLL





WLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQE





LRAKQWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVN





DLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHELSTSVQGPWERAISPNKVPYY





INHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI





INCLITIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTG





FCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAA





ETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKER





TKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDS





ISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKG





LSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVS





SPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM





Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin region


(SEQ ID NO: 106)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRFDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKQPDLAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLMLEVPKDSTQ





WLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHM





ITENINASWRSIHKRVSEREAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQW





QDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNEKWSELRKKSLNIRSHLEASSDQWKRLGPASQ





HELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLS





AACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGII





SLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALF





LDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVE





YCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHS





RIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNODSPLSQPRS





Nterm-R1_R17_R22_H4 to 1st coiled coil


(SEQ ID NO: 107)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDRFDRSVEKWRR





FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL





GSLNLRWQEVCKQLSDRKKRLEEQKEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMK





QWQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLGPA





SQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLS





LSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRIRVLSFKTG





IISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASEGGSNIEPSVRSCFQFANNKPEIEAA





LFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPM





VEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDT





HSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERIL





ADLEEENRNLQAEYDRLKQQHEHKGLS





Nterm-R1_R16-R17_R21-R22_H4 to end of syntrophin region


(SEQ ID NO: 108)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY





MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDHTVREETMMVM





TEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAA





LQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGREDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHA





KYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKKDSTQW





LEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMI





TENINASWRSIHKRVSEREAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQ





DLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNEKWSELRKKSLNIRSHLEASSDQWKRLGPASQH





FLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSA





ACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRIRVLSFKTGIIS





LCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFL





DWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEY





CTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSR





IEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRS





A micro-dystrophin protein optimized for cardiac muscle is further understood to





include proteins that are variants of any one of SEQ ID Nos. 1-6, 86-91, and





106-108. Variant polypeptides include polypeptides that differ by one or more





(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or





more) amino acid substitutions, additions, or deletions, and







will, therefore, include amino acid sequences that differ from the amino acid sequences designated in any one of SEQ ID Nos. 1-6, 86-91, and 106-108.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle comprises an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6, 86-91, and 106-108.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle consists essentially of an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6, 86-91, and 106-108.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle consists of an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6, 86-91, and 106-108.


In some embodiments, functional variants or modified forms of micro-dystrophin proteins optimized for cardiac muscle include fusion proteins having at least a portion of the micro-dystrophin protein and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. Other fusion domains are particularly useful for increasing protein stability.


The disclosure also provides nucleic acids encoding any of the micro-dystrophins optimized for cardiac muscle described herein. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making micro-dystrophins or as direct therapeutic agents in a gene therapy approach.


Non-limiting examples of nucleotide sequences encoding human micro-dystrophin proteins optimized for cardiac muscle are described below.










Nterm-R1_R17-R19_R24 to trCterm:



(SEQ ID NO: 7)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAA





TATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCC





CACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTG





CGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCA





AGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAG





GAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCAT





CTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTIGACGT





TAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACA





AGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTA





CTTCAAGAGCTGAGGGCAAAGCAGTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGC





CACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCA





TTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGC





CACGTCAATGACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGA





CCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACT





TTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTG





CCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGC





TGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGG





ATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATC





CTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCT





CTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTT





TTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGT





TCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGA





AGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGA





TCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTG





GCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAG





GAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGC





ACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAAC





AAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGA





CAACATGGAAACTCCC





Nterm-R1_R17-R19_R24 to trCterm:


(SEQ ID NO: 92)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTC





AGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAAC





CTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGA





ATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACT





TGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTG





AAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTG





TGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGTTAAGGAAAC





TGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAAC





CAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAG





CTGAGGGCAAAGCAGTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGA





GCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTC





TCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAAT





GACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACAC





CAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAG





CATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTAT





ATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAA





TAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGA





GCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATT





ATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGA





TATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTG





GCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGA





TTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATC





CTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGG





CCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCA





GAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAA





GCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCA





TGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGA





ACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGA





AACTCCC





Nterm-R1_R17-R19_H4 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 8)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAA





TATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCC





CACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTG





CGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCA





AGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAG





GAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCAT





CTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGT





TAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACA





AGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTA





CTTCAAGAGCTGAGGGCAAAGCAGGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAG





AGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGA





CAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGA





CTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCA





AAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACA





ACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACA





GGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATA





CCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCC





AAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAA





TTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTG





GCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTC





CAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGA





GTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGA





CTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAG





TGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCT





GCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGA





AAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGC





ATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAG





AGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGA





CCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCC





AGAGTCCC





Nterm-R1_R17-R19_H4 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 93)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTC





AGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAAC





CTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGA





ATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACT





TGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTG





AAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTG





TGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTIGACGTTAAGGAAAC





TGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAAC





CAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAG





CTGAGGGCAAAGCAGGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTC





GCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCT





ACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAG





GCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCA





GCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGG





TCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATC





CGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAA





GCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAA





GACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAAT





AATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGT





CCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTG





GATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAA





GGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAA





GGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTG





TCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCC





CCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAA





TGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCC





AAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAA





AGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAA





GCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCC





Nterm-R1_R17-R19_H4 to end of syntrophin region:


(SEQ ID NO: 9)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAA





TATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCC





CACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTG





CGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCA





AGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAG





GAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCAT





CTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGT





TAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACA





AGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTA





CTTCAAGAGCTGAGGGCAAAGCAGGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAG





AGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGA





CAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGA





CTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCA





AAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACA





ACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACA





GGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATA





CCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCC





AAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAA





TTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTG





GCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTC





CAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGA





GTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGA





CTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAG





TGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCT





GCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGA





AAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGC





ATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGT





Nterm-R1_R17-R19_H4 to end of syntrophin region:


(SEQ ID NO: 94)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTC





AGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAAC





CTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGA





ATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACT





TGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTG





AAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTG





TGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGTTAAGGAAAC





TGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAAC





CAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAG





CTGAGGGCAAAGCAGGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTC





GCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCT





ACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAG





GCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCA





GCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGG





TCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATC





CGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAA





GCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAA





GACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAAT





AATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGT





CCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTG





GATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAA





GGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAA





GGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTG





TCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCC





CCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAA





TGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCC





AAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGT





Nterm-R1_R17-R19_R24 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 10)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAA





TATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCC





CACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTG





CGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCA





AGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAG





GAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCAT





CTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTIGACGT





TAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACA





AGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTA





CTTCAAGAGCTGAGGGCAAAGCAGTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGC





CACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCA





TTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGC





CACGTCAATGACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGA





CCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACT





TTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTG





CCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGC





TGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGG





ATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATC





CTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCT





CTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTT





TTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGT





TCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGA





AGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGA





TCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTG





GCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAG





GAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGC





ACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAAC





AAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGA





CAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCAC





ACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTA





AATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCA





GGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAG





AGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAA





CATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCC





Nterm-R1_R17-R19_R24 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 95)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTC





AGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAAC





CTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGA





ATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACT





TGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTG





AAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTG





TGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGTTAAGGAAAC





TGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAAC





CAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAG





CTGAGGGCAAAGCAGTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGA





GCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTC





TCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAAT





GACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACAC





CAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAG





CATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTAT





ATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAA





TAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGA





GCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATT





ATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGA





TATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTG





GCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGA





TTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATC





CTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGG





CCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCA





GAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAA





GCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCA





TGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGA





ACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGA





AACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATA





CTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGC





ATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCC





CCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCC





TAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGC





CTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCC





Nterm-R1_R17-R19_H4 to end of dystrophin:


(SEQ ID NO: 11)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAA





TATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCC





CACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTG





CGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCA





AGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAG





GAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCAT





CTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGT





TAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACA





AGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTA





CTTCAAGAGCTGAGGGCAAAGCAGGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAG





AGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGA





CAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGA





CTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCA





AAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACA





ACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACA





GGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATA





CCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCC





AAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAA





TTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTG





GCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTC





CAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGA





GTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGA





CTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAG





TGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCT





GCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGA





AAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGC





ATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAG





AGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGA





CCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCC





AGAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATG





CAAATCCTGGAAGACCACAATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGC





AGAGGCCAAAGTGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGC





TGCTCCGAGTGGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGC





ACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCC





AATGAGAGAGGACACAATG





Nterm-R1_R17-R19_H4 to end of dystrophin:


(SEQ ID NO: 96)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTC





AGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAAC





CTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGA





ATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACT





TGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTG





AAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTG





TGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTIGACGTTAAGGAAAC





TGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAAC





CAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAG





CTGAGGGCAAAGCAGGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTC





GCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCT





ACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAG





GCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCA





GCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGG





TCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATC





CGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAA





GCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAA





GACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAAT





AATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGT





CCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTG





GATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAA





GGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAA





GGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTG





TCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCC





CCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAA





TGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCC





AAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAA





AGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAA





GCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCC





GGGATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTG





GAAGACCACAATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAA





AGTGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAG





TGGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTTA





GAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAGA





GGACACAATG





Nterm-R1_R17-R19_R24 to end of dystrophin:


(SEQ ID NO: 12)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAA





TATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCC





CACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTG





CGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCA





AGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAG





GAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCAT





CTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTIGACGT





TAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACA





AGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTA





CTTCAAGAGCTGAGGGCAAAGCAGTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGC





CACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCA





TTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGC





CACGTCAATGACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGA





CCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACT





TTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTG





CCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGC





TGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGG





ATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATC





CTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCT





CTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTT





TTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGT





TCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGA





AGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGA





TCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTG





GCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAG





GAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGC





ACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAAC





AAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGA





CAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCAC





ACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTA





AATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCA





GGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAG





AGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAA





CATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCT





CATTGCTGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATA





AACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGCACA





ACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAGTGGTTGGCAGTCA





AACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTTAGAGGAGGTGATGG





AGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAGAGGACACAATG





Nterm-R1_R17-R19_R24 to end of dystrophin:


(SEQ ID NO: 97)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTC





AGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAAC





CTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGA





ATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACT





TGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTG





AAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTG





TGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGTTAAGGAAAC





TGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAAC





CAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAG





CTGAGGGCAAAGCAGTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGA





GCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTC





TCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAAT





GACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACAC





CAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAG





CATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTAT





ATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAA





TAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGA





GCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATT





ATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGA





TATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTG





GCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGA





TTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATC





CTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGG





CCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCA





GAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAA





GCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCA





TGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGA





ACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGA





AACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATA





CTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGC





ATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCC





CCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCC





TAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGC





CTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGCTGA





GGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGG





AGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGCACAACGGTGTCC





TCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAGTGGTTGGCAGTCAAACTTCGGA





CTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCA





ACAACTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAGAGGACACAATG





Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin region


(SEQ ID NO: 109)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGCA





GCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCTCTCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGG





TTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTAAGGATTCAACACAA





TGGCTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAGGGTCC





CTATACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAGCAGTTGGCCAAAGACCTCCGCCAGTGGCAGACAA





ATGTAGATGTGGCAAATGACTTGGCCCTGAAACTTCTCCGGGATTATTCTGCAGATGATACCAGAAAAGTCCACATG





ATAACAGAGAATATCAATGCCTCTTGGAGAAGCATTCATAAAAGGGTGAGTGAGCGAGAGGCTGCTTTGGAAGAAAC





TCATAGATTACTGCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAACTGCCA





ATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAACAATGG





CAAGACCTCCAAGGTGAAATTGAAGCTCACACAGATGTTTATCACAACCTGGATGAAAACAGCCAAAAAATCCTGAG





ATCCCTGGAAGGTTCCGATGATGCAGTCCTGTTACAAAGACGTTTGGATAACATGAACTTCAAGTGGAGTGAACTTC





GGAAAAAGTCTCTCAACATTAGGTCCCATTTGGAAGCCAGTTCTGACCAGTGGAAGCGTCTGGGTCCAGCATCTCAG





CACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCA





CGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCA





GATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCA





GCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTG





TTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTC





TGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATT





TCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGA





CCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGG





GCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTC





CTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGC





CAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTA





ATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAA





TATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAG





GTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCG





TTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCA





CGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCC





TAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCC





AGCCTCGTAGT





Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin region


Codon optimized sequence


(SEQ ID NO: 110)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGG





TTCCACTACGACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGA





GAACTGGGAGCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGC





GGACCCTGAACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTG





GGCAGCCTGAACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGCA





GCCCGACCTGGCCCCCGGCCTGACCACCATCGGCGCCAGCCCCACCCAGACCGTGACCCTGGTGACCCAGCCCGTGG





TGACCAAGGAGACCGCCATCAGCAAGCTGGAGATGCCCAGCAGCCTGATGCTGGAGGTGCCCAAGGACAGCACCCAG





TGGCTGGAGGCCAAGGAGGAGGCCGAGCAGGTGCTGGGCCAGGCCCGGGCCAAGCTGGAGAGCTGGAAGGAGGGCCC





CTACACCGTGGACGCCATCCAGAAGAAGATCACCGAGACCAAGCAGCTGGCCAAGGACCTGCGGCAGTGGCAGACCA





ACGTGGACGTGGCCAACGACCTGGCCCTGAAGCTGCTGCGGGACTACAGCGCCGACGACACCCGGAAGGTGCACATG





ATCACCGAGAACATCAACGCCAGCTGGCGGAGCATCCACAAGCGGGTGAGCGAGCGGGAGGCCGCCCTGGAGGAGAC





CCACCGGCTGCTGCAGCAGTTCCCCCTGGACCTGGAGAAGTTCCTGGCCTGGCTGACCGAGGCCGAGACCACCGCCA





ACGTGCTGCAGGACGCCACCCGGAAGGAGCGGCTGCTGGAGGACAGCAAGGGCGTGAAGGAGCTGATGAAGCAGTGG





CAGGACCTGCAGGGCGAGATCGAGGCCCACACCGACGTGTACCACAACCTGGACGAGAACAGCCAGAAGATCCTGCG





GAGCCTGGAGGGCAGCGACGACGCCGTGCTGCTGCAGCGGCGGCTGGACAACATGAACTTCAAGTGGAGCGAGCTGC





GGAAGAAGAGCCTGAACATCCGGAGCCACCTGGAGGCCAGCAGCGACCAGTGGAAGCGGCTGGGCCCCGCCAGCCAG





CACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCA





CGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGC





GGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGC





GCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTG





CCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCC





TGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATC





AGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGA





CCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCG





GCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTC





CTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGC





CAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCA





ACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAG





TACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCG





GTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCCG





TGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAGC





CGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCCC





CAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGCC





AGCCCCGGAGC





Nterm-R1_R17_R22_H4 to 1st coiled coil


(SEQ ID NO: 111)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCGATTTGACAGATCTGTTGAGAAATGGCGGCGT





TTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGA





GAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCA





GAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTG





GGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGGA





AGAAACTCATAGATTACTGCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAA





CTGCCAATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAA





CAATGGCAAGACCTCCAAGGTGAAATTGAAGCTCACACAGATGTTTATCACAACCTGGATGAAAACAGCCAAAAAAT





CCTGAGATCCCTGGAAGGTTCCGATGATGCAGTCCTGTTACAAAGACGTTTGGATAACATGAACTTCAAGTGGAGTG





AACTTCGGAAAAAGTCTCTCAACATTAGGTCCCATTTGGAAGCCAGTTCTGACCAGTGGAAGCGTCTGGGTCCAGCA





TCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATAT





CAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATA





ATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGC





CTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTAT





TAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATA





TGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGC





ATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATT





TTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCT





TTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCC





CTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGA





AACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGC





ACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATG





GTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAAC





CAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAA





CTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACT





CATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCAT





CTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCC





TGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTA





GCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCT





GTCC





Nterm-R1_R17_R22_H4 to 1st coiled coil


Codon optimized sequence


(SEQ ID NO: 112)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGG





TTCCACTACGACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGA





GAACTGGGAGCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGC





GGACCCTGAACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTG





GGCAGCCTGAACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGGA





GGAGACCCACCGGCTGCTGCAGCAGTTCCCCCTGGACCTGGAGAAGTTCCTGGCCTGGCTGACCGAGGCCGAGACCA





CCGCCAACGTGCTGCAGGACGCCACCCGGAAGGAGCGGCTGCTGGAGGACAGCAAGGGCGTGAAGGAGCTGATGAAG





CAGTGGCAGGACCTGCAGGGCGAGATCGAGGCCCACACCGACGTGTACCACAACCTGGACGAGAACAGCCAGAAGAT





CCTGCGGAGCCTGGAGGGCAGCGACGACGCCGTGCTGCTGCAGCGGCGGCTGGACAACATGAACTTCAAGTGGAGCG





AGCTGCGGAAGAAGAGCCTGAACATCCGGAGCCACCTGGAGGCCAGCAGCGACCAGTGGAAGCGGCTGGGCCCCGCC





AGCCAGCACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACAT





CAACCACGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACA





ACGTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGC





CTGAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCAT





CAACTGCCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACA





TGTGCCTGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGC





ATCATCAGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTT





CTGCGACCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCT





TCGGCGGCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCC





CTGTTCCTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGA





GACCGCCAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGC





ACTTCAACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATG





GTGGAGTACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGAC





CAAGCGGTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGA





CCCCCGTGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACC





CACAGCCGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCAT





CAGCCCCAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCC





TGAGCCAGCCCCGGAGCCCCGCCCAGATCCTGATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTG





GCCGACCTGGAGGAGGAGAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCT





GAGC





Nterm-R1_R16-R17_R21-R22_H4 to end of syntrophin region


(SEQ ID NO: 113)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCACACTGTCCGTGAAGAAACGATGATGGTGATG





ACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCACAAGCCCT





ATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAAGAGGAGT





CTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGACAGCAGCA





TTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGGAAAAAGT





TAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAA





AGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCT





AAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAAC





TGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGC





GGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGAAGGATTCAACACAATGG





CTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAGGGTCCCTA





TACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAGCAGTTGGCCAAAGACCTCCGCCAGTGGCAGACAAATG





TAGATGTGGCAAATGACTTGGCCCTGAAACTTCTCCGGGATTATTCTGCAGATGATACCAGAAAAGTCCACATGATA





ACAGAGAATATCAATGCCTCTTGGAGAAGCATTCATAAAAGGGTGAGTGAGCGAGAGGCTGCTTTGGAAGAAACTCA





TAGATTACTGCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAACTGCCAATG





TCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAACAATGGCAA





GACCTCCAAGGTGAAATTGAAGCTCACACAGATGTTTATCACAACCTGGATGAAAACAGCCAAAAAATCCTGAGATC





CCTGGAAGGTTCCGATGATGCAGTCCTGTTACAAAGACGTTTGGATAACATGAACTTCAAGTGGAGTGAACTTCGGA





AAAAGTCTCTCAACATTAGGTCCCATTTGGAAGCCAGTTCTGACCAGTGGAAGCGTCTGGGTCCAGCATCTCAGCAC





TTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGA





GACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGAT





TCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCT





GCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTT





GACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGA





ACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCC





CTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCA





GCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCA





GTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTA





GACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAA





GCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATT





ATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATAT





TGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTA





TTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTA





CTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGC





ATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAA





TGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGC





CTCGTAGT





Nterm-R1_R16-R17_R21-R22_H4 to end of syntrophin region


Codon optimized sequence


(SEQ ID NO: 114)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCACACCGTGCGGGAGGAGACCATGATGGTGATG





ACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCCAGGCCCT





GCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAGGAGGAGA





GCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGACCGCCGCC





CTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGGAGAAGGT





GAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTACGACATCA





AGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGAGCACGCC





AAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGAACGCCAC





CGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTGAACCTGC





GGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGAAGGACAGCACCCAGTGG





CTGGAGGCCAAGGAGGAGGCCGAGCAGGTGCTGGGCCAGGCCCGGGCCAAGCTGGAGAGCTGGAAGGAGGGCCCCTA





CACCGTGGACGCCATCCAGAAGAAGATCACCGAGACCAAGCAGCTGGCCAAGGACCTGCGGCAGTGGCAGACCAACG





TGGACGTGGCCAACGACCTGGCCCTGAAGCTGCTGCGGGACTACAGCGCCGACGACACCCGGAAGGTGCACATGATC





ACCGAGAACATCAACGCCAGCTGGCGGAGCATCCACAAGCGGGTGAGCGAGCGGGAGGCCGCCCTGGAGGAGACCCA





CCGGCTGCTGCAGCAGTTCCCCCTGGACCTGGAGAAGTTCCTGGCCTGGCTGACCGAGGCCGAGACCACCGCCAACG





TGCTGCAGGACGCCACCCGGAAGGAGCGGCTGCTGGAGGACAGCAAGGGCGTGAAGGAGCTGATGAAGCAGTGGCAG





GACCTGCAGGGCGAGATCGAGGCCCACACCGACGTGTACCACAACCTGGACGAGAACAGCCAGAAGATCCTGCGGAG





CCTGGAGGGCAGCGACGACGCCGTGCTGCTGCAGCGGCGGCTGGACAACATGAACTTCAAGTGGAGCGAGCTGCGGA





AGAAGAGCCTGAACATCCGGAGCCACCTGGAGGCCAGCAGCGACCAGTGGAAGCGGCTGGGCCCCGCCAGCCAGCAC





TTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGA





GACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGCGGT





TCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGCGCC





GCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTGCCT





GACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGA





ACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATCAGC





CTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGACCA





GCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCGGCA





GCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTG





GACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGCCAA





GCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCAACT





ACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAGTAC





TGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCGGTA





CTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCCGTGA





CCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAGCCGG





ATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCCCCAA





CGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGCCAGC





CCCGGAGC






A nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle is further understood to include nucleotide sequences that are variants of any one of SEQ ID Nos. 7-12, 92-97, and 109-114. Variant nucleotide sequences include sequences that differ by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotide substitutions, additions or deletions, such as allelic variants, and will, therefore, include coding sequences that differ from the nucleotide sequence of the coding sequence designated in any one of SEQ ID Nos. 7-12, 92-97, and 109-114.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle is encoded by a nucleic acid comprising a nucleotide sequence that is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 7-12, 92-97, and 109-114.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle is encoded by a nucleic acid consisting essentially of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 7-12, 92-97, and 109-114.


In some embodiments, a micro-dystrophin protein optimized for cardiac muscle is encoded by a nucleic acid consisting of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 7-12, 92-97, and 109-114.


In some embodiments, it is desirable to avoid overexpression of micro-dystrophin proteins in the heart. Thus, in some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle may be codon optimized for lower protein expression. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) are substituted for a rare codon.


Skeletal Muscle Micro-Dystrophins

The present disclosure provides micro-dystrophin genes and proteins optimized for skeletal muscle. The inventors have found that certain combinations of domains from full-length dystrophin are beneficial for skeletal muscle.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle does not comprise spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin. In some embodiments, a micro-dystrophin protein does not comprise spectrin-like repeat 2. In some embodiments, a micro-dystrophin protein does not comprise spectrin-like repeat 3. In some embodiments, a micro-dystrophin protein does not comprise spectrin-like repeat 2 and spectrin-like repeat 3.


In some embodiments, a micro-dystrophin protein comprises optimized for skeletal muscle spectrin-like repeat 1 and spectrin-like repeat 2. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 1, spectrin-like repeat 2, and spectrin-like repeat 3.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 2, and spectrin-like repeat 17 of dystrophin. In some embodiments, the micro-dystrophin protein comprises spectrin-like repeat 16. In some embodiments, the micro-dystrophin protein does not comprise spectrin-like repeat 3 of dystrophin. In some embodiments, the micro-dystrophin protein comprises spectrin-like repeat 3 of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises one or more of: hinge domain 3, spectrin-like repeat 22, spectrin-like repeat 23, spectrin-like repeat 24, hinge domain 4, and the dystroglycan binding site of dystrophin. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 23 and spectrin-like repeat 24. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 23 and hinge domain 4. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 23 and the dystroglycan binding site. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 24 and hinge domain 4. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 24 and the dystroglycan binding site. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises hinge domain 4 and the dystroglycan binding site. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 23, spectrin-like repeat 24 and hinge domain 4. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 23, spectrin-like repeat 24 and the dystroglycan binding site. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 23, hinge domain 4, and the dystroglycan binding site. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 24, hinge domain 4, and the dystroglycan binding site. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 23, spectrin-like repeat 24, hinge domain 4, and the dystroglycan binding site. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises hinge domain 3, spectrin-like repeat 22, hinge domain 4, and the dystroglycan binding site.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises one or more syntrophin binding domains of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises one or more coiled coil domains of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 2 directly coupled to spectrin like repeat 16.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 2 directly coupled to spectrin like repeat 17.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 2 directly coupled to spectrin like repeat 23.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 1 directly coupled spectrin-like repeat 15.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 17 directly coupled to hinge domain 3. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 17 directly coupled to spectrin-like repeat 24. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises spectrin-like repeat 17 directly coupled to hinge domain 4.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises the region of dystrophin that is C-terminal to the cysteine-rich portion of the C-terminal region (FIG. 2). In some embodiments, a micro-dystrophin optimized for skeletal muscle comprises region of dystrophin that is C-terminal to the dystroglycan binding site of dystrophin.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises the region of dystrophin from spectrin-like repeat 24 to the C-terminus. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises the region of dystrophin from spectrin-like repeat 24 to the end of the proline rich region following the first coiled coil domain.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises the region of dystrophin from hinge domain 4 to the C-terminus. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises the region of dystrophin from hinge domain 4 to the end of the proline rich region following the first coiled coil domain. In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises the region of dystrophin from hinge domain 4 to the end of the second syntrophin binding domain.


In some embodiments, a micro-dystrophin protein of the disclosure comprises an amino-terminal actin-binding domain and hinge domain 1 region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 1 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 2 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 3 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 16 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 16 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44 without the linker sequence. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 17 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, a micro-dystrophin protein of the disclosure comprises spectrin-like repeats 17-19 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, a micro-dystrophin protein of the disclosure comprises spectrin-like repeats 17-19 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, a micro-dystrophin protein of the disclosure comprises a hinge domain 3 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 21 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 22 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 22 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 121 without the linker sequence. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 23 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 23 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, a micro-dystrophin protein of the disclosure comprises a spectrin-like repeat 24 comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, a micro-dystrophin protein of the disclosure comprises a hinge domain 4 and dystroglycan binding site (also referred to herein as “trCterm”) comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49. In some embodiments, a micro-dystrophin protein of the disclosure comprises a syntrophin binding region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a micro-dystrophin protein of the disclosure comprises a 1st coiled coil region and proline rich region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, a micro-dystrophin protein of the disclosure comprises a 1st coiled coil region comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51 not including the proline rich region. In some embodiments, a micro-dystrophin protein of the disclosure comprises a region comprising the 2nd coiled coil region to end of dystrophin comprising an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, a micro-dystrophin protein of the disclosure comprises a an amino acid sequence that has up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions or deletions relative to a sequence provided herein (e.g., any one of SEQ ID NOs. 40-52, 81, 82, and 119-121).


Non-limiting examples of human micro-dystrophin proteins optimized for skeletal muscle are described below.










Nterm-R2_R16-R17_R24 to trCterm:



(SEQ ID NO: 13)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ


EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGREDRSVEKWRRFHY


DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL


NLRWQEVCKQLSDRKKRLEEQKWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVK


ALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS


VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDAL


DQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRIRVLSFKTGIISLCKAH


LEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRL


EPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTT


SGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETP





Nterm-R2_R16-R17_H4 to end of syntrophin region:


(SEQ ID NO: 14)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ


EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHY


DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL


NLRWQEVCKQLSDRKKRLEEQKGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN


VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDM


CLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASF


GGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKH


FNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMET


PVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPL


SQPRS





Nterm-R2_R16-R17_H4 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 15)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ


EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHY


DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL


NLRWQEVCKQLSDRKKRLEEQKGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN


VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDM


CLNWLLNVYDTGRIGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASF


GGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKH


FNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMET


PVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPL


SQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSP





Nterm-R2_R16-R17_R24 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 16)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ


EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHY


DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL


NLRWQEVCKQLSDRKKRLEEQKWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVK


ALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS


VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDAL


DQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAH


LEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRL


EPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTT


SGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYA


SRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENR


NLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSP





Nterm-R2_R23_R24 to end of dystrophin:


(SEQ ID NO: 17)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHLSLQELLVWLQLKDDELSRQAPIGGDEPAVQKQNDVHRAFKRELKTKEPVIMSTLETVR


IFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELD


LKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRW


KLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNV


RFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMC


LNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFG


GSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHF


NYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETP


VTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLS


QPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAK


LLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSM


GEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM





Nterm-R2_R23_R24 to end of dystrophin:


(SEQ ID NO: 98)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPV


IMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQE


LQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLS


TLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELY


QSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLV


NVPLCVDMCLNWLLNVYDTGRIGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPR


QLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIG


FRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTV


LEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQ


SLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPR


DAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRV


VGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM





Nterm-R2_R16-R17_H4 to end of dystrophin:


(SEQ ID NO: 18)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ


EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHY


DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRILNATGEEIIQQSSKTDASILQEKLGSL


NLRWQEVCKQLSDRKKRLEEQKGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN


VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDM


CLNWLLNVYDTGRIGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASF


GGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKH


FNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMET


PVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPL


SQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEA


KLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDS


MGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM





Nterm-R1_R16-R17_R24 to end of dystrophin:


(SEQ ID NO: 19)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDHTVREETMMVM


TEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAA


LQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHA


KYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKWQRKID


ETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGI


QLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWD


HPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRL


EQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLL


HDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNI


CKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRM


GYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEH


LLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMM


PTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDS


SQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM





Nterm-R2_R16-R17_R24 to end of dystrophin:


(SEQ ID NO: 20)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ


EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHY


DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL


NLRWQEVCKQLSDRKKRLEEQKWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVK


ALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS


VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDAL


DQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAH


LEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRL


EPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTT


SGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYA


SRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENR


NLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQ


LLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRG


RNTPGKPMREDTM





Nterm-R2_R16-R17_R24 to end of syntrophin region:


(SEQ ID NO: 21)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ


EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHY


DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSL


NLRWQEVCKQLSDRKKRLEEQKWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVK


ALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS


VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDAL


DQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAH


LEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRL


EPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTT


SGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYA


SRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRS





Nterm-R2_R17_H3_R22_H4 to end of syntrophin region:


(SEQ ID NO: 115)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV



NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV


NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY


ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT


TSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY


MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND


WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDR


WANICRWTEDRWVLLQDRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQT


VVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKQPDLAPGLTTIGASPTQTVTLVTQ


PVVTKETAISKLEMPSSLMLEVPEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQW


QDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLGPASQ


HFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLS


AACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGII


SLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALF


LDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVE


YCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHS


RIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRS






A micro-dystrophin protein optimized for skeletal muscle is further understood to include proteins that are variants of any one of SEQ ID Nos. 13-21, 98, and 115. Variant polypeptides include polypeptides that differ by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acid substitutions, additions, or deletions, and will, therefore, include amino acid sequences that differ from the amino acid sequences designated in any one of SEQ ID Nos. 13-21, 98, and 115.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle comprises an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID Nos. 13-21, 98, and 115.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle consists essentially of an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID Nos. 13-21, 98, and 115.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle consists of an amino acid sequence that is at least 65%, at least 68%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID Nos. 13-21, 98, and 115.


In some embodiments, functional variants or modified forms of micro-dystrophin proteins optimized for skeletal muscle include fusion proteins having at least a portion of the micro-dystrophin protein and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. Other fusion domains are particularly useful for increasing protein stability.


The disclosure also provides nucleic acids encoding any of the micro-dystrophins optimized for skeletal muscle described herein. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making micro-dystrophins or as direct therapeutic agents in a gene therapy approach.


Non-limiting examples of nucleotide sequences encoding human micro-dystrophin proteins optimized for skeletal muscle are described below.










Nterm-R2_R16-R17_R24 to trCterm:



(SEQ ID NO: 22)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACACTGTCCGTGAAGAAACGATGAT





GGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCAC





AAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAA





GAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGAC





AGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGG





AAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGTGGCAGAGAAA





AATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTG





AGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAG





GCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTT





GGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCG





TCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCT





GTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTG





CTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGA





CTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTG





GACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGA





CCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATG





TTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACAT





TTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCT





CCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAA





GTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTG





GAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATG





TAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAA





GCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACA





TCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCC





CCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCC





Nterm-R2_R16-R17_H4 to end of syntrophin region:


(SEQ ID NO: 23)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACACTGTCCGTGAAGAAACGATGAT





GGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCAC





AAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAA





GAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGAC





AGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGG





AAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGGGTCCAGCATC





TCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCA





ACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAAT





GTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCT





GTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTA





ATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATG





TGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCAT





CATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTT





GTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTT





GGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCT





CTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAA





CTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCAC





TTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGT





GGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCA





AAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACT





CCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCA





TTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCT





CTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTG





AGCCAGCCTCGTAGT





Nterm-R2_R16-R17_H4 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 24)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACACTGTCCGTGAAGAAACGATGAT





GGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCAC





AAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAA





GAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGAC





AGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGG





AAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGGGTCCAGCATC





TCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCA





ACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAAT





GTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCT





GTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTA





ATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATG





TGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCAT





CATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTT





GTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTT





GGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCT





CTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAA





CTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCAC





TTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGT





GGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCA





AAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACT





CCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCA





TTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCT





CTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTG





AGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGC





AGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGT





CCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCC





Nterm-R2_R16-R17_R24 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 25)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACACTGTCCGTGAAGAAACGATGAT





GGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCAC





AAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAA





GAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGAC





AGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGG





AAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGTGGCAGAGAAA





AATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTG





AGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAG





GCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTT





GGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCG





TCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCT





GTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTG





CTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGA





CTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTG





GACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGA





CCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATG





TTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACAT





TTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCT





CCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAA





GTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTG





GAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATG





TAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAA





GCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACA





TCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCC





CCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCT





GGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCT





AGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGA





TGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCC





AGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGG





AATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGA





AATGATGCCCACCTCTCCCCAGAGTCCC





Nterm-R2_R23_R24 to end of dystrophin:


(SEQ ID NO: 26)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACCTTTCTCTGCAGGAACTTCTGGT





GTGGCTACAGCTGAAAGATGATGAATTAAGCCGGCAGGCACCTATTGGAGGCGACTTTCCAGCAGTTCAGAAGCAGA





ACGATGTACATAGGGCCTTCAAGAGGGAATTGAAAACTAAAGAACCTGTAATCATGAGTACTCTTGAGACTGTACGA





ATATTTCTGACAGAGCAGCCTTTGGAAGGACTAGAGAAACTCTACCAGGAGCCCAGAGAGCTGCCTCCTGAGGAGAG





AGCCCAGAATGTCACTCGGCTTCTACGAAAGCAGGCTGAGGAGGTCAATACTGAGTGGGAAAAATTGAACCTGCACT





CCGCTGACTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGAGCTGGAC





CTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGA





TCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTG





CTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGG





AAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCATCTCA





GCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACC





ACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTC





AGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTC





AGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATT





GTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGT





CTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCAT





TTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTG





ACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGG





GGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTT





CCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTG





CCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTT





AATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGA





ATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAA





GGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCC





GTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTC





ACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTC





CTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGC





CAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGA





TCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCC





CACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCCAAG





CTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGGAGTCACA





GTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGCACAACGGTGTCCTCTCCTT





CTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAGTGGTTGGCAGTCAAACTTCGGACTCCATG





GGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTC





CTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAGAGGACACAATG





Nterm-R2_R23_R24 to end of dystrophin:


(SEQ ID NO: 99)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACGCCAGTTCTGACCAGTGGAAGCGTCT





GCACCTTTCTCTGCAGGAACTTCTGGTGTGGCTACAGCTGAAAGATGATGAATTAAGCCGGCAGGCACCTATTGGAG





GCGACTTTCCAGCAGTTCAGAAGCAGAACGATGTACATAGGGCCTTCAAGAGGGAATTGAAAACTAAAGAACCTGTA





ATCATGAGTACTCTTGAGACTGTACGAATATTTCTGACAGAGCAGCCTTTGGAAGGACTAGAGAAACTCTACCAGGA





GCCCAGAGAGCTGCCTCCTGAGGAGAGAGCCCAGAATGTCACTCGGCTTCTACGAAAGCAGGCTGAGGAGGTCAATA





CTGAGTGGGAAAAATTGAACCTGCACTCCGCTGACTGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAA





CTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGG





CGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAG





AGAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGC





ACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGC





CCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGC





CAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTAC





CAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGC





CCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGC





CCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTC





AACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCG





TGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGC





AAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGA





CAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAA





TAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCC





TGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGA





TTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGG





CCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGG





TACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTC





TTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCC





TCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATG





GATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAA





AGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAG





AGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGC





AGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGG





GATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGA





AGACCACAATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAG





TGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAGTG





GTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTTAGA





GGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAGAGG





ACACAATG





Nterm-R2_R16-R17_H4 to end of dystrophin:


(SEQ ID NO: 27)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACACTGTCCGTGAAGAAACGATGAT





GGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCAC





AAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAA





GAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGAC





AGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGG





AAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGGGTCCAGCATC





TCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCA





ACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAAT





GTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCT





GTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTA





ATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATG





TGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCAT





CATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTT





GTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTT





GGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCT





CTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAA





CTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCAC





TTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGT





GGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCA





AAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACT





CCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCA





TTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCT





CTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTG





AGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGC





AGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGT





CCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCC





AAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGGAGTC





ACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGCACAACGGTGTCCTCTC





CTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAGTGGTTGGCAGTCAAACTTCGGACTCC





ATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAA





CTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAGAGGACACAATG





Nterm-R1_R16-R17_R24 to end of dystrophin:


(SEQ ID NO: 28)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCACACTGTCCGTGAAGAAACGATGATGGTGATG





ACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCACAAGCCCT





ATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAAGAGGAGT





CTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGACAGCAGCA





TTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGGAAAAAGT





TAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAA





AGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCT





AAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAAC





TGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGC





GGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGTGGCAGAGAAAAATAGAT





GAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTGAGGTGAT





CAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAGGCACTTC





GAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTTGGGCATT





CAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCGTCGAGGA





CCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGG





GTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTGCTGGGAC





CATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCAT





GAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGC





ACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTG





GAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGA





TACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAG





ACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTG





CATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCG





GAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCC





AGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATC





TGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTT





TTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAG





AAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATG





GGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGT





AGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGC





TAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACAT





TTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTT





GATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCTGC





AAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATG





CCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCT





GGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGG





AGCAACCCCAGGCAGAGGCCAAAGTGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGC





AGTCAGCCTATGCTGCTCCGAGTGGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCC





CCAGGACACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGAAGAAATA





CCCCTGGAAAGCCAATGAGAGAGGACACAATG





Nterm-R2_R16-R17_R24 to end of dystrophin:


(SEQ ID NO: 29)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACACTGTCCGTGAAGAAACGATGAT





GGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCAC





AAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAA





GAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGAC





AGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGG





AAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGTGGCAGAGAAA





AATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTG





AGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAG





GCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTT





GGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCG





TCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCT





GTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTG





CTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGA





CTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTG





GACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGA





CCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATG





TTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACAT





TTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCT





CCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAA





GTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTG





GAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATG





TAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAA





GCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACA





TCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCC





CCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCT





GGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCT





AGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGA





TGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCC





AGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGG





AATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGA





AATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACAAAG





GCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAG





CTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTC





CGACAGCAGTCAGCCTATGCTGCTCCGAGTGGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCA





GTCCTCCCCAGGACACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGA





AGAAATACCCCTGGAAAGCCAATGAGAGAGGACACAATG





Nterm-R2_R16-R17_R24 to end of syntrophin region:


(SEQ ID NO: 30)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCACACTGTCCGTGAAGAAACGATGAT





GGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCAC





AAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTGAAGATCTCTTTAAGCAA





GAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGAC





AGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGG





AAAAAGTTAACAAAATGTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTAT





GATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGA





ACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGA





ATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG





AATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAACAAAAGTGGCAGAGAAA





AATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCTGCGCCAAGCTG





AGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAGAAAGTCAAG





GCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTT





GGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCG





TCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTCCACGTCT





GTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACTTG





CTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGA





CTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTG





GACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGA





CCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATG





TTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGCACAT





TTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGCCT





CCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAA





GTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTG





GAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATG





TAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAA





GCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTACA





TCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGCATCC





CCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTTCT





GGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCT





AGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGA





TGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGT





Nterm-R2_R17_H3_R22_H4 to end of syntrophin region


(SEQ ID NO: 116)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTGAAGACAAGTCATTTGGCAGTTCATT





GATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGTATTATCGTGGCTTCTTTCTGCTGAGG





ACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTAC





ATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCTGATTGGAACAGGAAA





ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGAATGCCTCAGGGTAG





CTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAATGAC





TGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACG





CCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAATTCTCTCACTCACATGG





TGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTGGAAGAACAACTTAAGGTATTGGGAGATCGA





TGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGACCGATTTGACAGATCTGTTGAGAAATG





GCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAA





TTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCATTGGGCAGCGGCAAACT





GTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGA





AAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAAC





AAAAGCAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCTCTCCTACTCAGACTGTTACTCTGGTGACACAA





CCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGAAGAAAC





TCATAGATTACTGCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAACTGCCA





ATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAACAATGG





CAAGACCTCCAAGGTGAAATTGAAGCTCACACAGATGTTTATCACAACCTGGATGAAAACAGCCAAAAAATCCTGAG





ATCCCTGGAAGGTTCCGATGATGCAGTCCTGTTACAAAGACGTTTGGATAACATGAACTTCAAGTGGAGTGAACTTC





GGAAAAAGTCTCTCAACATTAGGTCCCATTTGGAAGCCAGTTCTGACCAGTGGAAGCGTCTGGGTCCAGCATCTCAG





CACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCCCTACTATATCAACCA





CGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTGACCTGAATAATGTCA





GATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCTGTCA





GCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTG





TTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTC





TGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATT





TCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTGA





CCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGCATCCTTTGGGG





GCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTC





CTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGC





CAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTA





ATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAA





TATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAG





GTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTCCCG





TTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTCA





CGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTCC





TAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCC





AGCCTCGTAGT






Non-limiting examples of codon optimized nucleotide sequences encoding human micro-dystrophin proteins optimized for skeletal muscle are described below.










Nterm-R2_R16-R17_R24 to trCterm:



(SEQ ID NO: 31)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACACCGTGCGGGAGGAGACCATGAT





GGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCC





AGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAG





GAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGAC





CGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGG





AGAAGGTGAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTAC





GACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGA





GCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGA





ACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTG





AACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGTGGCAGCGGAA





GATCGACGAGACCCTGGAGCGGCTGCAGGAGCTGCAGGAGGCCACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCG





AGGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGACCACCTGGAGAAGGTGAAG





GCCCTGCGGGGCGAGATCGCCCCCCTGAAGGAGAACGTGAGCCACGTGAACGACCTGGCCCGGCAGCTGACCACCCT





GGGCATCCAGCTGAGCCCCTACAACCTGAGCACCCTGGAGGACCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCG





TGGAGGACCGGGTGCGGCAGCTGCACGAGGCCCACCGGGACTTCGGCCCCGCCAGCCAGCACTTCCTGAGCACCAGC





GTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGAGACCCAGACCACCTG





CTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGCGGTTCAGCGCCTACCGGA





CCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGCGCCGCCTGCGACGCCCTG





GACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTGCCTGACCACCATCTACGA





CCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGAACTGGCTGCTGAACG





TGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCAC





CTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGACCAGCGGCGGCTGGGCCT





GCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCGGCAGCAACATCGAGCCCA





GCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTG





GAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGCCAAGCACCAGGCCAAGTG





CAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCAACTACGACATCTGCCAGA





GCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAGTACTGCACCCCCACCACC





AGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCGGTACTTCGCCAAGCACCC





CCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCC





Nterm-R2_R16-R17_H4 to end of syntrophin region:


(SEQ ID NO: 32)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACACCGTGCGGGAGGAGACCATGAT





GGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCC





AGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAG





GAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGAC





CGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGG





AGAAGGTGAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTAC





GACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGA





GCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGA





ACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTG





AACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGGGCCCCGCCAG





CCAGCACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCA





ACCACGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAAC





GTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCT





GAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCA





ACTGCCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATG





TGCCTGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCAT





CATCAGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCT





GCGACCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTC





GGCGGCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCT





GTTCCTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGA





CCGCCAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCAC





TTCAACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGT





GGAGTACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCA





AGCGGTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACC





CCCGTGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCA





CAGCCGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCA





GCCCCAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTG





AGCCAGCCCCGGAGC





Nterm-R2_R16-R17_H4 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 33)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACACCGTGCGGGAGGAGACCATGAT





GGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCC





AGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAG





GAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGAC





CGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGG





AGAAGGTGAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTAC





GACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGA





GCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGA





ACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTG





AACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGGGCCCCGCCAG





CCAGCACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCA





ACCACGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAAC





GTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCT





GAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCA





ACTGCCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATG





TGCCTGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCAT





CATCAGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCT





GCGACCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTC





GGCGGCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCT





GTTCCTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGA





CCGCCAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCAC





TTCAACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGT





GGAGTACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCA





AGCGGTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACC





CCCGTGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCA





CAGCCGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCA





GCCCCAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTG





AGCCAGCCCCGGAGCCCCGCCCAGATCCTGATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTGGC





CGACCTGGAGGAGGAGAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGA





GCCCCCTGCCCAGCCCCCCCGAGATGATGCCCACCAGCCCCCAGAGCCCC





Nterm-R2_R16-R17_R24 to 1st coiled coil and proline rich region that follows:


(SEQ ID NO: 34)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACACCGTGCGGGAGGAGACCATGAT





GGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCC





AGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAG





GAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGAC





CGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGG





AGAAGGTGAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTAC





GACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGA





GCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGA





ACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTG





AACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGTGGCAGCGGAA





GATCGACGAGACCCTGGAGCGGCTGCAGGAGCTGCAGGAGGCCACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCG





AGGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGACCACCTGGAGAAGGTGAAG





GCCCTGCGGGGCGAGATCGCCCCCCTGAAGGAGAACGTGAGCCACGTGAACGACCTGGCCCGGCAGCTGACCACCCT





GGGCATCCAGCTGAGCCCCTACAACCTGAGCACCCTGGAGGACCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCG





TGGAGGACCGGGTGCGGCAGCTGCACGAGGCCCACCGGGACTTCGGCCCCGCCAGCCAGCACTTCCTGAGCACCAGC





GTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGAGACCCAGACCACCTG





CTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGCGGTTCAGCGCCTACCGGA





CCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGCGCCGCCTGCGACGCCCTG





GACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTGCCTGACCACCATCTACGA





CCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGAACTGGCTGCTGAACG





TGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCAC





CTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGACCAGCGGCGGCTGGGCCT





GCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCGGCAGCAACATCGAGCCCA





GCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTG





GAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGCCAAGCACCAGGCCAAGTG





CAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCAACTACGACATCTGCCAGA





GCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAGTACTGCACCCCCACCACC





AGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCGGTACTTCGCCAAGCACCC





CCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCCGTGACCCTGATCAACTTCT





GGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAGCCGGATCGAGCACTACGCC





AGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCCCCAACGAGAGCATCGACGA





CGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGCCAGCCCCGGAGCCCCGCCC





AGATCCTGATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTGGCCGACCTGGAGGAGGAGAACCGG





AACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCCAGCCCCCCCGA





GATGATGCCCACCAGCCCCCAGAGCCCC





Nterm-R2_R23_R24 to end of dystrophin:


(SEQ ID NO: 35)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACCTGAGCCTGCAGGAGCTGCTGGT





GTGGCTGCAGCTGAAGGACGACGAGCTGAGCCGGCAGGCCCCCATCGGCGGCGACTTCCCCGCCGTGCAGAAGCAGA





ACGACGTGCACCGGGCCTTCAAGCGGGAGCTGAAGACCAAGGAGCCCGTGATCATGAGCACCCTGGAGACCGTGCGG





ATCTTCCTGACCGAGCAGCCCCTGGAGGGCCTGGAGAAGCTGTACCAGGAGCCCCGGGAGCTGCCCCCCGAGGAGCG





GGCCCAGAACGTGACCCGGCTGCTGCGGAAGCAGGCCGAGGAGGTGAACACCGAGTGGGAGAAGCTGAACCTGCACA





GCGCCGACTGGCAGCGGAAGATCGACGAGACCCTGGAGCGGCTGCAGGAGCTGCAGGAGGCCACCGACGAGCTGGAC





CTGAAGCTGCGGCAGGCCGAGGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGA





CCACCTGGAGAAGGTGAAGGCCCTGCGGGGCGAGATCGCCCCCCTGAAGGAGAACGTGAGCCACGTGAACGACCTGG





CCCGGCAGCTGACCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGAGCACCCTGGAGGACCTGAACACCCGGTGG





AAGCTGCTGCAGGTGGCCGTGGAGGACCGGGTGCGGCAGCTGCACGAGGCCCACCGGGACTTCGGCCCCGCCAGCCA





GCACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACC





ACGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTG





CGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAG





CGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACT





GCCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGC





CTGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCAT





CAGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCG





ACCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGC





GGCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTT





CCTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCG





CCAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTC





AACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGA





GTACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGC





GGTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCC





GTGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAG





CCGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCC





CCAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGC





CAGCCCCGGAGCCCCGCCCAGATCCTGATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTGGCCGA





CCTGGAGGAGGAGAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGAGCC





CCCTGCCCAGCCCCCCCGAGATGATGCCCACCAGCCCCCAGAGCCCCCGGGACGCCGAGCTGATCGCCGAGGCCAAG





CTGCTGCGGCAGCACAAGGGCCGGCTGGAGGCCCGGATGCAGATCCTGGAGGACCACAACAAGCAGCTGGAGAGCCA





GCTGCACCGGCTGCGGCAGCTGCTGGAGCAGCCCCAGGCCGAGGCCAAGGTGAACGGCACCACCGTGAGCAGCCCCA





GCACCAGCCTGCAGCGGAGCGACAGCAGCCAGCCCATGCTGCTGCGGGTGGTGGGCAGCCAGACCAGCGACAGCATG





GGCGAGGAGGACCTGCTGAGCCCCCCCCAGGACACCAGCACCGGCCTGGAGGAGGTGATGGAGCAGCTGAACAACAG





CTTCCCCAGCAGCCGGGGCCGGAACACCCCCGGCAAGCCCATGCGGGAGGACACCATG





Nterm-R2_R23_R24 to end of dystrophin:


(SEQ ID NO: 118)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACGCCAGCAGCGACCAGTGGAAGCGGCT





GCACCTGAGCCTGCAGGAGCTGCTGGTGTGGCTGCAGCTGAAGGACGACGAGCTGAGCCGGCAGGCCCCCATCGGCG





GCGACTTCCCCGCCGTGCAGAAGCAGAACGACGTGCACCGGGCCTTCAAGCGGGAGCTGAAGACCAAGGAGCCCGTG





ATCATGAGCACCCTGGAGACCGTGCGGATCTTCCTGACCGAGCAGCCCCTGGAGGGCCTGGAGAAGCTGTACCAGGA





GCCCCGGGAGCTGCCCCCCGAGGAGCGGGCCCAGAACGTGACCCGGCTGCTGCGGAAGCAGGCCGAGGAGGTGAACA





CCGAGTGGGAGAAGCTGAACCTGCACAGCGCCGACTGGCAGCGGAAGATCGACGAGACCCTGGAGCGGCTGCAGGAG





CTGCAGGAGGCCACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCGAGGTGATCAAGGGCAGCTGGCAGCCCGTGGG





CGACCTGCTGATCGACAGCCTGCAGGACCACCTGGAGAAGGTGAAGGCCCTGCGGGGCGAGATCGCCCCCCTGAAGG





AGAACGTGAGCCACGTGAACGACCTGGCCCGGCAGCTGACCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGAGC





ACCCTGGAGGACCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCGTGGAGGACCGGGTGCGGCAGCTGCACGAGGC





CCACCGGGACTTCGGCCCCGCCAGCCAGCACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCC





CCAACAAGGTGCCCTACTACATCAACCACGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTAC





CAGAGCCTGGCCGACCTGAACAACGTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGC





CCTGTGCCTGGACCTGCTGAGCCTGAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGC





CCATGGACATCCTGCAGATCATCAACTGCCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTG





AACGTGCCCCTGTGCGTGGACATGTGCCTGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCG





GGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGC





AGGTGGCCAGCAGCACCGGCTTCTGCGACCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGG





CAGCTGGGCGAGGTGGCCAGCTTCGGCGGCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAA





CAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGC





TGCACCGGGTGGCCGCCGCCGAGACCGCCAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGC





TTCCGGTACCGGAGCCTGAAGCACTTCAACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGG





CCACAAGATGCACTACCCCATGGTGGAGTACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGG





TGCTGAAGAACAAGTTCCGGACCAAGCGGTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTG





CTGGAGGGCGACAACATGGAGACCCCCGTGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCC





CCAGCTGAGCCACGACGACACCCACAGCCGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACG





GCAGCTACCTGAACGACAGCATCAGCCCCAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAG





AGCCTGAACCAGGACAGCCCCCTGAGCCAGCCCCGGAGCCCCGCCCAGATCCTGATCAGCCTGGAGAGCGAGGAGCG





GGGCGAGCTGGAGCGGATCCTGGCCGACCTGGAGGAGGAGAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGC





AGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCCAGCCCCCCCGAGATGATGCCCACCAGCCCCCAGAGCCCCCGG





GACGCCGAGCTGATCGCCGAGGCCAAGCTGCTGCGGCAGCACAAGGGCCGGCTGGAGGCCCGGATGCAGATCCTGGA





GGACCACAACAAGCAGCTGGAGAGCCAGCTGCACCGGCTGCGGCAGCTGCTGGAGCAGCCCCAGGCCGAGGCCAAGG





TGAACGGCACCACCGTGAGCAGCCCCAGCACCAGCCTGCAGCGGAGCGACAGCAGCCAGCCCATGCTGCTGCGGGTG





GTGGGCAGCCAGACCAGCGACAGCATGGGCGAGGAGGACCTGCTGAGCCCCCCCCAGGACACCAGCACCGGCCTGGA





GGAGGTGATGGAGCAGCTGAACAACAGCTTCCCCAGCAGCCGGGGCCGGAACACCCCCGGCAAGCCCATGCGGGAGG





ACACCATG





Nterm-R2_R16-R17_H4 to end of dystrophin:


(SEQ ID NO: 36)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACACCGTGCGGGAGGAGACCATGAT





GGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCC





AGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAG





GAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGAC





CGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGG





AGAAGGTGAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTAC





GACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGA





GCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGA





ACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTG





AACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGGGCCCCGCCAG





CCAGCACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCA





ACCACGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAAC





GTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCT





GAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCA





ACTGCCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATG





TGCCTGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCAT





CATCAGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCT





GCGACCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTC





GGCGGCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCT





GTTCCTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGA





CCGCCAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCAC





TTCAACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGT





GGAGTACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCA





AGCGGTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACC





CCCGTGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCA





CAGCCGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCA





GCCCCAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTG





AGCCAGCCCCGGAGCCCCGCCCAGATCCTGATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTGGC





CGACCTGGAGGAGGAGAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGA





GCCCCCTGCCCAGCCCCCCCGAGATGATGCCCACCAGCCCCCAGAGCCCCCGGGACGCCGAGCTGATCGCCGAGGCC





AAGCTGCTGCGGCAGCACAAGGGCCGGCTGGAGGCCCGGATGCAGATCCTGGAGGACCACAACAAGCAGCTGGAGAG





CCAGCTGCACCGGCTGCGGCAGCTGCTGGAGCAGCCCCAGGCCGAGGCCAAGGTGAACGGCACCACCGTGAGCAGCC





CCAGCACCAGCCTGCAGCGGAGCGACAGCAGCCAGCCCATGCTGCTGCGGGTGGTGGGCAGCCAGACCAGCGACAGC





ATGGGCGAGGAGGACCTGCTGAGCCCCCCCCAGGACACCAGCACCGGCCTGGAGGAGGTGATGGAGCAGCTGAACAA





CAGCTTCCCCAGCAGCCGGGGCCGGAACACCCCCGGCAAGCCCATGCGGGAGGACACCATG





Nterm-R1_R16-R17_R24 to end of dystrophin:


(SEQ ID NO: 37)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCACACCGTGCGGGAGGAGACCATGATGGTGATG





ACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCCAGGCCCT





GCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAGGAGGAGA





GCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGACCGCCGCC





CTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGGAGAAGGT





GAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTACGACATCA





AGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGAGCACGCC





AAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGAACGCCAC





CGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTGAACCTGC





GGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGTGGCAGCGGAAGATCGAC





GAGACCCTGGAGCGGCTGCAGGAGCTGCAGGAGGCCACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCGAGGTGAT





CAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGACCACCTGGAGAAGGTGAAGGCCCTGC





GGGGCGAGATCGCCCCCCTGAAGGAGAACGTGAGCCACGTGAACGACCTGGCCCGGCAGCTGACCACCCTGGGCATC





CAGCTGAGCCCCTACAACCTGAGCACCCTGGAGGACCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCGTGGAGGA





CCGGGTGCGGCAGCTGCACGAGGCCCACCGGGACTTCGGCCCCGCCAGCCAGCACTTCCTGAGCACCAGCGTGCAGG





GCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGAGACCCAGACCACCTGCTGGGAC





CACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGCGGTTCAGCGCCTACCGGACCGCCAT





GAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGCGCCGCCTGCGACGCCCTGGACCAGC





ACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTGCCTGACCACCATCTACGACCGGCTG





GAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGAACTGGCTGCTGAACGTGTACGA





CACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCACCTGGAGG





ACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGACCAGCGGCGGCTGGGCCTGCTGCTG





CACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCGGCAGCAACATCGAGCCCAGCGTGCG





GAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTGGAGCCCC





AGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGCCAAGCACCAGGCCAAGTGCAACATC





TGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCAACTACGACATCTGCCAGAGCTGCTT





CTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAGTACTGCACCCCCACCACCAGCGGCG





AGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCGGTACTTCGCCAAGCACCCCCGGATG





GGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCCGTGACCCTGATCAACTTCTGGCCCGT





GGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAGCCGGATCGAGCACTACGCCAGCCGGC





TGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCCCCAACGAGAGCATCGACGACGAGCAC





CTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGCCAGCCCCGGAGCCCCGCCCAGATCCT





GATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTGGCCGACCTGGAGGAGGAGAACCGGAACCTGC





AGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCCAGCCCCCCCGAGATGATG





CCCACCAGCCCCCAGAGCCCCCGGGACGCCGAGCTGATCGCCGAGGCCAAGCTGCTGCGGCAGCACAAGGGCCGGCT





GGAGGCCCGGATGCAGATCCTGGAGGACCACAACAAGCAGCTGGAGAGCCAGCTGCACCGGCTGCGGCAGCTGCTGG





AGCAGCCCCAGGCCGAGGCCAAGGTGAACGGCACCACCGTGAGCAGCCCCAGCACCAGCCTGCAGCGGAGCGACAGC





AGCCAGCCCATGCTGCTGCGGGTGGTGGGCAGCCAGACCAGCGACAGCATGGGCGAGGAGGACCTGCTGAGCCCCCC





CCAGGACACCAGCACCGGCCTGGAGGAGGTGATGGAGCAGCTGAACAACAGCTTCCCCAGCAGCCGGGGCCGGAACA





CCCCCGGCAAGCCCATGCGGGAGGACACCATG





Nterm-R2_R16-R17_R24 to end of dystrophin:


(SEQ ID NO: 38)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACACCGTGCGGGAGGAGACCATGAT





GGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCC





AGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAG





GAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGAC





CGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGG





AGAAGGTGAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTAC





GACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGA





GCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGA





ACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTG





AACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGTGGCAGCGGAA





GATCGACGAGACCCTGGAGCGGCTGCAGGAGCTGCAGGAGGCCACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCG





AGGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGACCACCTGGAGAAGGTGAAG





GCCCTGCGGGGCGAGATCGCCCCCCTGAAGGAGAACGTGAGCCACGTGAACGACCTGGCCCGGCAGCTGACCACCCT





GGGCATCCAGCTGAGCCCCTACAACCTGAGCACCCTGGAGGACCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCG





TGGAGGACCGGGTGCGGCAGCTGCACGAGGCCCACCGGGACTTCGGCCCCGCCAGCCAGCACTTCCTGAGCACCAGC





GTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGAGACCCAGACCACCTG





CTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGCGGTTCAGCGCCTACCGGA





CCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGCGCCGCCTGCGACGCCCTG





GACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTGCCTGACCACCATCTACGA





CCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGAACTGGCTGCTGAACG





TGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCAC





CTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGACCAGCGGCGGCTGGGCCT





GCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCGGCAGCAACATCGAGCCCA





GCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTG





GAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGCCAAGCACCAGGCCAAGTG





CAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCAACTACGACATCTGCCAGA





GCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAGTACTGCACCCCCACCACC





AGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCGGTACTTCGCCAAGCACCC





CCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCCGTGACCCTGATCAACTTCT





GGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAGCCGGATCGAGCACTACGCC





AGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCCCCAACGAGAGCATCGACGA





CGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGCCAGCCCCGGAGCCCCGCCC





AGATCCTGATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTGGCCGACCTGGAGGAGGAGAACCGG





AACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCCAGCCCCCCCGA





GATGATGCCCACCAGCCCCCAGAGCCCCCGGGACGCCGAGCTGATCGCCGAGGCCAAGCTGCTGCGGCAGCACAAGG





GCCGGCTGGAGGCCCGGATGCAGATCCTGGAGGACCACAACAAGCAGCTGGAGAGCCAGCTGCACCGGCTGCGGCAG





CTGCTGGAGCAGCCCCAGGCCGAGGCCAAGGTGAACGGCACCACCGTGAGCAGCCCCAGCACCAGCCTGCAGCGGAG





CGACAGCAGCCAGCCCATGCTGCTGCGGGTGGTGGGCAGCCAGACCAGCGACAGCATGGGCGAGGAGGACCTGCTGA





GCCCCCCCCAGGACACCAGCACCGGCCTGGAGGAGGTGATGGAGCAGCTGAACAACAGCTTCCCCAGCAGCCGGGGC





CGGAACACCCCCGGCAAGCCCATGCGGGAGGACACCATG





Nterm-R2_R16-R17_R24 to end of syntrophin region:


(SEQ ID NO: 39)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCACACCGTGCGGGAGGAGACCATGAT





GGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGAGCC





AGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGACCTGTTCAAGCAG





GAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAGAC





CGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGCCCTGAGCCAGCTGGACTTCCAGTGGG





AGAAGGTGAACAAGATGTACAAGGACCGGCAGGGCCGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTAC





GACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGA





GCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGA





ACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTG





AACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGCAGAAGTGGCAGCGGAA





GATCGACGAGACCCTGGAGCGGCTGCAGGAGCTGCAGGAGGCCACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCG





AGGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGACCACCTGGAGAAGGTGAAG





GCCCTGCGGGGCGAGATCGCCCCCCTGAAGGAGAACGTGAGCCACGTGAACGACCTGGCCCGGCAGCTGACCACCCT





GGGCATCCAGCTGAGCCCCTACAACCTGAGCACCCTGGAGGACCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCG





TGGAGGACCGGGTGCGGCAGCTGCACGAGGCCCACCGGGACTTCGGCCCCGCCAGCCAGCACTTCCTGAGCACCAGC





GTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGAGACCCAGACCACCTG





CTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGCGGTTCAGCGCCTACCGGA





CCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGCGCCGCCTGCGACGCCCTG





GACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTGCCTGACCACCATCTACGA





CCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGAACTGGCTGCTGAACG





TGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCAC





CTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGACCAGCGGCGGCTGGGCCT





GCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCGGCAGCAACATCGAGCCCA





GCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTG





GAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGCCAAGCACCAGGCCAAGTG





CAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCAACTACGACATCTGCCAGA





GCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAGTACTGCACCCCCACCACC





AGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCGGTACTTCGCCAAGCACCC





CCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCCGTGACCCTGATCAACTTCT





GGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAGCCGGATCGAGCACTACGCC





AGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCCCCAACGAGAGCATCGACGA





CGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGCCAGCCCCGGAGC





Nterm-R2_R17_H3_R22_H4 to end of syntrophin region:


(SEQ ID NO: 117)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCCGAGGACAAGAGCTTCGGCAGCAGCCT





GATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGTGCTGAGCTGGCTGCTGAGCGCCGAGG





ACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGGACCAGTTCCACACCCACGAGGGCTAC





ATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTGATCGGCACCGGCAA





GCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAACAGCCGGTGGGAGTGCCTGCGGGTGG





CCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGACCTGCAGAACCAGAAGCTGAAGGAGCTGAACGAC





TGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGAGCCCCTGGGCCCCGACCTGGAGGACCTGAAGCG





GCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGGAGCAGGTGCGGGTGAACAGCCTGACCCACATGG





TGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCCCTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGG





TGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCTGCAGGACCGGTTCGACCGGAGCGTGGAGAAGTG





GCGGCGGTTCCACTACGACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGCGGAAGACCCAGA





TCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGCAGGACGGCATCGGCCAGCGGCAGACC





GTGGTGCGGACCCTGAACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAGGA





GAAGCTGGGCAGCCTGAACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAGCGACCGGAAGAAGCGGCTGGAGGAGC





AGAAGCAGCCCGACCTGGCCCCCGGCCTGACCACCATCGGCGCCAGCCCCACCCAGACCGTGACCCTGGTGACCCAG





CCCGTGGTGACCAAGGAGACCGCCATCAGCAAGCTGGAGATGCCCAGCAGCCTGATGCTGGAGGTGCCCGAGGAGAC





CCACCGGCTGCTGCAGCAGTTCCCCCTGGACCTGGAGAAGTTCCTGGCCTGGCTGACCGAGGCCGAGACCACCGCCA





ACGTGCTGCAGGACGCCACCCGGAAGGAGCGGCTGCTGGAGGACAGCAAGGGCGTGAAGGAGCTGATGAAGCAGTGG





CAGGACCTGCAGGGCGAGATCGAGGCCCACACCGACGTGTACCACAACCTGGACGAGAACAGCCAGAAGATCCTGCG





GAGCCTGGAGGGCAGCGACGACGCCGTGCTGCTGCAGCGGCGGCTGGACAACATGAACTTCAAGTGGAGCGAGCTGC





GGAAGAAGAGCCTGAACATCCGGAGCCACCTGGAGGCCAGCAGCGACCAGTGGAAGCGGCTGGGCCCCGCCAGCCAG





CACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCA





CGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAACGTGC





GGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGACCTGCTGAGCCTGAGC





GCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCTGCAGATCATCAACTG





CCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGTGCGTGGACATGTGCC





TGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTCAAGACCGGCATCATC





AGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAGCACCGGCTTCTGCGA





CCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGGTGGCCAGCTTCGGCG





GCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTC





CTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGCCGCCGCCGAGACCGC





CAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGAGCCTGAAGCACTTCA





ACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCACTACCCCATGGTGGAG





TACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAAGTTCCGGACCAAGCG





GTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACAACATGGAGACCCCCG





TGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAGC





CGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCCC





CAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGCC





AGCCCCGGAGC






A nucleotide sequence encoding a micro-dystrophin protein optimized for skeletal muscle is further understood to include nucleotide sequences that are variants of any one of SEQ ID Nos. 22-39, 99, and 116-118. Variant nucleotide sequences include sequences that differ by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotide substitutions, additions or deletions, such as allelic variants, and will, therefore, include coding sequences that differ from the nucleotide sequence of the coding sequence designated in any one of SEQ ID Nos. 22-39, 99, and 116-118.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle is encoded by a nucleic acid comprising a nucleotide sequence that is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 22-39, 99, and 116-118.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle is encoded by a nucleic acid consisting essentially of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 22-39, 99, and 116-118.


In some embodiments, a micro-dystrophin protein optimized for skeletal muscle is encoded by a nucleic acid consisting of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 22-39, 99, and 116-118.


In some embodiments, it is desirable to overexpress micro-dystrophin proteins in skeletal muscle. Thus, in some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for skeletal muscle may be codon optimized for higher expression. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) are substituted.


The “percent identity” of two amino acid sequences or nucleic acid sequences may be determined by any method known in the art.


In some embodiments, the percent identity of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12, to obtain guide sequences homologous to a target nucleic acid. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.


For the purposes of comparing two or more amino acid sequences, the percentage of “sequence identity” between a first amino acid sequence and a second amino acid sequence (also referred to herein as “amino acid identity”) may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence—compared to the first amino acid sequence—is considered as a difference at a single amino acid residue (position), i.e., as an “amino acid difference” as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm (e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. (1970) 48:443, by the search for similarity method of Pearson and Lipman. Proc. Natl. Acad. Sci. USA (1998) 85:2444, or by computerized implementations of algorithms available as Blast, Clustal Omega, or other sequence alignment algorithms) and, for example, using standard settings. Usually, for the purpose of determining the percentage of “sequence identity” between two amino acid sequences in accordance with the calculation method outlined hereinabove, the amino acid sequence with the greatest number of amino acid residues will be taken as the “first” amino acid sequence, and the other amino acid sequence will be taken as the “second” amino acid sequence.


Recombinant Nucleic Acids

The disclosure provides recombinant nucleic acids comprising a nucleotide sequence encoding a micro-dystrophin protein of the disclosure. A recombinant nucleic acid is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) from multiple sources.


A recombinant nucleic acid may comprise DNA (e.g., genomic DNA, cDNA or a combination of genomic DNA and cDNA), RNA or a hybrid molecule, for example, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine.


Recombinant nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, nucleic acids are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D. G. et al. Nature Methods, 343-345, 2009; and Gibson, D. G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5′ exonuclease, the 3′extension activity of a DNA polymerase and DNA ligase activity. The 5′ exonuclease activity chews back the 5′ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed domains. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. Other methods of producing engineered nucleic acids may be used in accordance with the present disclosure.


Expression of the micro-dystrophin protein may be controlled using one or more regulatory sequences such as enhancers and promoters, operably linked to the nucleotide sequences encoding the micro-dystrophin protein.


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


Promoters that may be used in accordance with the present disclosure may comprise any suitable promoter that can drive the expression of the nucleotide sequences encoding the micro-dystrophin proteins.


In some embodiments, a promoter is naturally associated with dystrophin, and may be obtained by isolating the 5′ non-coding sequence upstream of the coding segment and/or exon of dystrophin. Such a promoter may be referred to as an endogenous promoter or a native promoter.


In some embodiments, the promoter is a chimeric promoter comprising sequence elements from two or more different promoters.


In some embodiments, the promoter is a constitutively active promoter. Constitutive promoters include any constitutive promoter described herein or known to one of ordinary skill in the art. Non-limiting examples of constitutive promoters include the immediate early cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as mammalian gene promoters such as, but not limited to, the elongation Factor-la (EF-1a) the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.


Inducible promoters are also contemplated herein. An “inducible promoter” refers to a promoter that is characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by an inducer signal.


In some embodiments, the promoter is a tissue-specific promoter. A “tissue-specific promoter”, as used herein, refers to promoters that preferentially or selectively function in a specific type of tissue. In some embodiments, a tissue-specific promoter is not able to drive the expression of the genes in other types of tissues. In some embodiments, a promoter that may be used in accordance with the present disclosure is a skeletal muscle-specific promoter. In some embodiments, the skeletal muscle-specific promoter is the CK6 promoter, CK8 promoter, or skeletal α-actin promoter. In some embodiments, the skeletal muscle-specific promoter comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80. In some embodiments, a promoter that may be used in accordance with the present disclosure is a cardiac-specific promoter. In some embodiments, the cardiac-specific promoter is the cardiac troponin C promoter, the cardiac troponin I promoter, or the cardiac troponin T (cTnT) promoter. In some embodiments, the cardiac-specific promoter is the cTnT promoter.


In some embodiments, the engineered nucleic acids of the present disclosure further comprise one or more enhancer elements. In some embodiments, an enhancer element is a skeletal muscle alpha-actin enhancer. In some embodiments, the promoter is a skeletal muscle-specific promoter with skeletal muscle alpha-actin enhancer elements.


In some embodiments, the recombinant nucleic acids of the present disclosure further comprise additional regulatory sequences, including, without limitation, a 3′ untranslated region (3′UTR), and/or a poly-adenylation (polyA) signal sequence.


In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle is operably linked to a cardiac-specific promoter. In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for skeletal muscle is operably linked to a skeletal muscle-specific promoter. In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle is operably linked to a weaker promoter. In some embodiments, the expression of a micro-dystrophin protein optimized for cardiac muscle is 5%, 10%, 155, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower than the expression of a micro-dystrophin protein optimized for skeletal muscle.


Skeletal Muscle-Specific Promoter

In one aspect, the present disclosure provides minimal skeletal muscle-specific promoters based on the skeletal alpha-actin promoter. In some embodiments, a skeletal muscle-specific promoter of the disclosure does not comprise any element that mediates a response in cardiac muscle.


In some embodiments, a skeletal muscle-specific promoter of the disclosure is less than about 600 bp in length. In some embodiments, the skeletal muscle-specific promoter is less than about 550 bp in length. In some embodiments, the skeletal muscle-specific promoter is less than about 500 bp in length. In some embodiments, the skeletal muscle-specific promoter is less than about 400 bp in length.


In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises one or more (e.g., 1, 2, 3, or more) copies of the skeletal muscle alpha-actin enhancer. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises one or more (e.g., 1, 2, 3, or more) copies of an enhancer that comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to nucleotides 8-106 of SEQ ID NO: 76. In some embodiments, the skeletal alpha-actin enhancer comprises nucleotides 8-106 of SEQ ID NO: 76. In some embodiments, the skeletal alpha-actin enhancer consists essentially of nucleotides 8-106 of SEQ ID NO: 76. In some embodiments, the skeletal alpha-actin enhancer consists of nucleotides 8-106 of SEQ ID NO: 76. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises one or more (e.g., 1, 2, 3, or more) copies of an enhancer that comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 76. In some embodiments, the skeletal alpha-actin enhancer comprises SEQ ID NO: 76. In some embodiments, the skeletal alpha-actin enhancer consists essentially of SEQ ID NO: 76. In some embodiments, the skeletal alpha-actin enhancer consists of SEQ ID NO: 76.


In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises two copies of the skeletal muscle alpha-actin enhancer. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to nucleotides 8-205 of SEQ ID NO: 79. In some embodiments, the skeletal alpha-actin enhancer comprises nucleotides 8-205 of SEQ ID NO: 79. In some embodiments, the skeletal alpha-actin enhancer consists essentially of nucleotides 8-205 of SEQ ID NO: 79. In some embodiments, the skeletal alpha-actin enhancer consists of nucleotides 8-205 of SEQ ID NO: 79. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises an enhancer that comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 79. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises an enhancer that consists essentially of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 79. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises an enhancer that consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 79.


In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises a core promoter sequence that comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to nucleotides 1-274 of SEQ ID NO: 77. In some embodiments, the core promoter sequence comprises nucleotides 1-274 of SEQ ID NO: 77. In some embodiments, the core promoter sequence consists essentially of nucleotides 1-274 of SEQ ID NO: 77. In some embodiments, the core promoter sequence consists of nucleotides 1-274 of SEQ ID NO: 77. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises a core promoter sequence plus Kozak consensus sequence that comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 77. In some embodiments, the core promoter sequence plus Kozak consensus sequence comprises SEQ ID NO: 77. In some embodiments, the core promoter sequence plus a Kozak consensus sequence consists essentially of SEQ ID NO: 77. In some embodiments, the core promoter sequence plus a Kozak consensus sequence consists of SEQ ID NO: 77.


In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80. In some embodiments, a skeletal muscle-specific promoter of the disclosure consists essentially of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80. In some embodiments, a skeletal muscle-specific promoter of the disclosure consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80. In some embodiments, a skeletal muscle-specific promoter of the disclosure comprises the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80. In some embodiments, a skeletal muscle-specific promoter of the disclosure consists essentially of the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80. In some embodiments, a skeletal muscle-specific promoter of the disclosure consists of the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80.


A skeletal muscle-specific promoter of the disclosure may be used to express any gene of interest in skeletal muscle. Thus, a skeletal muscle-specific promoter of the disclosure may be operably linked to any gene of interest, to express the gene of interest in skeletal muscle. In some embodiments, the gene of interest is a micro-dystrophin gene of the disclosure.


A skeletal muscle-specific promoter of the disclosure may be used may be used for gene therapy for skeletal muscle disorders. In some embodiments, the skeletal muscle disorder is a muscular dystrophy. In some embodiments, the muscular dystrophy is DMD.


Vectors

In some aspects, the present disclosure provides vectors comprising the recombinant nucleic acids described herein. A vector is any nucleic acid that may be used as a vehicle to deliver exogenous (foreign) genetic material to a cell. A vector, in some embodiments, is a DNA sequence that includes an insert (e.g., a nucleotide sequence encoding a micro-dystrophin protein and a larger sequence that serves as the backbone of the vector. Non-limiting examples of vectors include plasmids, viruses/viral vectors, phagemids, cosmids (comprising a plasmid and Lambda phage cos sequences), and artificial chromosomes, any of which may be used as provided herein. In some embodiments, the vector is a viral vector, such as a viral particle. In some embodiments, the viral vector is an adenovirus, adeno associated virus (AAV), 7-retrovirus, HSV, lentivirus, or Sendai virus vector. In some embodiments, the viral vector is an recombinant AAV (rAAV) vector.


In some embodiments, a nucleic acid of the disclosure is flanked by AAV ITRs for packaging into an rAAV vector. The phrase “rAAV vector” can include a rAAV genome comprising the gene of interest flanked by AAV ITRs, and an rAAV particle comprising an rAAV genome encapsidated with rAAV capsid proteins.


The ITR sequences may be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or may be derived from more than one serotype. In some embodiments, the ITR sequences are the same serotype as the capsid (e.g., AAV8 ITR sequences and AAV8 capsid, etc.). In some embodiments, the ITR sequences are of a different serotype from the capsid.


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


Further provided herein are rAAV viral particles or rAAV preparations containing such particles. The rAAV particles comprise a viral capsid and an rAAV genome comprising the gene of interest flanked by AAV ITRs, which is encapsidated by the viral capsid. Methods of producing rAAV particles are known in the art and are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Application Publication Numbers US 2007/0015238 and US 2012/0322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing the rAAV genome comprising the gene of interest flanked by AAV ITRs may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP3 region as described herein), and transfected into a producer cell line such that the rAAV particle can be packaged and subsequently purified.


The rAAV particles or particles within an rAAV preparation disclosed herein, may be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). As used herein, the serotype of an rAAV an rAAV particle refers to the serotype of the capsid proteins of the recombinant virus. Non-limiting examples of derivatives and pseudotypes include AAVrh.10, AAVrh.74, AAV2/1, AAV2/5, AAV2/6, AAV2/8, AAV2/9, AAV2-AAV3 hybrid, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives/pseudotypes, and methods of producing such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan. 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A1, Schaffer D V, Samulski R J). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001). In some embodiments, the capsid of any of the herein disclosed rAAV particles is of the serotype AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAVrh.74, AAVrh.10, AAV2/6 or AAV9.


In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle and a nucleotide sequence encoding a micro-dystrophin protein optimized for skeletal muscle are packaged in rAAV particles of the same serotype.


In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle and a nucleotide sequence encoding a micro-dystrophin protein optimized for skeletal muscle are packaged in rAAV particles of different serotypes.


In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle is packaged in an rAAV particle of the serotype AAV1, AAV8, AAV9, AAVrh.74, or AAVrh.10. In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for cardiac muscle is packaged in an rAAV particle of the serotype AAV9, AAVrh.74, or AAVrh.10. In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for skeletal muscle is packaged in an rAAV particle of the serotype AAV1, AAV6, AAV7, AAV8, or AAV9. In some embodiments, a nucleotide sequence encoding a micro-dystrophin protein optimized for skeletal muscle is packaged in an rAAV particle of the serotype AAV8.


In some aspects, the methods described herein comprise expressing a micro-dystrophin protein in cardiac or skeletal muscle. The vectors provided herein may be used for gene therapy for treating skeletal muscle disorders in a subject in need thereof. In some embodiments, the vectors provided herein may be used for gene therapy for treating a muscular dystrophy (e.g., DMD) in a subject in need thereof.


Methods of Use

In some aspects, the present disclosure provides methods of treating DMD.


In one aspect, the present disclosure provides a method of treating DMD in a subject in need thereof, comprising administering to the subject an effective amount of a micro-dystrophin protein of the disclosure.


In another aspect, the present disclosure provides a gene therapy for treating DMD in a subject in need thereof. Accordingly, the present disclosure provides a method of treating DMD in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid molecule encoding a micro-dystrophin protein.


In some embodiments, the present disclosure provides a method of treating DMD in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid molecule encoding a micro-dystrophin protein optimized for cardiac muscle and an effective amount of a nucleic acid molecule encoding a micro-dystrophin protein optimized for skeletal muscle.


In some embodiments, the present disclosure provides a method of treating DMD in a subject in need thereof, comprising administering to the subject an effective amount of a nucleic acid molecule encoding a micro-dystrophin protein optimized for cardiac muscle to cardiac muscle and an effective amount of a nucleic acid molecule encoding a micro-dystrophin protein optimized for skeletal muscle to skeletal muscle.


In some embodiments, the present disclosure provides a method of treating DMD in a subject in need thereof, the method comprising delivering to the subject a first gene therapy vector (e.g., a first rAAV particle) comprising a nucleotide sequence encoding a first micro-dystrophin protein to cardiac muscle and a second gene therapy vector (e.g., a second rAAV particle) comprising a nucleotide sequence encoding a second micro-dystrophin to skeletal muscle.


The present disclosure thus contemplates methods of expressing one or more micro-dystrophin proteins in a subject for treating DMD, the method comprising administering to a subject in need thereof an effective amount of one or more nucleic acids of the disclosure.


In some embodiments, additional gene therapy approaches are combined with micro-dystrophin.


The terms “subject,” and “patient,” are used interchangeably herein. In some embodiments, a subject is a mammal, such as a human, a nonhuman primate, a dog, a cat, a horse, a sheep, a poultry, a cow, a pig, a mouse, a rat, a rodent, or a goat. In some embodiments, the subject and mammal is a human.


An “effective amount” of the compositions of the disclosure generally refers to an amount sufficient to elicit the desired biological response, e.g., express the micro-dystrophin protein in a target cell, treat DMD, etc. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent described herein may vary depending on such factors as the condition being treated, the mode of administration, and the age, body composition, and health of the subject. Suitable dosage ranges are readily determinable by one skilled in the art.


The terms “treat”, “treating”, “treatment”, and “therapy” encompass an action that occurs while a subject is suffering from a condition which reduces the severity of the condition (or a symptom associated with the condition) or retards or slows the progression of the condition (or a symptom associated with the condition).


Compositions

In some aspects, the present disclosure provides compositions comprising the polypeptides, nucleic acids, or vectors disclosed herein. For administration to a subject, the polypeptides, engineered nucleic acids, or vectors disclosed herein may be formulated in a composition. In some embodiments, the composition further comprises additional agents (e.g., for specific delivery, increasing half-life, or other therapeutic agents).


In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. A “pharmaceutically acceptable carrier” is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.


Nucleic acids, in some embodiments, may be formulated in a non-viral delivery vehicle. Non-limiting examples of non-viral delivery vehicles include nanoparticles, such as nanocapsules and nanospheres. See, e.g., Sing, R et al. Exp Mol Pathol. 2009; 86(3):215-223. A nanocapsule is often comprised of a polymeric shell encapsulating an agent. Nanospheres are often comprised of a solid polymeric matrix throughout which the agent is dispersed. In some embodiments, the nanoparticle is a lipid particle, such as a liposome. See, e.g., Puri, A et al. Crit Rev Ther Drug Carrier Syst. 2009; 26(6):523-80. The term ‘nanoparticle’ also encompasses microparticles, such as microcapsules and microspheres.


Methods developed for making particles for delivery of encapsulated agents are described in the literature (for example, please see Doubrow, M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release 5:13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283,1987; Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774, 1988; each of which is incorporated herein by reference).


General considerations in the formulation and/or manufacture of pharmaceutical agents, such as compositions comprising any of the engineered nucleic acids disclosed herein may be found, for example, in Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa (1990) (incorporated herein by reference in its entirety).


Methods of Administration

Any of the polypeptides, nucleic acids, vectors, or compositions disclosed herein may be administered to a subject.


Suitable routes of administration include, without limitation, intravenous, intranasal, intramuscular, intrathecal, or subcutaneous. In some embodiments, a polypeptide, engineered nucleic acid, vector, or composition of the disclosure is administered intravenously, subcutaneously, intramuscularly intrathecally or intranasally. In some embodiments, a polypeptide, nucleic acid, vector, or composition of the disclosure is administered directly (e.g., by direct injection) to one or more cells (e.g., cardiac or skeletal muscle cells), tissues (e.g., cardiac or skeletal muscle), or organs (e.g., heart). Other routes of administration are contemplated herein. The administration route can be changed depending on a number of factors, including the desired cell, tissue, or organ.


Formulations comprising pharmaceutically-acceptable excipients and/or carrier solutions are well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intraarticular, and intramuscular administration and formulation.


In one aspect, the present disclosure relates to methods of achieving localized delivery of a gene therapy vector (e.g., an rAAV vector) to a muscle of a subject. Localized delivery to a muscle can be achieved through different methods including, but not limited to, catheter-based methods, local injections, local injection devices, microneedles or drug-eluting stents or drug-eluting implants. In one aspect, the present disclosure relates to catheter-based methods of delivering gene therapy vectors to a muscle. Examples of catheters include, but are not limited to, guiding catheters, microporous infusion catheters, balloon catheters, porous balloon catheters, microporous balloon catheters, retractable-needle catheters, over-the-needle (OTN) catheters, iontophoretic catheters or butterfly catheters. In some embodiments, the muscle is cardiac muscle. In some embodiments, the muscle is skeletal muscle. Non-limiting examples of skeletal muscles include arm muscles, hand muscles, shoulder muscles, chest muscles, neck muscles, muscles of the larynx, scalp muscles, eye muscles, hip muscles, leg muscles and thigh muscles.


In some embodiments, catheter delivery allows the combined delivery of one or more gene therapy vectors to the cardiac muscle and to one or more skeletal muscle(s). In some embodiments, the gene therapy vector delivered to the cardiac muscle is the same as the gene therapy vector delivered to the skeletal muscle(s). In some embodiments, the gene therapy vector delivered to the cardiac muscle is different from the gene therapy vector delivered to the skeletal muscle(s). In some embodiments, the catheter has multiple lumens for the delivery of the different gene therapy vectors. In some embodiments, the catheter has a single lumen. In some embodiments, the different gene therapy vectors are sequentially introduced as the catheter is moved to different arteries. In some embodiments, the combined delivery to cardiac and skeletal muscles comprises advancing a catheter to heart, delivering a gene therapy vector into the left and right coronary arteries, retracting the catheter to the aortic arch, and delivering a gene therapy vector to arteries that irrigate the skeletal muscles (e.g., the subclavian and/or carotid arteries). In some embodiments, the method further comprises retracting the catheter into the descending aorta and delivering the gene therapy to skeletal muscle via descending aortic branches.


In some embodiments, the present disclosure relates to the delivery of a gene therapy vector to an artery via a catheter. Non-limiting examples of arteries include a femoral artery, a subclavian artery, a carotid artery, an axillary artery, a brachial artery, a radial artery, an ulnar artery, an iliac artery, a popliteal artery, a tibial artery, a dorsalis pedis artery and an aorta. In some embodiments, the gene therapy vector is delivered to a subclavian and/or a carotid artery.


In some embodiments, the catheter is first introduced into the femoral artery and advanced to the subclavian and/or carotid arteries. In some embodiments, the catheter is then retracted into the descending aorta to deliver the gene therapy vector to skeletal muscle via descending aortic branches.


In some embodiments, the methods described herein relate to the delivery of a first rAAV to cardiac muscle and a second rAAV to a skeletal muscle, comprising: (i) introducing a catheter into the femoral artery and advancing to the heart; (ii) delivering the first gene therapy vector into the left and right coronary arteries; (iii) retracting the catheter to the aortic arch; and (iv) delivering the second gene therapy vector to the subclavian and/or carotid arteries. In some embodiments, the first and second rAAVs have different capsid serotypes. In some embodiments, the first and second rAAVs have the same capsid serotype. In some embodiments, the first and second rAAVs carry the same therapeutic gene. In some embodiments, the first and second rAAVs carry different therapeutic genes. In some embodiments, a first rAAV carries a micro-dystrophin gene optimized for cardiac muscle and the second rAAV carries a micro-dystrophin gene optimized for skeletal muscle.


In some embodiments, a vasodilator is administered to the subject prior to, or simultaneously with, the introduction of the catheter into an artery for gene therapy vector delivery. Vasodilators are medications that open (dilate) blood vessels. Non-limiting examples of vasodilators are arterial dilators, venous dilators, mixed dilators, nitroprusside, nitroglycerin, nitric oxide, hydralazine, allicin, nitrates, isosorbide dinitrate, isosorbide mononitrate, erythrityl tetranitrate, pentaerythritol tetranitrate, sodium nitroprusside, alpha-adrenoceptor antagonists (alpha blockers), al-adrenoceptor antagonists (e.g., prazosin, terazosin, doxazosin, trimazosin), phentolamine, phenoxybenzamine, sympatholytics, angiotensin converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, quinapril, ramipril), angiotensin receptor blockers (ARBs) (e.g., candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, azilsartan, valsartan), alpha-2-agonists (e.g., α-methyldopa, clonidine), alpha agonist of the alpha-2 adrenergic receptor (e.g., guanabenz, guanfacine) beta2-adrenoceptor agonists (β2-agonists), beta-adrenoceptor agonist (isoprenaline), beta-1-adrenergic agonist (e.g., dobutamine), calcium-channel blockers (CCBs) (e.g., amlodipine, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nitrendipine, verapamil, diltiazem), centrally acting sympatholytics, direct acting vasodilators, endothelin receptor antagonists (e.g., bosentan, ambrisentan), ganglionic blockers (e.g. trimethaphan camsylate), nitrodilators, phosphodiesterase inhibitors, potassium-channel openers (e.g., minoxidil), renin inhibitors (e.g., aliskiren), PDE3 inhibitors (e.g., milrinone, inamrinone, amrinone, cilostazol), PDE5 inhibitors (e.g., sildenafil, tadalafil), epinephrine, norepinephrine, or dopamine. In some embodiments, the vasodilator is adenosine. In some embodiments, the vasodilator is a PDE5 inhibitor. In some embodiments, the PDE5 inhibitor is sildenafil or tadalafil. In some embodiments, the PDE5 inhibitor is sildenafil.


In some embodiments, the vasodilator is administered 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150 or 180 minutes prior to the delivery of the gene therapy vector.


In some embodiments, administration of the vasodilator is through intravascular injection. In some embodiments, administration of the vasodilator is through intramuscular injection. In some embodiments, administration of the vasodilator is through ingestion. In some embodiments, administration of the vasodilator is through topical application. s In some embodiments, the methods of the present disclosure further comprise inflating a blood pressure cuff on each limb of the subject at the time of delivery of the gene therapy vector.


In some embodiments, the nucleic acids of the disclosure are delivered via an AAV vector. In some embodiments, the number of AAV particles administered to a subject may be on the order ranging from about 109 to about 1016 particles, or any values in between, such as for example, about 109, 1010, 1011, 1012, 1013, 1014, 1015, or 1016 particles. In some embodiments, the number of AAV particles administered to a subject may be on the order ranging from about 109 to about 1016 vector genomes (vgs), or any values in between, such as for example, about 109, 1010, 1011, 1012, 1013, 1014, 1015, or 1016 vgs. The AAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy. In some embodiments, the two or more administrations are within 24 hours of each other.


EXAMPLES
Example 1. Micro-Dystrophin Constructs that are Optimized for Either Skeletal Muscle or Cardiac Muscle

Based on studies in mice with micro-dystrophin constructs that are currently in clinical trials, two discoveries were made. First, it was found that the affinity of the micro-dystrophin for its membrane complex is modulated by the inclusion of the first three spectrin-like repeats (FIG. 1). The inclusion of only R1 leads to relatively weak binding, while the presence of R1+R2 or R1+R2+R3 leads to a much higher apparent affinity for the complex.


Secondly, it was discovered that overexpression of a micro-dystrophin containing either R1+R2 or R1+R2+R3 leads to outcompeting utrophin at the cardiac membrane, while not affecting utrophin in skeletal muscle. This displacement of utrophin in the heart leads to a much more rapid onset of cardiomyopathy and heart failure than if no dystrophin is present. On the other hand, a R1 only containing micro-dystrophin does not outcompete utrophin for its membrane binding in the heart to the same extent. Accordingly, a micro-dystrophin that will be beneficial to the heart, can be designed based on including only R1 of the first three repeats.


In the case of skeletal muscle, it was found that the greater the affinity for the complex, the better, since utrophin localization is unaffected. Thus, for skeletal muscle, the ideal constructs contain either R1+R2 or R1+R2+R3. However, in some embodiments, in order to accommodate the full C-terminus, it may be necessary to exclude R2 and only include R1.


Lastly, the loss of regulation of ion channels, including the TRPC1,3,6 channels, leads to residual pathology in skeletal and cardiac muscles that the current micro-dystrophins do not address. Accordingly, in some embodiments, regions in the C-terminus of dystrophin, including the syntrophin binding sites and/or the coiled coil regions that interact with dystrobrevin (FIG. 1), are included in micro-dystrophin.


The following exemplary micro-dystrophin constructs were designed for AAV delivery in view of the above findings. In some embodiments, the micro-dystrophin constructs designed for skeletal muscle are used with a promoter that expresses only in skeletal muscle and not in the heart. In some embodiments, cardiac-specific expression is achieved with the truncated cTnT promoter. In some embodiments, a smaller cTnT promoter is generated to allow use of the largest cardiac micro-dystrophins described below. FIGS. 2A and 2B shows the order of Repeats (R) and hinges (H), as well as the cysteine-rich portion of the C-terminus (CR), which is commonly included in micro-dystrophins and referred to as the truncated C-terminus, which is then followed by the rest of the C-terminus (CT).









TABLE 1







Cardiac micro-dystrophins









Size



(amino



acid


Elements included in construct
number)





Nterm-R1_R17-R19_R24 to trCterm
1263/1260


Nterm-R1_R17-R19_H4 to 1st coiled coil and proline
1286/1283


rich region that follows



Nterm-R1_R17-R19_H4 to end of syntrophin region
1223/1220


Nterm-R1_R17-R19_R24 to 1st coiled coil and proline
1407/1404


rich region that follows



Nterm-R1_R17-R19_H4 to end of dystrophin
1418/1415


Nterm-R1_R17-R19_R24 to end of dystrophin
1539/1536


Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin
1287


region



Nterm-R1_R17_R22_H4 to 1st coiled coil
1198


Nterm-R1_R16-R17_R21-R22_H4 to end of syntrophin
1363


region
















TABLE 2







Skeletal Muscle micro-dystrophins











Size




(amino




acid



Elements included in construct
number)







Nterm-R2_R16-R17_R24 to trCterm
1277



Nterm-R2_R16-R17_H4 to end of syntrophin region
1237



Nterm-R2_R16-R17_H4 to 1st coiled coil and proline
1300



rich region that follows




Nterm-R2_R16-R17_R24 to 1st coiled coil and proline
1421



rich region that follows




Nterm-R2_R23, R24 to end of dystrophin
1431



Nterm-R2_R16-R17_H4 to end of dystrophin
1432



Nterm-R1_R16-R17_R24 to end of dystrophin
1448



Nterm-R2_R16-R17_R24 to end of dystrophin
1553



Nterm-R2_R16-R17_R24 to end of syntrophin region
1358



Nterm-R2_R17_H3_R22_H4 to end of syntrophin region
1287










“-” is used to show the end points of domains that are continuously linked while “_” is used to indicate a deletion between the domains. For example, Nterm-R1_R17-R19_R24 to trCterm indicates that the micro-dystrophin comprises the N-terminus region up to and including R1 linked to R17 up to and including R19 linked to R24 up to and including the truncated C-terminus of dystrophin (i.e., the region between R1 and R17, the region between R19 and R24, and the region after the truncated C-terminus are deleted). The amino acid sequences of modules to build the micro-dystrophin proteins described in this example are provided below. The amino acids of the micro-dystrophin constructs in Tables 1 and 2 are provided as SEQ ID Nos. 1-6 and 13-21 elsewhere in the description.










N-terminus through Hinge 1 (325 amino acids)



(SEQ ID NO: 40)



MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNV






NKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQV





NVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMY





ITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVT





TSDPTRSPFPSQHLEAP





Repeat 1 (126 amino acids)


(SEQ ID NO: 41)



EDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNILQL






GSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMD





Repeat 2 (105 amino acids)


(SEQ ID NO: 42)



LQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAA






LEEQLKVLGDRWANICRWTEDRWVLLQD





Repeat 3 + 4 residues of H2 (118 amino acids)


(SEQ ID NO: 43)



ILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKN






KSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQP





Repeat 16 + linker (114 + 9 amino acids = 123 amino acids)


(SEQ ID NO: 44)



HTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGR






IDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDROG





Repeat 17 (113 amino acids)


(SEQ ID NO: 45)



RFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSS






KTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQK





Repeats 17-19 (327 aa)


(SEQ ID NO: 46)



RFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSS






KTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLE





QVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEK





KLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLED





LSSEWKAVNRLLQELRAKQ





Repeats 17-19 (324 aa)


(SEQ ID NO: 81)



RSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEII






QQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEP





GKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALP





EKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVE





EILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRAKQ





Hinge 3 (47 aa)


(SEQ ID NO: 119)



QPDLAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLMLEVP






Repeat 21 (105 aa)


(SEQ ID NO: 120)



KDSTQWLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDT






RKVHMITENINASWRSIHKRVSEREAAL





Repeat 22 + linker (126 aa)


(SEQ ID NO: 121)



EETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAHTDVYHNLDENSQK






ILRSLEGSDDAVLLORRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRL





Repeat 23 (114 aa)


(SEQ ID NO: 47)



HLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQE






PRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSAD





Repeat 23 (123 amino acids)


(SEQ ID NO: 82)



ASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPL






EGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSAD





Repeat 24 (121 amino acids)


(SEQ ID NO: 48)



WQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQ






LTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDF





Hinge 4 + truncated C terminus (364 amino acids)


(SEQ ID NO: 49)



GPASQHELSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRESAYRTAMKLRRLQKALCLD






LLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRIGRIRVLSF





KTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASEGGSNIEPSVRSCFQFANNKPEI





EAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMH





YPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETP





Syntrophin binding sites (81 amino acids)


(SEQ ID NO: 50)



VTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLS






QPRS





Coiled coil region (195 amino acids)


1st coiled coil adds 47 aa; including proline rich region adds 16 more aa


(total of 63 aa)


(SEQ ID NO: 51)



PAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSP






2nd coiled coil region to end of dystrophin; including 2nd coiled coil adds 


another 61 aa and region after adds 71 more aa (total of 132 aa)


(SEQ ID NO: 52)



RDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLR






VVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM






The nucleotide sequences of modules to build the micro-dystrophin genes described in this example are provided below. The nucleotide sequences of the micro-dystrophin constructs in Tables 1 and 2 are provided as SEQ ID Nos. 7-12, 22-39, 92-97, 109-114, and 116-118 elsewhere in the description.










N-terminus through Hinge 1



Human sequence without codon alterations:


(SEQ ID NO: 53)



ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACATTCACAAAATGGGTAAA






TGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAG





ACCTCCTCGAAGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGAGTTCATGCCCTGAACAATGTC





AACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATTTAGTGAATATTGGAAGTACTGACATCGTAGATGGAAA





TCATAAACTGACTCTTGGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAATATCATGG





CTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATTATCCACAGGTT





AATGTAATCAACTTCACCACCAGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGACCT





ATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGGAACATGCATTCAACATCGCCAGATATC





AATTAGGCATAGAGAAACTACTCGATCCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTAC





ATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCC





ACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAGATCACGGTCAGTCTAG





CACAGGGATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACC





ACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCT





Human sequence with codon optimization:


(SEQ ID NO: 54)



ATGCTGTGGTGGGAGGAGGTGGAGGACTGCTACGAGCGGGAGGACGTGCAGAAGAAGACCTTCACCAAGTGGGTGAA






CGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGACGGCCGGCGGCTGCTGG





ACCTGCTGGAGGGCCTGACCGGCCAGAAGCTGCCCAAGGAGAAGGGCAGCACCCGGGTGCACGCCCTGAACAACGTG





AACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGACATCGTGGACGGCAA





CCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTGAAGAACGTGATGAAGAACATCATGG





CCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGGCAGAGCACCCGGAACTACCCCCAGGTG





AACGTGATCAACTTCACCACCAGCTGGAGCGACGGCCTGGCCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCT





GTTCGACTGGAACAGCGTGGTGTGCCAGCAGAGCGCCACCCAGCGGCTGGAGCACGCCTTCAACATCGCCCGGTACC





AGCTGGGCATCGAGAAGCTGCTGGACCCCGAGGACGTGGACACCACCTACCCCGACAAGAAGAGCATCCTGATGTAC





ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGAGCATCGAGGCCATCCAGGAGGTGGAGATGCTGCCCCGGCC





CCCCAAGGTGACCAAGGAGGAGCACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTGAGCCTGG





CCCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC





ACCAGCGACCCCACCCGGAGCCCCTTCCCCAGCCAGCACCTGGAGGCCCCC





Repeat 1


Human sequence without codon alterations:


(SEQ ID NO: 55)



GAAGACAAGTCATTTGGCAGTTCATTGATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGAAGT






ATTATCGTGGCTTCTTTCTGCTGAGGACACATTGCAAGCACAAGGAGAGATTTCTAATGATGTGGAAGTGGTGAAAG





ACCAGTTTCATACTCATGAGGGGTACATGATGGATTTGACAGCCCATCAGGGCCGGGTTGGTAATATTCTACAATTG





GGAAGTAAGCTGATTGGAACAGGAAAATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAA





TTCAAGATGGGAATGCCTCAGGGTAGCTAGCATGGAAAAACAAAGCAATTTACATAGAGTTTTAATGGAT





Human sequence with codon optimization:


(SEQ ID NO: 56)



GAGGACAAGAGCTTCGGCAGCAGCCTGATGGAGAGCGAGGTGAACCTGGACCGGTACCAGACCGCCCTGGAGGAGGT






GCTGAGCTGGCTGCTGAGCGCCGAGGACACCCTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAGGTGGTGAAGG





ACCAGTTCCACACCCACGAGGGCTACATGATGGACCTGACCGCCCACCAGGGCCGGGTGGGCAACATCCTGCAGCTG





GGCAGCAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAGGAGACCGAGGTGCAGGAGCAGATGAACCTGCTGAA





CAGCCGGTGGGAGTGCCTGCGGGTGGCCAGCATGGAGAAGCAGAGCAACCTGCACCGGGTGCTGATGGAC





Repeat 2


Human sequence without codon alterations:


(SEQ ID NO: 57)



CTCCAGAATCAGAAACTGAAAGAGTTGAATGACTGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGGAGGAAGA






GCCTCTTGGACCTGATCTTGAAGACCTAAAACGCCAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACAAG





AACAAGTCAGGGTCAATTCTCTCACTCACATGGTGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCT





TTGGAAGAACAACTTAAGGTATTGGGAGATCGATGGGCAAACATCTGTAGATGGACAGAAGACCGCTGGGTTCTTTT





ACAAGAC





Human sequence with codon optimization:


(SEQ ID NO: 58)



CTGCAGAACCAGAAGCTGAAGGAGCTGAACGACTGGCTGACCAAGACCGAGGAGCGGACCCGGAAGATGGAGGAGGA






GCCCCTGGGCCCCGACCTGGAGGACCTGAAGCGGCAGGTGCAGCAGCACAAGGTGCTGCAGGAGGACCTGGAGCAGG





AGCAGGTGCGGGTGAACAGCCTGACCCACATGGTGGTGGTGGTGGACGAGAGCAGCGGCGACCACGCCACCGCCGCC





CTGGAGGAGCAGCTGAAGGTGCTGGGCGACCGGTGGGCCAACATCTGCCGGTGGACCGAGGACCGGTGGGTGCTGCT





GCAGGAC





Repeat 16 + linker


Human sequence without codon alterations:


(SEQ ID NO: 59)



CACACTGTCCGTGAAGAAACGATGATGGTGATGACTGAAGACATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTA






TTTGACTGAAATCACTCATGTCTCACAAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTA





AGGACTTTGAAGATCTCTTTAAGCAAGAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCTCAGGTCGG





ATTGACATTATTCATAGCAAGAAGACAGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGC





TCTCTCCCAGCTTGATTTCCAATGGGAAAAAGTTAACAAAATGTACAAGGACCGACAAGGG





Human sequence with codon optimization:


(SEQ ID NO: 60)



CACACCGTGCGGGAGGAGACCATGATGGTGATGACCGAGGACATGCCCCTGGAGATCAGCTACGTGCCCAGCACCTA






CCTGACCGAGATCACCCACGTGAGCCAGGCCCTGCTGGAGGTGGAGCAGCTGCTGAACGCCCCCGACCTGTGCGCCA





AGGACTTCGAGGACCTGTTCAAGCAGGAGGAGAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGAGCAGCGGCCGG





ATCGACATCATCCACAGCAAGAAGACCGCCGCCCTGCAGAGCGCCACCCCCGTGGAGCGGGTGAAGCTGCAGGAGGC





CCTGAGCCAGCTGGACTTCCAGTGGGAGAAGGTGAACAAGATGTACAAGGACCGGCAGGGC





Repeat 17


Human sequence without codon alterations:


(SEQ ID NO: 61)



CGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGC






TGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCC





AGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCA





AAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTC





AGACAGAAAAAAGAGGCTAGAAGAACAAAAG





Human sequence with codon optimization:


(SEQ ID NO: 62)



CGGTTCGACCGGAGCGTGGAGAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTCAACCAGTGGCTGACCGAGGC






CGAGCAGTTCCTGCGGAAGACCCAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGGTACCTGAAGGAGCTGC





AGGACGGCATCGGCCAGCGGCAGACCGTGGTGCGGACCCTGAACGCCACCGGCGAGGAGATCATCCAGCAGAGCAGC





AAGACCGACGCCAGCATCCTGCAGGAGAAGCTGGGCAGCCTGAACCTGCGGTGGCAGGAGGTGTGCAAGCAGCTGAG





CGACCGGAAGAAGCGGCTGGAGGAGCAGAAG





Repeats 17-19 (encoding 327 aa)


Human sequence without codon alterations:


(SEQ ID NO: 63)



CGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGC






TGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCC





AGGATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCA





AAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTC





AGACAGAAAAAAGAGGCTAGAAGAACAAAAGAATATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTAT





GGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAG





CAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGT





GCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAAGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGA





TAAAGGTTTCCAGAGCTTTACCTGAGAAACAAGGAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAA





AAGCTTGAAGACCTTGAAGAGCAGTTAAATCATCTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTA





TAACCAACCAAACCAAGAAGGACCATTTGACGTTAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGG





AAGAGATTTTGTCTAAAGGGCAGCATTTGTACAAGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGAT





CTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAG





Repeats 17-19 (encoding 324 aa)


Human sequence without codon alterations:


(SEQ ID NO: 83)



AGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTT






TCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGCA





TTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGAT





GCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAA





AAAGAGGCTAGAAGAACAAAAGAATATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGG





AAGCAGATAACATTGCTAGTATCCCACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAG





TTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGAATTCTCAAACAATTAAATGAAACTGGAGGACCCGTGCTTGTAAG





TGCTCCCATAAGCCCAGAAGAGCAAGATAAACTTGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTT





CCAGAGCTTTACCTGAGAAACAAGGAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAAAAGCTTGAA





GACCTTGAAGAGCAGTTAAATCATCTGCTGCTGTGGTTATCTCCTATTAGGAATCAGTTGGAAATTTATAACCAACC





AAACCAAGAAGGACCATTTGACGTTAAGGAAACTGAAATAGCAGTTCAAGCTAAACAACCGGATGTGGAAGAGATTT





TGTCTAAAGGGCAGCATTTGTACAAGGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCT





GAGTGGAAGGCGGTAAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAG





Hinge 3


Human sequence without codon alterations:


(SEQ ID NO: 100)



CAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCTCTCCTACTCAGACTGTTACTCTGGTGACACAACCTGT






GGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCT





Human Sequences with codon optimization:


(SEQ ID NO: 101)



CAGCCCGACCTGGCCCCCGGCCTGACCACCATCGGCGCCAGCCCCACCCAGACCGTGACCCTGGTGACCCAGCCCGT






GGTGACCAAGGAGACCGCCATCAGCAAGCTGGAGATGCCCAGCAGCCTGATGCTGGAGGTGCCC





Repeat 21


Human sequence without codon alterations:


(SEQ ID NO: 102)



AAGGATTCAACACAATGGCTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTC






ATGGAAGGAGGGTCCCTATACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAGCAGTTGGCCAAAGACCTCC





GCCAGTGGCAGACAAATGTAGATGTGGCAAATGACTTGGCCCTGAAACTTCTCCGGGATTATTCTGCAGATGATACC





AGAAAAGTCCACATGATAACAGAGAATATCAATGCCTCTTGGAGAAGCATTCATAAAAGGGTGAGTGAGCGAGAGGC





TGCTTTG





Human Sequences with codon optimization:


(SEQ ID NO: 103)



AAGGACAGCACCCAGTGGCTGGAGGCCAAGGAGGAGGCCGAGCAGGTGCTGGGCCAGGCCCGGGCCAAGCTGGAGAG






CTGGAAGGAGGGCCCCTACACCGTGGACGCCATCCAGAAGAAGATCACCGAGACCAAGCAGCTGGCCAAGGACCTGC





GGCAGTGGCAGACCAACGTGGACGTGGCCAACGACCTGGCCCTGAAGCTGCTGCGGGACTACAGCGCCGACGACACC





CGGAAGGTGCACATGATCACCGAGAACATCAACGCCAGCTGGCGGAGCATCCACAAGCGGGTGAGCGAGCGGGAGGC





CGCCCTG





Repeat 22 + linker


Human sequence without codon alterations:


(SEQ ID NO: 104)



GAAGAAACTCATAGATTACTGCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAAC






AACTGCCAATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGA





AACAATGGCAAGACCTCCAAGGTGAAATTGAAGCTCACACAGATGTTTATCACAACCTGGATGAAAACAGCCAAAAA





ATCCTGAGATCCCTGGAAGGTTCCGATGATGCAGTCCTGTTACAAAGACGTTTGGATAACATGAACTTCAAGTGGAG





TGAACTTCGGAAAAAGTCTCTCAACATTAGGTCCCATTTGGAAGCCAGTTCTGACCAGTGGAAGCGTCTG





Human Sequences with codon optimization:


(SEQ ID NO: 105)



GAGGAGACCCACCGGCTGCTGCAGCAGTTCCCCCTGGACCTGGAGAAGTTCCTGGCCTGGCTGACCGAGGCCGAGAC






CACCGCCAACGTGCTGCAGGACGCCACCCGGAAGGAGCGGCTGCTGGAGGACAGCAAGGGCGTGAAGGAGCTGATGA





AGCAGTGGCAGGACCTGCAGGGCGAGATCGAGGCCCACACCGACGTGTACCACAACCTGGACGAGAACAGCCAGAAG





ATCCTGCGGAGCCTGGAGGGCAGCGACGACGCCGTGCTGCTGCAGCGGCGGCTGGACAACATGAACTTCAAGTGGAG





CGAGCTGCGGAAGAAGAGCCTGAACATCCGGAGCCACCTGGAGGCCAGCAGCGACCAGTGGAAGCGGCTG





Repeat 23


Human sequence without codon alterations:


(SEQ ID NO: 64)



CACCTTTCTCTGCAGGAACTTCTGGTGTGGCTACAGCTGAAAGATGATGAATTAAGCCGGCAGGCACCTATTGGAGG






CGACTTTCCAGCAGTTCAGAAGCAGAACGATGTACATAGGGCCTTCAAGAGGGAATTGAAAACTAAAGAACCTGTAA





TCATGAGTACTCTTGAGACTGTACGAATATTTCTGACAGAGCAGCCTTTGGAAGGACTAGAGAAACTCTACCAGGAG





CCCAGAGAGCTGCCTCCTGAGGAGAGAGCCCAGAATGTCACTCGGCTTCTACGAAAGCAGGCTGAGGAGGTCAATAC





TGAGTGGGAAAAATTGAACCTGCACTCCGCTGAC





Human Sequences with codon optimization:


(SEQ ID NO: 65)



CACCTGAGCCTGCAGGAGCTGCTGGTGTGGCTGCAGCTGAAGGACGACGAGCTGAGCCGGCAGGCCCCCATCGGCGG






CGACTTCCCCGCCGTGCAGAAGCAGAACGACGTGCACCGGGCCTTCAAGCGGGAGCTGAAGACCAAGGAGCCCGTGA





TCATGAGCACCCTGGAGACCGTGCGGATCTTCCTGACCGAGCAGCCCCTGGAGGGCCTGGAGAAGCTGTACCAGGAG





CCCCGGGAGCTGCCCCCCGAGGAGCGGGCCCAGAACGTGACCCGGCTGCTGCGGAAGCAGGCCGAGGAGGTGAACAC





CGAGTGGGAGAAGCTGAACCTGCACAGCGCCGAC





Repeat 23 (encoding 123 aa)





Human sequence without codon alterations:


(SEQ ID NO: 84)



GCCAGTTCTGACCAGTGGAAGCGTCTGCACCTTTCTCTGCAGGAACTTCTGGTGTGGCTACAGCTGAAAGATGATGA






ATTAAGCCGGCAGGCACCTATTGGAGGCGACTTTCCAGCAGTTCAGAAGCAGAACGATGTACATAGGGCCTTCAAGA





GGGAATTGAAAACTAAAGAACCTGTAATCATGAGTACTCTTGAGACTGTACGAATATTTCTGACAGAGCAGCCTTTG





GAAGGACTAGAGAAACTCTACCAGGAGCCCAGAGAGCTGCCTCCTGAGGAGAGAGCCCAGAATGTCACTCGGCTTCT





ACGAAAGCAGGCTGAGGAGGTCAATACTGAGTGGGAAAAATTGAACCTGCACTCCGCTGAC





Human sequence with codon optimization:


(SEQ ID NO: 85)



GCCAGCAGCGACCAGTGGAAGCGGCTGCACCTGAGCCTGCAGGAGCTGCTGGTGTGGCTGCAGCTGAAGGACGACGA






GCTGAGCCGGCAGGCCCCCATCGGCGGCGACTTCCCCGCCGTGCAGAAGCAGAACGACGTGCACCGGGCCTTCAAGC





GGGAGCTGAAGACCAAGGAGCCCGTGATCATGAGCACCCTGGAGACCGTGCGGATCTTCCTGACCGAGCAGCCCCTG





GAGGGCCTGGAGAAGCTGTACCAGGAGCCCCGGGAGCTGCCCCCCGAGGAGCGGGCCCAGAACGTGACCCGGCTGCT





GCGGAAGCAGGCCGAGGAGGTGAACACCGAGTGGGAGAAGCTGAACCTGCACAGCGCCGAC





Repeat 24


Human Sequences without codon alterations:


(SEQ ID NO: 66)



TGGCAGAGAAAAATAGATGAGACCCTTGAAAGACTCCGGGAACTTCAAGAGGCCACGGATGAGCTGGACCTCAAGCT






GCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCG





AGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGAGAACGTGAGCCACGTCAATGACCTTGCTCGCCAG





CTTACCACTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCT





GCAGGTGGCCGTCGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTT





Human Sequences with codon optimization:


(SEQ ID NO: 67)



TGGCAGCGGAAGATCGACGAGACCCTGGAGCGGCTGCAGGAGCTGCAGGAGGCCACCGACGAGCTGGACCTGAAGCT






GCGGCAGGCCGAGGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGACCACCTGG





AGAAGGTGAAGGCCCTGCGGGGCGAGATCGCCCCCCTGAAGGAGAACGTGAGCCACGTGAACGACCTGGCCCGGCAG





CTGACCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGAGCACCCTGGAGGACCTGAACACCCGGTGGAAGCTGCT





GCAGGTGGCCGTGGAGGACCGGGTGCGGCAGCTGCACGAGGCCCACCGGGACTTC





Hinge 4 + truncated C terminus


Human Sequences without codon alterations:


(SEQ ID NO: 68)



GGTCCAGCATCTCAGCACTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACAAAGTGCC






CTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAGCTG





ACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGAT





CTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCT





GCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCT





GCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTT





AAAACTGGCATCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTC





AACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAG





TTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATC





GAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGC





TGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGA





GTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCAC





TATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAA





ATTTCGAACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACA





ACATGGAAACTCCC





Human Sequences with codon optimization:


(SEQ ID NO: 69)



GGCCCCGCCAGCCAGCACTTCCTGAGCACCAGCGTGCAGGGCCCCTGGGAGCGGGCCATCAGCCCCAACAAGGTGCC






CTACTACATCAACCACGAGACCCAGACCACCTGCTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCCG





ACCTGAACAACGTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGCGGCTGCAGAAGGCCCTGTGCCTGGAC





CTGCTGAGCCTGAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGACATCCT





GCAGATCATCAACTGCCTGACCACCATCTACGACCGGCTGGAGCAGGAGCACAACAACCTGGTGAACGTGCCCCTGT





GCGTGGACATGTGCCTGAACTGGCTGCTGAACGTGTACGACACCGGCCGGACCGGCCGGATCCGGGTGCTGAGCTTC





AAGACCGGCATCATCAGCCTGTGCAAGGCCCACCTGGAGGACAAGTACCGGTACCTGTTCAAGCAGGTGGCCAGCAG





CACCGGCTTCTGCGACCAGCGGCGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCCGGCAGCTGGGCGAGG





TGGCCAGCTTCGGCGGCAGCAACATCGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATC





GAGGCCGCCCTGTTCCTGGACTGGATGCGGCTGGAGCCCCAGAGCATGGTGTGGCTGCCCGTGCTGCACCGGGTGGC





CGCCGCCGAGACCGCCAAGCACCAGGCCAAGTGCAACATCTGCAAGGAGTGCCCCATCATCGGCTTCCGGTACCGGA





GCCTGAAGCACTTCAACTACGACATCTGCCAGAGCTGCTTCTTCAGCGGCCGGGTGGCCAAGGGCCACAAGATGCAC





TACCCCATGGTGGAGTACTGCACCCCCACCACCAGCGGCGAGGACGTGCGGGACTTCGCCAAGGTGCTGAAGAACAA





GTTCCGGACCAAGCGGTACTTCGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACCGTGCTGGAGGGCGACA





ACATGGAGACCCCC





Syntrophin binding sites


Human Sequences without codon alterations:


(SEQ ID NO: 70)



GTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACGATGATACTCATTC






ACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAATGATAGCATCTCTC





CTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGC





CAGCCTCGTAGT





Human Sequences with codon optimization:


(SEQ ID NO: 71)



GTGACCCTGATCAACTTCTGGCCCGTGGACAGCGCCCCCGCCAGCAGCCCCCAGCTGAGCCACGACGACACCCACAG






CCGGATCGAGCACTACGCCAGCCGGCTGGCCGAGATGGAGAACAGCAACGGCAGCTACCTGAACGACAGCATCAGCC





CCAACGAGAGCATCGACGACGAGCACCTGCTGATCCAGCACTACTGCCAGAGCCTGAACCAGGACAGCCCCCTGAGC





CAGCCCCGGAGC





Coiled coil region


1st coiled coil plus proline rich region


Human sequence without codon alterations:


(SEQ ID NO: 72)



CCTGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGA






AAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCC





CTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCC





Human sequence with codon optimization:


(SEQ ID NO: 73)



CCCGCCCAGATCCTGATCAGCCTGGAGAGCGAGGAGCGGGGCGAGCTGGAGCGGATCCTGGCCGACCTGGAGGAGGA






GAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCCAGCC





CCCCCGAGATGATGCCCACCAGCCCCCAGAGCCCC





2nd coiled coil region to end of dystrophin


Human sequence without codon alterations:


(SEQ ID NO: 74)



CGGGATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCT






GGAAGACCACAATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCA





AAGTGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGA





GTGGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCACAGGGTT





AGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGAAGAAATACCCCTGGAAAGCCAATGAGAG





AGGACACAATG





Human sequence with codon optimization:


(SEQ ID NO: 75)



CGGGACGCCGAGCTGATCGCCGAGGCCAAGCTGCTGCGGCAGCACAAGGGCCGGCTGGAGGCCCGGATGCAGATCCT






GGAGGACCACAACAAGCAGCTGGAGAGCCAGCTGCACCGGCTGCGGCAGCTGCTGGAGCAGCCCCAGGCCGAGGCCA





AGGTGAACGGCACCACCGTGAGCAGCCCCAGCACCAGCCTGCAGCGGAGCGACAGCAGCCAGCCCATGCTGCTGCGG





GTGGTGGGCAGCCAGACCAGCGACAGCATGGGCGAGGAGGACCTGCTGAGCCCCCCCCAGGACACCAGCACCGGCCT





GGAGGAGGTGATGGAGCAGCTGAACAACAGCTTCCCCAGCAGCCGGGGCCGGAACACCCCCGGCAAGCCCATGCGGG





AGGACACCATG






Additional Studies

Four AAV vectors, AAVRH10 CK8.pDys_Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin region, AAVRH10-CK8. μDys_Nterm-R2_R17_H3_R22_H4 to end of syntrophin region; AAVRH10-CK8. pDys_Nterm-R1_R17_R22_H4 to 1st coiled coil; and AAVRH10-CK8. μDys_Nterm-R1_R16-R17_R21-R22_H4 to end of syntrophin region) were injected (via tail vein) at a dosage of 2×1014 vector genomes/kg into 5-week-old male D2.mdx mice (dystrophin-deficient mice in a DBA2/J genetic background). In parallel, D2.mdx. mice were also treated with three other micro-dystrophins—PF-06939926 (Pfizer), SRP-9001-10 (Sarepta), and SGT-001 (Solid Bio)—that are currently in clinical trials. A subset of mice were sacrificed at 9 weeks of age to analyze the degree of skeletal muscle protection and rescue (skeletal muscle functional loss of function is apparent by this time). Cardiac functional deficits are not apparent until >9 months of age. Therefore, two groups of mice were analyzed for preservation of cardiac function (cardiac output and normal ventricular diameter) at 12 months of age.


Skeletal muscle analyses revealed that AAVRH10-CK8. μDys_Nterm-R2_R17_H3_R22_H4 to end of syntrophin region improved maintenance of force production in diaphragm and EDL muscles (FIG. 3A). This AAV vector also exhibited enhanced protection of skeletal muscles from contraction-induced injury (see FIG. 3B). AAVRH10-CK8. μDys_Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin region also exhibited improved muscle function and resistance to injury (FIGS. 3A-3B).


Heart analyses revealed that AAVRH10-CK8. μDys_Nterm-R1_R16-R17_R21-R22_H4 to end of syntrophin region improved cardiac output (see. FIG. 4) and ventricular end diastolic volumes (EDV) (see FIG. 5) of D2.mdx mice. Administration of AAVRH10-CK8. μDys_Nterm-R1_R17_R22_H4 to 1st coiled coil and AAVRH10-CK8. μDys_Nterm-R1_R17_H3_R21-R22_H4 to end of syntrophin region also improved cardiac output (see FIG. 4) and EDV (see FIG. 5) in D2.mdx mice. These results demonstrate that micro-dystrophins of the present disclosure improve skeletal muscle and cardiac function in a manner that is comparable to or superior to the known micro-dystrophins that are currently in clinical trials.


Example 2. New Skeletal Muscle-Specific Promoter

The finding that the heart and skeletal muscle may need different micro-dystrophin constructs delivered to each muscle type, motivated the need for a small, skeletal muscle-specific promoter. Elements from the skeletal muscle alpha-actin promoter were taken to construct such a promoter. It consists of one or two copies of the 99 bp skeletal muscle alpha-actin enhancer and 274 bases of the core promoter elements, resulting in a 380 bp construct (or 479 bp with 2 enhancers).


Sequences of the promoter are provided below.


a) 380 bp Construct









Enhancer


(XbaI site for cutting 5′ end)


(SEQ ID NO: 76)


TCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAA


CCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAG


CCCGGC





Core Promoter + KOZAK


(SEQ ID NO: 77)


AGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGCG


TTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGTCAACGCAGGGGACC


CGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGGGGGGCTCTAGTGCCC


AACACCCAAATATGGCTCGAGAAGGGCAGCGACATTCCTGCGGGGTGGCG


CGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGCGG


CCACCGCAGCGGACAGCGCCACC ATGG








    • ATG+G—initiation codon+G of coding sequence follows and completes the optimal KOZAK and creates a NcoI site (CCATGG) to cut the full promoter. The G after the ATG ia not necessary if other cloning strategies are preferred.





The promoter is 380 bp in length:









(SEQ ID NO: 78)


TCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAA





CCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAG





CCCGGCAGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGG





CCCGCGTTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGTCAACGCAG





GGGACCCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCT





AGTGCCCAACACCCAAATATGGCTCGAGAAGGGCAGCGACATTCCTGCGG





GGTGGCGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGA





CAAGCGGCCACCGCAGCGGACAGCGCCACC






b) 479 bp Construct (Stronger Promoter)









Back to back Enhancers


(XbaI site for cutting 5′ end)


(SEQ ID NO: 79)


TCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAA





CCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAG





CCCGGCAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAAC





CTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAGC





CCGGC





Core Promoter + KOZAK


(SEQ ID NO: 77)


AGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGCG





TTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGTCAACGCAGGGGACC





CGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGCC





CAACACCCAAATATGGCTCGAGAAGGGCAGCGACATTCCTGCGGGGTGGC





GCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGCG





GCCACCGCAGCGGACAGCGCCACCATGG






The promoter is 479 bp in length:









(SEQ ID NO: 80)


TCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAG





AACCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAG





GCAGCCCGGCAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAA





TAGAACCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGG





GAGGCAGCCCGGCAGGAGGGGCAAACCCGCTAGGGAGACACTCCATAT





ACGGCCCGGCCCGCGTTACCTGGGACCGGGCCAACCCGCTCCTTCTTT





GGTCAACGCAGGGGACCCGGGCGGGGGCCCAGGCCGCGAACCGGCCGA





GGGAGGGGGCTCTAGTGCCCAACACCCAAATATGGCTCGAGAAGGGCA





GCGACATTCCTGCGGGGTGGCGCGGAGGGAATGCCCGCGGGCTATATA





AAACCTGAGCAGAGGGACAAGCGGCCACCGCAGCGGACAGCGCCACC









Example 3. New Approach for rAAV Delivery to Muscle

If both the heart and skeletal muscle are to be optimally targeted in rAAV gene delivery, then it would be best to deliver the cardiac rAAV and the skeletal muscle rAAV via the specific arterial beds for the muscles, rather than the intravenous delivery as is the common practice. This is accomplished using a drug delivery catheter that is introduced into the femoral artery and first advanced the heart. The cardiac rAAV is first delivered into the left and right coronary arteries. Following the cardiac rAAV delivery, the catheter is retracted to the aortic arch, where skeletal muscle-targeted rAAV is then delivered to the subclavian arteries and, if desired, the carotid arteries. The catheter is then retracted into the descending aorta, if desired, and the rest of the skeletal muscle rAAV is delivered via various descending aortic branches, depending on the desired skeletal muscle distribution.


To further enhance distribution of rAAV to skeletal muscle, a PDE5 inhibitor, such as sildenafil or tadalafil, is given 1 hour prior to rAAV delivery to increase blood flow to resting skeletal muscle. Additionally, inflation of a blood pressure cuff on each limb at a pressure below the systolic pressure and above the diastolyic pressure improves retention of virus in the limbs at the time of viral delivery.


EQUIVALENTS AND SCOPE

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.


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


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


The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”


The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.


As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.


As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.


It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.


In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

Claims
  • 1. A recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein comprising: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, and spectrin-like repeat 17, wherein the micro-dystrophin protein does not contain spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin, and wherein the micro-dystrophin protein optionally further comprises spectrin-like repeat 18 and/or spectrin-like repeat 19.
  • 2. The recombinant nucleic acid of claim 1, wherein the micro-dystrophin protein further comprises one or more of: spectrin-like repeat 16, hinge domain 3, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin.
  • 3. The recombinant nucleic acid of claim 1 or 2, wherein the micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin and/or one or more coiled coil domains of dystrophin.
  • 4. The recombinant nucleic acid of any one of claims 1-3, wherein the spectrin-like repeat 1 is directly coupled to the spectrin-like repeat 17.
  • 5. The recombinant nucleic acid of any one of claims 1-4, wherein the spectrin-like repeat 19 is present and is directly coupled to spectrin-like repeat 24.
  • 6. The recombinant nucleic acid of any one of claims 1-4, wherein the spectrin-like repeat 19 is present and is directly coupled to hinge domain 4.
  • 7. The method of any one of claims 1-4, wherein the spectrin-like repeat 17 is directly coupled to hinge domain 3 or spectrin-like repeat 22.
  • 8. A recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein comprising: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 2, and spectrin-like repeat 17, wherein the micro-dystrophin protein does not contain spectrin-like repeat 3 of dystrophin, and wherein the micro-dystrophin protein optionally further comprises spectrin-like repeat 16.
  • 9. The recombinant nucleic acid of claim 8, wherein the micro-dystrophin protein further comprises one or more of: hinge domain 3, spectrin-like repeat 22, spectrin-like repeat 23, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin.
  • 10. The recombinant nucleic acid of claim 8 or 9, wherein the micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin.
  • 11. The recombinant nucleic acid of any one of claims 8-10, wherein the micro-dystrophin protein further comprises one or more coiled coil domains of dystrophin.
  • 12. The recombinant nucleic acid of any one of claims 8-11, wherein the spectrin-like repeat 2 is directly coupled to spectrin-like repeat 16 or spectrin-like 17.
  • 13. The recombinant nucleic acid of any one of claims 8-12, wherein the spectrin-like repeat 17 is directly coupled to spectrin-like repeat 24.
  • 14. The recombinant nucleic acid of any one of claims 8-12, wherein the spectrin-like repeat 17 is directly coupled to hinge domain 3 or hinge domain 4.
  • 15. The recombinant nucleic acid of any preceding claim, wherein the micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the C-terminus.
  • 16. The recombinant nucleic acid of any one of claims 1-14, wherein the micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the end of the proline rich region following the first coiled coil domain.
  • 17. The recombinant nucleic acid of any one of claims 1-14, wherein the micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the C-terminus.
  • 18. The recombinant nucleic acid of any one of claims 1-14, wherein the micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the proline rich region following the first coiled coil domain.
  • 19. The recombinant nucleic acid of any one of claims 1-14, wherein the micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the second syntrophin binding domain.
  • 20. The recombinant nucleic acid of any preceding claim, wherein the recombinant nucleic acid is less than 5 kb in length.
  • 21. The recombinant nucleic acid of any preceding claim, wherein a promoter is operably linked to the nucleotide sequence encoding the micro-dystrophin protein.
  • 22. The recombinant nucleic acid of claim 21, wherein the promoter is a cardiac-specific promoter.
  • 23. The recombinant nucleic acid of claim 22, wherein the cardiac-specific promoter is a cardiac troponin T (cTnT) promoter.
  • 24. The recombinant nucleic acid of claim 21, wherein the promoter is a skeletal muscle-specific promoter.
  • 25. The recombinant nucleic acid of claim 24, wherein the skeletal muscle-specific promoter is a skeletal muscle alpha-actin promoter.
  • 26. The recombinant nucleic acid of any preceding claim, wherein the skeletal-muscle specific promoter comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 78 or SEQ ID NO: 80.
  • 27. A recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein, wherein the nucleotide sequence encoding the micro-dystrophin protein comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of any one of SEQ ID Nos. 7-12, 22-39, 92-97, 109-114, and 116-118.
  • 28. A recombinant nucleic acid comprising a nucleotide sequence encoding a micro-dystrophin protein that comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6, 13-21, 86-91, 98, 106-108, and 115.
  • 29. A micro-dystrophin protein encoded by the recombinant nucleic acid of any one of claims 1-28.
  • 30. A micro-dystrophin protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6 and 13-21, 86-91, 98, 106-108, and 115.
  • 31. A recombinant adeno-associated virus (rAAV) vector comprising the recombinant nucleic acid of any one of claims 1-28.
  • 32. An rAAV particle comprising the rAAV vector of claim 31 encapsidated in an AAV capsid.
  • 33. The rAAV particle of claim 32, wherein the AAV capsid comprises a capsid protein derived from AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAVrh.74, AAVrh.10, AAV2/6 or AAV9 serotypes.
  • 34. A composition comprising the rAAV particle of claim 32 or 33.
  • 35. A method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, comprising administering to the subject the recombinant nucleic acid of any one of claims 1-28, the micro-dystrophin protein of claim 29 or 30, the rAAV particle of claim 32 or 33, or the composition of claim 34.
  • 36. An isolated nucleic acid comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80.
  • 37. The isolated nucleic acid of claim 36, wherein the nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80 is operably linked to a nucleotide sequence that is at least 80% identical to the nucleotide sequence of any one of SEQ ID Nos. 7-12, 22-39, 92-97, 109-114, and 116-118.
  • 38. The isolated nucleic acid of claim 36, wherein the nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 78 or SEQ ID NO: 80 is operably linked to a nucleotide sequence that encodes a micro-dystrophin protein that comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID Nos. 1-6, 13-21, 86-91, 98, 106-108, and 115.
  • 39. A vector comprising the isolated nucleic acid of any one of claims 36-38.
  • 40. The vector of claim 39, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.
  • 41. An rAAV particle comprising the rAAV vector of claim 40 encapsidated in an AAV capsid.
  • 42. The rAAV particle of claim 41, wherein the AAV capsid comprises a capsid protein derived from AAV1, AAV2, AAV3, AAV6, AAV8, AAVrh.74, AAVrh.10, AAV2/6 or AAV9 serotypes.
  • 43. A composition comprising the rAAV particle of claim 41 or 42.
  • 44. A method of treating a skeletal muscle disorder in a subject in need thereof, comprising administering to the subject the isolated nucleic acid of any one of claims 36-38, the rAAV particle of claim 41 or 42, or the composition of claim 43.
  • 45. A method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, comprising administering to the subject the isolated nucleic acid of any one of claims 36-38, the rAAV particle of claim 41 or 42, or the composition of claim 43.
  • 46. A method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising delivering to the subject a first recombinant adeno-associated virus (rAAV) particle comprising a nucleotide sequence encoding a first micro-dystrophin protein to cardiac muscle and a second rAAV particle comprising a nucleotide sequence encoding a second micro-dystrophin to skeletal muscle.
  • 47. The method of claim 46, wherein the first rAAV particle comprises a cardiac-specific promoter operably linked to the nucleotide sequence encoding the first micro-dystrophin protein.
  • 48. The method of claim 46, wherein the second rAAV particle comprises a skeletal muscle-specific promoter operably linked to the nucleotide sequence encoding the second micro-dystrophin protein.
  • 49. The method of any one of claims 46-48, wherein the first rAAV particle comprises a capsid protein derived from AAV9, AAVrh.74, or AAVrh.10.
  • 50. The method of any one of claims 46-49, wherein the second rAAV particle comprises a capsid protein derived from AAV8.
  • 51. The method of any one of claims 46-50, wherein the first and second rAAV particles are delivered by the same method.
  • 52. The method of any one of claims 46-50, wherein the first and second rAAV particles are delivered by different methods.
  • 53. The method of any one of claims 46-51, wherein the first and second rAAV particles are delivered via one or more catheters.
  • 54. The method of claim 53, comprising: (i) introducing a catheter into the femoral artery and advancing to the heart;(ii) delivering the first rAAV particle into the left and right coronary arteries;(iii) retracting the catheter to the aortic arch; and(iv) delivering the second rAAV particle to the subclavian and/or carotid arteries.
  • 55. The method of claim 54, further comprising (v) retracting the catheter into the descending aorta and delivering the second rAAV particle to skeletal muscle via descending aortic branches.
  • 56. The method of any one of claims 46-55, further comprising administering a vasodilator to the subject prior to the delivery of the first and second rAAV particles.
  • 57. The method of claim 56, wherein the vasodilator is a PDE5 inhibitor.
  • 58. The method of claim 57, wherein the PDE5 inhibitor is sildenafil or tadalafil.
  • 59. The method of any one of claims 56-58, wherein the vasodilator is administered 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour prior to the delivery of first rAAV and/or second rAAV.
  • 60. The method of any one of claims 46-59, wherein a blood pressure cuff is inflated on each limb at the time of delivery of the first rAAV particle and/or the second rAAV particle.
  • 61. The method of any one of claims 46-59, wherein the first micro-dystrophin protein comprises: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, and spectrin-like repeat 17, wherein the micro-dystrophin protein does not contain spectrin-like repeat 2 and/or spectrin-like repeat 3 of dystrophin, and wherein the micro-dystrophin protein optionally further comprises spectrin-like repeat 18, and/or spectrin-like repeat 19.
  • 62. The method of claim 61, wherein the first micro-dystrophin protein further comprises one or more of: spectrin-like repeat 16, hinge domain 3, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin.
  • 63. The method of claim 61 or 62, wherein the first micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin and/or one or more coiled coil domains of dystrophin.
  • 64. The method of any one of claims 61-63, wherein the spectrin-like repeat 1 of the first micro-dystrophin protein is directly coupled to the spectrin-like repeat 17.
  • 65. The method of any one of claims 61-64, wherein the spectrin-like repeat 19 is present in the first micro-dystrophin protein and is directly coupled to spectrin-like repeat 24.
  • 66. The method of any one of claims 61-64, wherein the spectrin-like repeat 19 is present in the first micro-dystrophin protein and is directly coupled to hinge domain 4.
  • 67. The method of any one of claims 61-64, wherein the spectrin-like repeat 17 is directly coupled to hinge domain 3 or spectrin-like repeat 22.
  • 68. The method of any one of claims 46-67, wherein the second micro-dystrophin protein comprises: an amino-terminal actin-binding domain, hinge domain 1, spectrin-like repeat 1, spectrin-like repeat 2, and spectrin-like repeat 17, wherein the micro-dystrophin protein does not contain spectrin-like repeat 3 of dystrophin and wherein the micro-dystrophin protein optionally further comprises spectrin-like repeat 16.
  • 69. The method of claim 68, wherein the second micro-dystrophin protein further comprises one or more of: hinge domain 3, spectrin-like repeat 22, spectrin-like repeat 23, spectrin-like repeat 24, hinge domain 4, and dystroglycan binding site of dystrophin.
  • 70. The method of claim 68 or 69, wherein the second micro-dystrophin protein further comprises one or more syntrophin binding domains of dystrophin.
  • 71. The method of any one of claims 68-70, wherein the second micro-dystrophin protein further comprises one or more coiled coil domains of dystrophin.
  • 72. The method of any one of claims 68-71, wherein the spectrin-like repeat 2 of the second micro-dystrophin protein is directly coupled to spectrin-like repeat 16 or spectrin-like repeat 17.
  • 73. The method of any one of claims 68-72, wherein the spectrin-like repeat 17 of the second micro-dystrophin protein is directly coupled to spectrin-like repeat 24.
  • 74. The method of any one of claims 68-72, wherein the spectrin-like repeat 17 of the second micro-dystrophin protein is directly coupled to hinge domain 3 or hinge domain 4.
  • 75. The method of any one of 46-74, wherein the first or second micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the C-terminus.
  • 76. The method of any one of claims 46-74, wherein the first or second micro-dystrophin protein comprises the region of dystrophin from spectrin-like repeat 24 to the end of the proline rich region following the first coiled coil domain.
  • 77. The method of any one of claims 46-74, wherein the first or second micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the C-terminus.
  • 78. The method of any one of claims 46-74, wherein the first or second micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the proline rich region following the first coiled coil domain.
  • 79. The method of any one of claims 46-74, wherein the first or second micro-dystrophin protein comprises the region of dystrophin from hinge domain 4 to the end of the second syntrophin binding domain.
  • 80. A method of delivering an rAAV particle to a skeletal muscle in a subject, the method comprising delivering the rAAV particle via a catheter.
  • 81. The method of claim 80, comprising delivering the rAAV particle to the subclavian and/or carotid arteries.
  • 82. The method of claim 80 or 81, wherein the catheter is first introduced into the femoral artery and advanced to the subclavian and/or carotid arteries.
  • 83. The method of any one of claims 80-82, further comprising retracting the catheter into the descending aorta and delivering the rAAV particle to skeletal muscle via descending aortic branches.
  • 84. The method of any one of claims 80-83, comprising administering a vasodilator.
  • 85. The method of 84, wherein the vasodilator is a PDE5 inhibitor.
  • 86. The method of claim 85, wherein the PDE5 inhibitor is sildenafil or tadalafil.
  • 87. The method of any one of claims 84-86, wherein the vasodilator is administered 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour prior to the delivery of the rAAV particle.
  • 88. The method of any one of claims 80-87, further comprising inflating a blood pressure cuff on each limb at the time of delivery of the rAAV particle.
  • 89. The method of any one of claims 80-88, wherein the skeletal muscle is fast-twitch or slow-twitch.
RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application Ser. No. 63/197,976, filed Jun. 7, 2021, entitled “GENE THERAPY FOR DUCHENNE MUSCULAR DYSTROPHY”, the entire contents of which are incorporated herein by reference.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2022/032570 6/7/2022 WO
Provisional Applications (1)
Number Date Country
63197976 Jun 2021 US