Patent Application
20220023384
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 6, 2021, is named C123370191WO00-SEQ-RE and is 257,367 bytes in size.
Many forms of heart disease and heart arrhythmia are caused directly or indirectly by improper regulation of Ca2+ release in heart muscle cells. Ca2+ release in heart muscle cells occurs in specialized structures known as dyads. A key regulator of Ca2+ release is RYR2 (ryanodine receptor type 2), a Ca2+ channel through which Ca2+ is released from the sarcoplasmic reticulum into the cytoplasm. One example of heart arrhythmia caused abnormal Ca2+ release is CPVT (Catecholaminergic Polymorphic Ventricular Tachycardia), a malignant inherited arrhythmia in which patients are at risk for lethal arrhythmias during exercise. CPVT has an estimated prevalence of 1:10000 and causes about 15% of autopsy negative cases of sudden unexplained death in the young. 60% of CPVT cases are caused by mutations in RYR2. Within RYR2, over 160 different mutations, clustered within 4 “hotspot” regions of the coding sequence, cause CPVT. Currently CPVT is not adequately treated by available options, and patients continue to suffer from sudden death or aborted sudden death, as well as morbidities arising from current therapies. Other forms of arrhythmia, such as atrial fibrillation, involve abnormal regulation of Ca2+ release from RYR2. Abnormal Ca2+ release from RYR2 can also contribute to contractile dysfunction in inherited and acquired forms of heart failure.
The present disclosure is based, at least in part, on the surprising finding of an interaction between the C-terminus of an endogenous cardiac protein MYBPC3 and RYR2, and that overexpression of this interacting domain suppressed aberrant RYR2 activity and alleviated arrhythmia. In some aspects, the present disclosure provides compositions and methods for treating a disorder associated with abnormal RYR2 function (e.g., arrhythmia or heart failure that are either inherited or acquired). In some embodiments, the subject treated using the methods described herein is a subject with arrhythmia whose response to existing medical management is sub-optimal.
Some aspects of the present disclosure provide methods of treating a disorder associated with abnormal ryanodine receptor type 2 (RYR2) function. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a polypeptide comprising a C-terminal domain of Cardiac Myosin binding protein C (MYBPC3). In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a C-terminal domain of Cardiac Myosin binding protein C (MYBPC3).
In some embodiments, the abnormal RYR2 function is caused by one or more mutations in RYR2. In some embodiments, the mutation in RYR2 causes excessive diastolic Ca2+ release in cardiomyocytes in the subject.
In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1-16 or 53-64. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-16 or 53-64.
In some embodiments, the nucleotide sequence is operably linked to a promoter. In some embodiments, the nucleic acid is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is selected from a lentiviral vector, a retroviral vector, or a recombinant adeno-associated virus (rAAV) vector.
In some embodiments, the viral vector is a rAAV vector further comprising two AAV inverted terminal repeats (ITRs) flanking the nucleotide sequence encoding the polypeptide and the promoter. In some embodiments, wherein the rAAV vector is packaged in a rAAV particle. In some embodiments, the rAAV particle further comprises a capsid protein. In some embodiments, the capsid protein is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh39, AAV.43, AAV2/2-66, AAV2/2-84, and AAV2/2-125, or a variant thereof. In some embodiments, the capsid protein is of a serotype AAV9. In some embodiments, the rAAV is a self-complementary AAV (scAAV). In some embodiments, the nucleotide sequence encoding the polypeptide is codon-optimized. In some embodiments, the nucleic acid is a messenger RNA (mRNA). In some embodiments, the mRNA is a modified mRNA.
In some embodiments, the polypeptide or the nucleic acid is delivered to a cardiomyocyte in the subject.
In some embodiments, the disorder is arrhythmia. In some embodiments, the arrhythmia is inherited or acquired. In some embodiments, the inherited arrhythmia is Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the acquired arrhythmia is a ventricular arrhythmia or a supraventricular arrhythmia. In some embodiments, the ventricular arrhythmia is ventricular tachycardia, ventricular fibrillation, or premature ventricular contraction. In some embodiments, the supraventricular arrhythmia is atrial fibrillation, atrial flutter, atrial tachycardia, premature atrial contraction, or paroxysmal supraventricular tachycardia. In some embodiments, the disorder is heart failure.
In some embodiments, administering the polypeptide or the nucleic acid reduces the excessive diastolic Ca2+ release in cardiomyocytes in the subject.
In some embodiments, the subject is human. In some embodiments, the administering is via injection.
Some aspects of the present disclosure provide methods of treating arrhythmia, the method comprises administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises a capsid protein of serotype AAV9 and a nucleotide sequence encoding a polypeptide comprising a C-terminal domain of Cardiac Myosin binding protein C (MYBPC3).
Other aspects of the present disclosure provide recombinant adeno-associated virus (rAAV) comprising a capsid protein and a nucleotide sequence encoding a polypeptide comprising a C-terminal domain of Cardiac Myosin binding protein C (MYBPC3).
In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-16 or 53-64.
Further provided herein are uses of the rAAV described herein in treating a disorder associated with abnormal ryanodine receptor type 2 (RYR2) function. In some embodiments, the disorder is arrhythmia. In some embodiments, the arrhythmia is inherited or acquired. In some embodiments, the inherited arrhythmia is Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the acquired arrhythmia is a ventricular arrhythmia or a supraventricular arrhythmia. In some embodiments, the ventricular arrhythmia is ventricular tachycardia, ventricular fibrillation, or premature ventricular contraction. In some embodiments, the supraventricular arrhythmia is atrial fibrillation, atrial flutter, atrial tachycardia, premature atrial contraction, or paroxysmal supraventricular tachycardia. In some embodiments, the disorder is heart failure.
The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. In the drawings:
CPVT (Catecholaminergic Polymorphic Ventricular Tachycardia) is a malignant inherited arrhythmia in which patients are at risk for lethal ventricular arrhythmias during exercise. CPVT is caused by mutations in cardiomyocyte Ca2+ handling genes. Over 60% of cases are caused by mutations in the gene RYR2 (ryanodine receptor type 2), which encodes the major intracellular Ca2+ release channel. We have discovered a novel interaction between the C-terminus of an endogenous cardiac protein and RYR2. Overexpression of this interacting domain suppressed aberrant RYR2 activity that is the root cause of arrhythmias in CPVT. This overexpression strategy normalized Ca2+ handling in human iPSC-derived cardiomyocytes, and suppressed arrhythmia in a mouse model of CPVT. Importantly, dysfunction of RYR2 is a final common pathway underlying diverse cardiac arrhythmias. Our findings on CPVT serve as a proof-of-concept. We believe that our therapeutic concept is likely applicable to other inherited and acquired arrhythmias.
The present disclosure, in some aspects, provides compositions and methods (e.g., gene therapy or protein therapy) for a disorder associated with abnormal RYR2 function. It was demonstrated herein that polypeptides comprising a C-terminal domain of Cardiac Myosin binding protein C (MYBPC3), or nucleic acids encoding such polypeptides are effective in treating arrhythmia. In some embodiments, the compositions and methods described herein can be used to treat arrhythmia or heart failure that are either inherited or acquired, including arterial fibrillation.
Accordingly, some aspects of the present disclosure provide methods of treating arrhythmia. In some embodiments, the method comprising administering to a subject in need thereof an effective amount of a polypeptide comprising a C-terminal domain of Cardiac Myosin binding protein C (MYBPC3). In some embodiments, the method comprising administering to a subject in need thereof an effective amount of a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a C-terminal domain of MYBPC3.
“Cardiac Myosin binding protein C (MYBPC3)” is found in cardiac muscle cells. In these cells, MYBPC3 is known to be associated with a structure called the sarcomere, which is the basic unit of muscle contraction. Sarcomeres are made up of thick and thin filaments. It was surprisingly found herein that, C-terminal domain fragments of the MYBPC3 protein localizes to dyads in the sarcomere, wherein the RYR2 protein is localized, while full-length MYBPC3 localizes to a different portion of the sarcomere. Human MYBPC3 protein sequence is provided under GenBank Accession No. NP_000247. Mouse MYBPC3 protein sequence is provided under GenBank Accession No. NP_032679.2. The domain structure of MYBPC3 is described in Sadayappan et al. (Biophys Rev. 2012 June; 4(2): 93-106, incorporated herein by references) and also illustrated in
In some embodiments, the polypeptide used in the methods described herein comprises a C-terminal domain (e.g., the C7-C8 domains as shown in
In some embodiments, the polypeptide used in the methods described herein comprises the full-length mouse MYBPC3 of SEQ ID NO: 1, consists essentially of the full-length mouse MYBPC3 of SEQ ID NO: 1 or consists of the full-length mouse MYBPC3 of SEQ ID NO: 1. In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C6-C7 (SEQ ID NO: 2), consists essentially of the mouse MYBPC3 C6-C7 (SEQ ID NO: 2) or consists of the mouse MYBPC3 C6-C7 (SEQ ID NO: 2). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C6-C8 (SEQ ID NO: 3), consists essentially of the mouse MYBPC3 C6-C8 (SEQ ID NO: 3) or consists of the mouse MYBPC3 C6-C8 (SEQ ID NO: 3). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C6-C9 (SEQ ID NO: 4), consists essentially of the mouse MYBPC3 C6-C9 (SEQ ID NO: 4) or consists of the mouse MYBPC3 C6-C9 (SEQ ID NO: 4). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C6-C10 (SEQ ID NO: 5), consists essentially of the mouse MYBPC3 C6-C10 (SEQ ID NO: 5) or consists of the mouse MYBPC3 C6-C10 (SEQ ID NO: 5). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C8-C10 (SEQ ID NO: 6), consists essentially of the mouse MYBPC3 C8-C10 (SEQ ID NO: 6) or consists of the mouse MYBPC3 C8-C10 (SEQ ID NO: 6). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C9-C10 (SEQ ID NO: 7), consists essentially of the mouse MYBPC3 C6-C7 (SEQ ID NO: 7) or consists of the mouse MYBPC3 C6-C7 (SEQ ID NO: 7). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C10 (SEQ ID NO: 8), consists essentially of the mouse MYBPC3 C10 (SEQ ID NO: 8) or consists of the mouse MYBPC3 C10 (SEQ ID NO: 8). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C7-C8 (SEQ ID NO: 59), consists essentially of the mouse MYBPC3 C7-C8 (SEQ ID NO: 59) or consists of the mouse MYBPC3 C7-C8 (SEQ ID NO: 59). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C7 (SEQ ID NO: 60), consists essentially of the mouse MYBPC3 C7 (SEQ ID NO: 60) or consists of the mouse MYBPC3 C7 (SEQ ID NO: 60). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C8 (SEQ ID NO: 61), consists essentially of the mouse MYBPC3 C8 (SEQ ID NO: 61) or consists of the mouse MYBPC3 C8 (SEQ ID NO: 61). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C7-C10 (SEQ ID NO: 62), consists essentially of the mouse MYBPC3 C7-C10 (SEQ ID NO: 62) or consists of the mouse MYBPC3 C7-C10 (SEQ ID NO: 62). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C6, C8-C10 (SEQ ID NO: 63), consists essentially of the mouse MYBPC3 C6, C8-C10 (SEQ ID NO: 63) or consists of the mouse MYBPC3 C6, C8-C10 (SEQ ID NO: 63). In some embodiments, the polypeptide used in the methods described herein comprises the mouse MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 64), consists essentially of the mouse MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 64) or consists of the mouse MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 64).
In some embodiments, the polypeptide used in the methods described herein comprises the full length human MYBPC3 of SEQ ID NO: 9, consists essentially of the full length human MYBPC3 of SEQ ID NO: 9 or consists of the full length human MYBPC3 of SEQ ID NO: 9. In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C6-C7 (SEQ ID NO: 10), consists essentially of the human MYBPC3 C6-C7 (SEQ ID NO: 10) or consists of the human MYBPC3 C6-C7 (SEQ ID NO: 10). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C6-C8 (SEQ ID NO: 11), consists essentially of the human MYBPC3 C6-C8 (SEQ ID NO: 11) or consists of the human MYBPC3 C6-C8 (SEQ ID NO: 11). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C6-C9 (SEQ ID NO: 12), consists essentially of the human MYBPC3 C6-C9 (SEQ ID NO: 12) or consists of the human MYBPC3 C6-C9 (SEQ ID NO: 12). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C6-C10 (SEQ ID NO: 13), consists essentially of the human MYBPC3 C6-C10 (SEQ ID NO: 13) or consists of the human MYBPC3 C6-C10 (SEQ ID NO: 13). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C8-C10 (SEQ ID NO: 14), consists essentially of the human MYBPC3 C8-C10 (SEQ ID NO: 14) or consists of the human MYBPC3 C8-C10 (SEQ ID NO: 14). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C9-C10 (SEQ ID NO: 15), consists essentially of the human MYBPC3 C6-C7 (SEQ ID NO: 15) or consists of the human MYBPC3 C6-C7 (SEQ ID NO: 15). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C10 (SEQ ID NO: 16), consists essentially of the human MYBPC3 C10 (SEQ ID NO: 16) or consists of the human MYBPC3 C10 (SEQ ID NO: 16). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C7-C8 (SEQ ID NO: 53) consists essentially of the human MYBPC3 C7-C8 (SEQ ID NO: 53) or consists of the human MYBPC3 C7-C8 (SEQ ID NO: 53). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C7 (SEQ ID NO: 54), consists essentially of the human MYBPC3 C7 (SEQ ID NO: 54) or consists of the human MYBPC3 C7 (SEQ ID NO: 54). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C8 (SEQ ID NO: 55), consists essentially of the human MYBPC3 C8 (SEQ ID NO: 55) or consists of the human MYBPC3 C8 (SEQ ID NO: 55). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C7-C10 (SEQ ID NO: 56), consists essentially of the human MYBPC3 C7-C10 (SEQ ID NO: 56) or consists of the human MYBPC3 C7-C10 (SEQ ID NO: 56). In some embodiments, the polypeptide used in the methods described herein comprises the human MYBPC3 C6, C8-C10 (SEQ ID NO: 57) consists essentially of the human MYBPC3 C6, C8-C10 (SEQ ID NO: 57) or consists of the human MYBPC3 C6, C8-C10 (SEQ ID NO: 57). In some embodiments, the polypeptide used in the methods described herein comprises the human MYPBC3 C6-C7, C9-C10 (SEQ ID NO: 58), consists essentially of the human MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 58) or consists of the human MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 58),In some embodiments, the polynucleotide used in the methods described herein comprises the full-length mouse MYBPC3 (SEQ ID NO: 17), consists essentially of the full-length mouse MYBPC3 (SEQ ID NO: 17) or consists of the full-length mouse MYBPC3 (SEQ ID NO: 17). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C6-C7 (SEQ ID NO: 18), consists essentially of the mouse MYBPC3 C6-C7 (SEQ ID NO: 18) or consists of the mouse MYBPC3 C6-C7 (SEQ ID NO: 18). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C6-C8 (SEQ ID NO: 19), consists essentially of the mouse MYBPC3 C6-C8 (SEQ ID NO: 19) or consists of the mouse MYBPC3 C6-C8 (SEQ ID NO: 19). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C6-C9 (SEQ ID NO: 20), consists essentially of the mouse MYBPC3 C6-C9 (SEQ ID NO: 20) or consists of the mouse MYBPC3 C6-C9 (SEQ ID NO: 20). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C6-C10 (SEQ ID NO: 21), consists essentially of the mouse MYBPC3 C6-C10 (SEQ ID NO: 21) or consists of the mouse MYBPC3 C6-C10 (SEQ ID NO: 21). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C8-C10 (SEQ ID NO: 22), consists essentially of the mouse MYBPC3 C8-C10 (SEQ ID NO: 22) or consists of the mouse MYBPC3 C8-C10 (SEQ ID NO: 22). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C9-C10 (SEQ ID NO: 23), consists essentially of the mouse MYBPC3 C6-C7 (SEQ ID NO: 23) or consists of the mouse MYBPC3 C6-C7 (SEQ ID NO: 23). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C10 (SEQ ID NO: 24), consists essentially of the mouse MYBPC3 C10 (SEQ ID NO: 24) or consists of the mouse MYBPC3 C10 (SEQ ID NO: 24). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C7-C8 (SEQ ID NO: 71), consists essentially of the mouse MYBPC3 C7-C8 (SEQ ID NO: 71) or consists of the mouse MYBPC3 C7-C8 (SEQ ID NO: 71). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C7 (SEQ ID NO: 72), consists essentially of the mouse MYBPC3 C7 (SEQ ID NO: 72) or consists of the mouse MYBPC3 C7 (SEQ ID NO: 72). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C8 (SEQ ID NO: 73), consists essentially of the mouse MYBPC3 C8 (SEQ ID NO: 73) or consists of the mouse MYBPC3 C8 (SEQ ID NO: 73). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C7-C10 (SEQ ID NO: 74), consists essentially of the mouse MYBPC3 C7-C10 (SEQ ID NO: 74) or consists of the mouse MYBPC3 C7-C10 (SEQ ID NO: 74). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C6, C8-C10 (SEQ ID NO: 75), consists essentially of the mouse MYBPC3 C6, C8-C10 (SEQ ID NO: 75) or consists of the mouse MYBPC3 C6, C8-C10 (SEQ ID NO: 75). In some embodiments, the polynucleotide used in the methods described herein comprises the mouse MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 76), consists essentially of the mouse MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 76) or consists of the mouse MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 76).
In some embodiments, the polynucleotide used in the methods described herein comprises the full length human MYBPC3 (SEQ ID NO: 25), consists essentially of the full length human MYBPC3 (SEQ ID NO: 25) or consists of the full length human MYBPC3 (SEQ ID NO: 25). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C6-C7 (SEQ ID NO: 26), consists essentially of the human MYBPC3 C6-C7 (SEQ ID NO: 26) or consists of the human MYBPC3 C6-C7 (SEQ ID NO: 26). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C6-C8 (SEQ ID NO: 27), consists essentially of the human MYBPC3 C6-C8 (SEQ ID NO: 27) or consists of the human MYBPC3 C6-C8 (SEQ ID NO: 27). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C6-C9 (SEQ ID NO: 28), consists essentially of the human MYBPC3 C6-C9 (SEQ ID NO: 28) or consists of the human MYBPC3 C6-C9 (SEQ ID NO: 28). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C6-C10 (SEQ ID NO: 29), consists essentially of the human MYBPC3 C6-C10 (SEQ ID NO: 29) or consists of the human MYBPC3 C6-C10 (SEQ ID NO: 29). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C8-C10 (SEQ ID NO: 30), consists essentially of the human MYBPC3 C8-C10 (SEQ ID NO: 30) or consists of the human MYBPC3 C8-C10 (SEQ ID NO: 30). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C9-C10 (SEQ ID NO: 31), consists essentially of the human MYBPC3 C6-C7 (SEQ ID NO: 31) or consists of the human MYBPC3 C6-C7 (SEQ ID NO: 31). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C10 (SEQ ID NO: 32), consists essentially of the human MYBPC3 C10 (SEQ ID NO: 32) or consists of the human MYBPC3 C10 (SEQ ID NO: 32). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C7-C8 (SEQ ID NO: 65) consists essentially of the human MYBPC3 C7-C8 (SEQ ID NO: 65) or consists of the human MYBPC3 C7-C8 (SEQ ID NO: 65). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C7 (SEQ ID NO: 66), consists essentially of the human MYBPC3 C7 (SEQ ID NO: 66) or consists of the human MYBPC3 C7 (SEQ ID NO: 66). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C8 (SEQ ID NO: 67), consists essentially of the human MYBPC3 C8 (SEQ ID NO: 67) or consists of the human MYBPC3 C8 (SEQ ID NO: 67). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C7-C10 (SEQ ID NO: 68), consists essentially of the human MYBPC3 C7-C10 (SEQ ID NO: 68) or consists of the human MYBPC3 C7-C10 (SEQ ID NO: 68). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYBPC3 C6, C8-C10 (SEQ ID NO: 69) consists essentially of the human MYBPC3 C6, C8-C10 (SEQ ID NO: 69) or consists of the human MYBPC3 C6, C8-C10 (SEQ ID NO: 69). In some embodiments, the polynucleotide used in the methods described herein comprises the human MYPBC3 C6-C7, C9-C10 (SEQ ID NO: 70), consists essentially of the human MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 70) or consists of the human MYBPC3 C6-C7, C9-C10 (SEQ ID NO: 70).
In some embodiments, the polypeptide used in the methods described herein comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to any one of SEQ ID NOs: 1-16 or 53-64. In some embodiments, the polypeptide used in the methods described herein comprises an amino acid sequence that is 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 1-16 or 53-64. In some embodiments, the polypeptide used in the methods described herein comprises the amino acid sequence of SEQ ID NOs: 1-16 or 53-64.
In some embodiments, the nucleic acid used in the methods described herein comprises a nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein). In some embodiments, the nucleic acid used in the methods described herein comprises a nucleotide sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to any one of SEQ ID NOs: 17-32 or 65-76. In some embodiments, the nucleic acid used in the methods described herein comprises a nucleotide sequence that is 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 17-32 or 65-76. In some embodiments, the nucleic acid used in the methods described herein comprises the nucleotide sequence of SEQ ID NOs: 17-32 or 65-76.
As used herein, “nucleic acids” may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof. The nucleic acids molecules of the present disclosure may be DNA or RNA. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the present disclosure will recite “T”s in a representative DNA sequence but where the sequence represents RNA, the “T”s would be substituted for “U”s.
In some embodiments, the nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein) is operably linked to a promoter.
A “promoter” is a control region of a nucleic acid at which initiation and rate of transcription of the remainder of a nucleic acid are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules, such as transcription factors, bind. Promoters of the present disclosure may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid that it regulates. A promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to the nucleic acid it regulates to control (“drive”) transcriptional initiation and/or expression of that nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter (also referred to as regulatable promoter).
Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter [Invitrogen]. In some embodiments, a promoter is an enhanced chicken β-actin promoter. In some embodiments, a promoter is a U6 promoter. In some embodiments, the promoter used in present disclosure is a CAG promoter (e.g., containing a CMV enhancer, a promoter and the first exon and the first intron from the chicken beta-actin gene, and a splice acceptor of the rabbit beta-globin gene, as described in Okabe et al., FEB S Lett. 1997 May 5; 407(3):313-9; and Alexopoulou et al., BMC Cell Biology 9: 2, 2008, incorporated herein by reference).
Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.
In some embodiments, inducible promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used (Yao et al., Human Gene Therapy; Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).
In some embodiments, the native promoter for MYBPC3 used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.
In some embodiments, the promoter is a tissue-specific promoter containing regulatory sequences that impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor α-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among others which will be apparent to the skilled artisan.
In some embodiments, the nucleic acid used in the method described herein is a messenger RNA (mRNA). A “messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. In some preferred embodiments, an mRNA is translated in vivo. The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “U.” One of ordinary skill in the art would understand how to identify an mRNA sequence based on the corresponding DNA sequence.
The basic components of an mRNA molecule typically include at least one coding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and a poly-A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.
In some embodiments, the mRNA described herein comprises one or more chemical modifications (e.g., comprises one or more modified nucleotides). The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties.
The mRNAs described herein, some embodiments, comprise various (more than one) different modifications. In some embodiments, a particular region of a mRNA contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified mRNA, introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified mRNA. In some embodiments, a modified mRNA introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).
Modifications of polynucleotides include, without limitation, those described herein. Modified mRNAs of the present disclosure may comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications. The mRNAs may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).
The mRNAs described herein, in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
In some embodiments, the modified mRNA comprises one or more modified nucleosides and nucleotides. A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
In some embodiments, modified nucleobases in the modified mRNA described herein are selected from the group consisting of pseudouridine (ψ), N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.
In some embodiments, the nucleic acid used in the methods described herein is a vector (e.g., a cloning vector or an expression vector). The vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.
An expression vector comprising the nucleic acid can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the polypeptides described herein. In some embodiments, the expression of the polypeptides described herein is regulated by a constitutive, an inducible or a tissue-specific promoter.
A variety of host-expression vector systems may be utilized in accordance with the present disclosure. Such host-expression systems represent vehicles by which the nucleotide sequences described herein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide sequences, express the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein) in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein); yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein); insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) containing the nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein); plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein); or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In some embodiments, the vector of the present disclosure is a viral vector. In some embodiments, the viral vector is suitable for mammalian expression of the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein). Suitable viral vectors include lentiviral vectors, retroviral vectors, or a recombinant adeno-associated virus (rAAV) vectors.
A “lentiviral vector” refers to a vector derived from a lentivirus genome (e.g., HIV). Lentiviral vectors have been commonly used in gene therapy, e.g., to insert beneficial genes into a host cell or organism, or to delete or modify a gene in a host cell or organism. Lentiviral vectors are efficient vehicles for gene transfer in mammalian cells due to their capacity to stably express a gene of interest in non-dividing and dividing cells.
A “retroviral vector” refers to a vector derived from a retrovirus genome. A retroviral vector consists of proviral sequences that can accommodate the gene of interest, to allow incorporation of both into the target cells. The vector also contains viral and cellular gene promoters, such as the CMV promoter, to enhance expression of the gene of interest in the target cells. Retroviral vectors have also been commonly used in gene therapy.
A “recombinant adeno-associated virus (rAAV) vector” is typically composed of, at a minimum, a transgene and its regulatory sequences (e.g., a promoter), and 5′ and 3′ AAV inverted terminal repeats (ITRs). The transgene may comprise, as disclosed elsewhere herein, a nucleotide sequence encoding, for example, a polypeptide comprising a C-terminal domain of MYBPC3, as described elsewhere in the disclosure.
Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the rAAV vectors described herein comprises two ITRs flanking (one ITR on each end of the sequence being flanked) the nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein). In some embodiments, the nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein) is operably linked to a promoter and the rAAV vectors described herein comprises two ITRs flanking (one ITR on each end of the sequence being flanked) the nucleotide sequence encoding the polypeptide (e.g., a polypeptide comprising a C-terminal domain of MYBPC3 described herein) and the promoter.
In some embodiments, the ITRs are of a serotype selected from AAV1, AAV2, AAV2i8, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2-125, and variants thereof. In some embodiments, the rAAV vector comprises ITRs of serotype AAV2. In some embodiments, the ITR used in the rAAV vector described herein comprises the nucleotide sequence of:
In some embodiments, the rAAV vector of the present disclosure is a self-complementary AAV vector (scAAV). A “self-complementary AAV vector” (scAAV) refers to a vector containing a double-stranded vector genome generated by the absence of a terminal resolution site (TR) from one of the ITRs of the AAV (e.g., as described in McCarthy (2008) Molecular Therapy 16(10):1648-1656, incorporated herein by reference). The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present. In general, scAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle. The instant invention is based, in part, on the recognition that DNA fragments encoding RNA hairpin structures (e.g. shRNA, miRNA, and AmiRNA) can serve a function similar to a mutant inverted terminal repeat (mTR) during viral genome replication, generating self-complementary AAV vector genomes. In some embodiments, the ITR used in the scAAV vector described herein comprises the nucleotide sequence of:
Further provided herein, in some aspects, are recombinant adeno-associated virus (rAAV) comprising a capsid protein and any one of the nucleic acid molecules described herein. In some embodiments, a “capsid protein” refers to structural proteins encoded by the CAP gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host.
In some embodiments, an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV2i8, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAV2/2-66, AAV2/2-84, AAV2/2-125. In some embodiments, an AAV capsid protein is of a serotype derived from a non-human primate, for example scAAV.rh8, AAV.rh39, or AAV.rh43 serotype. In some embodiments, an AAV capsid protein is of an AAV9 serotype. In some embodiments, an AAV capsid protein is of an AAV2i8 serotype. Non-limiting examples of the amino acid sequences of capsid proteins are provided as SEQ ID NOs: 35-52.
Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003/0138772), the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.
The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.
In some embodiments, recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the “AAV helper function” sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
In some aspects, the present disclosure provides rAAV vector transfected host cells. The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. In some embodiments, a host cell is a bacterial cell, yeast cell, insect cell (519), or a mammalian (e.g., human, rodent, non-human primate, etc.) cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. In some embodiments, the host cell in accordance with the present disclosure is a cardiomyocyte.
In some embodiments, the polypeptides or the nucleic acids (e.g., mRNAs, viral vectors, or rAAV) encoding the polypeptide are formulated in compositions (e.g., pharmaceutical compositions) for administration to a subject for treating arrhythmia. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
The phrase “pharmaceutically acceptable” is employed herein to refer 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. The phrase “pharmaceutically acceptable carrier” means 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 and not injurious to the tissue of the patient (e.g., physiologically compatible, sterile, physiologic pH, etc.). The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the composition (e.g., pharmaceutical composition) is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.
Typically, the compositions (e.g., pharmaceutical compositions) may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
In some embodiments, the compositions comprise any one of the rAAVs described herein. In some embodiments, these compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ˜1013 GC/ml or more). Methods for reducing aggregation of rAAVs are well known in the art and include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
The formulation of the pharmaceutical composition may dependent upon the route of administration. Injectable preparations suitable for parenteral administration or intratumoral, peritumoral, intralesional or perilesional administration include, for example, sterile injectable aqueous or oleaginous suspensions and may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 propanediol or 1,3 butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
For topical administration, the pharmaceutical composition can be formulated into ointments, salves, gels, or creams, as is generally known in the art. Topical administration can utilize transdermal delivery systems well known in the art. An example is a dermal patch.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the anti-inflammatory agent, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the anti-inflammatory agent is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
In some embodiments, the pharmaceutical compositions used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. The polypeptides, nucleic acids, rAAV, or pharmaceutical composition ordinarily will be stored in lyophilized form or as an aqueous solution if it is highly stable to thermal and oxidative denaturation. The pH of the preparations typically will be about from 6 to 8, although higher or lower pH values can also be appropriate in certain instances.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.
Sterile injectable solutions are prepared by incorporating the active agents in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the nucleic acids, proteins, or rAAVs may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids, proteins, or the rAAVs disclosed herein. The formation and use of liposomes are generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, containing an aqueous solution in the core.
Alternatively, nanocapsule formulations of the active agents may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the compositions to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).
The compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
Other aspects of the present disclosure provide uses of any one of the polypeptides, nucleic acids, the rAAV, or the composition described herein for use in treating arrhythmia. In some embodiments, the method of treating arrhythmia comprises administering to a subject in need thereof an effective amount of a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises a capsid protein (e.g., a capsid protein of serotype AAV9) and a nucleotide sequence encoding a polypeptide comprising a C-terminal domain of MYBPC3 (e.g., the polypeptide of any one of SEQ ID NOs: 1-16).
In its broadest sense, the terms “treatment” or “to treat” refer to both therapeutic and prophylactic treatments. If the subject is in need of treatment of a disease (e.g., arrhythmia), “treating the condition” refers to ameliorating, reducing or eliminating one or more symptoms associated with the or preventing any further progression of the disease (e.g., arrhythmia). If the subject in need of treatment is one who is at risk of having arrhythmia, then treating the subject refers to reducing the risk of the subject having arrhythmia or preventing the subject from developing arrhythmia.
A subject shall mean a human or vertebrate animal or mammal including but not limited to a rodent, e.g., a rat or a mouse, dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, and primate, e.g., monkey. The methods of the present disclosure are useful for treating a subject in need thereof.
The term “therapeutically effective amount” of the present disclosure refers to the amount necessary or sufficient to realize a desired biologic effect. For example, a therapeutically effective amount of the polypeptide or nucleic acid encoding such associated with the present disclosure may be that amount sufficient to ameliorate one or more symptoms of arrhythmia. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular therapeutic compounds being administered the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular therapeutic compound associated with the present disclosure without necessitating undue experimentation.
In some embodiments, an “effective amount” of an rAAV is an amount sufficient to target infect an animal, target a desired tissue (e.g., heart tissue). The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109 to 1016 genome copies. In some embodiments, a dosage between about 1013 to 1015 rAAV genome copies is appropriate.
The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., delivery to the heart), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.
The polypeptides, nucleic acids, rAAVs, and compositions comprising such of the disclosure may be delivered to a subject in compositions according to any appropriate methods known in the art. For example, an rAAV, preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some embodiments a host animal does not include a human.
Delivery of the polypeptides, nucleic acids, rAAVs, and compositions to a mammalian subject may be by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, the polypeptides, nucleic acids, rAAVs, and compositions as described in the disclosure are administered by intravenous injection. In some embodiments, the polypeptides, nucleic acids, rAAVs, and compositions are administered by intramuscular injection. In some embodiments, the polypeptides, nucleic acids, rAAVs, and compositions are administered by injection into the heart. In some embodiments, the polypeptides, nucleic acids, rAAVs, and compositions are delivered to a cardiomyocyte in the subject.
In some embodiments, a dose of the polypeptides, nucleic acids, rAAVs, or compositions are administered to a subject by intramuscular injection no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of the polypeptides, nucleic acids, rAAVs, or compositions are administered by intramuscular injection to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of the polypeptides, nucleic acids, rAAVs, or compositions is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of the polypeptides, nucleic acids, rAAVs, or compositions is administered to a subject no more than bi-weekly (e.g., once in a two-calendar week period). In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of the polypeptides, nucleic acids, rAAVs, or compositions is administered to a subject no more than once per six calendar months. In some embodiments, a dose of the polypeptides, nucleic acids, rAAVs, or compositions is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year). In some embodiments, a dose of the polypeptides, nucleic acids, rAAVs, or compositions is administered to a subject as single dose therapy.
The disorders that may be treated using the methods described herein are associated with abnormal ryanodine receptor type 2 (RYR2) function. In some embodiments, the abnormal RYR2 function is caused by one or more (e.g., 1, 2, 3, 4, 5, or more) mutations in RYR2. In some embodiments, the abnormal RYR2 function (e.g., caused by mutations in RYR2) is associated with excessive (e.g., at least 20%, at least 50%, at least 100%, at least 2-fold, at least 10-fold, at least 100-fold or more) diastolic Ca2+ release in cardiomyocytes in the subject. Mutations in RYR2 that cause excessive diastolic Ca2+ release in cardiomyocytes are known in the art, e.g., as described in Jiang et al., PNAS Aug. 31, 2004 101 (35) 13062-13067; Liu et al., PLoS One. 2017; 12(9): e0184177; and Postma et al., J Med Genet. November; 42(11):863-70, incorporated herein by reference.
In some embodiments, the disorder associated with abnormal RYR2 function is arrhythmia. In some embodiments, the arrhythmia is inherited or acquired. In some embodiments, the inherited arrhythmia is Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the CPVT is associated with a mutation in RYR2. In some embodiments, the acquired arrhythmia is a ventricular arrhythmia or a supraventricular arrhythmia. In some embodiments, the ventricular arrhythmia is ventricular tachycardia, ventricular fibrillation, or premature ventricular contraction. In some embodiments, the supraventricular arrhythmia is atrial fibrillation, atrial flutter, atrial tachycardia, premature atrial contraction, or paroxysmal supraventricular tachycardia. In some embodiments, the disorder associated with abnormal RYR2 function is heart failure.
In some embodiments, administering the polypeptide, the nucleic acid, or the rAAV reduces the excessive diastolic Ca2+ release (e.g., by at least 20%, at least 50%, or at least 90%) in cardiomyocytes in the subject. In some embodiments, administering the polypeptide, the nucleic acid, or the rAAV restores the diastolic Ca2+ release to a normal level in cardiomyocytes in the subject. In some embodiments, the normal level is the level of diastolic Ca2+ release in a healthy subject.
CPVT (Catecholaminergic Polymorphic Ventricular Tachycardia) is a malignant inherited arrhythmia in which patients are at risk for lethal arrhythmias during exercise1. CPVT has an estimated prevalence of 1:10000 and causes about 15% of autopsy negative cases of sudden unexplained death in the young2. 60% of CPVT cases are caused by mutations in ryanodine receptor type 2 (RYR2)1,3, the major intracellular Ca2+ release channel of cardiomyocytes. Within RYR2, over 160 different mutations, clustered within 4 “hotspot” regions of the coding sequence4, are known to cause CPVT. Currently CPVT is not adequately treated by available options, and patients continue to suffer from sudden death or aborted sudden death, as well as morbidities arising from current therapies5. Therefore the immediate proof-of-concept market space are patients with CPVT whose response to medical management is sub-optimal. Ultimately, it is anticipated that the gene therapy approach could become standard treatment for CPVT.
Mutations in CPVT interfere with normal cardiomyocyte Ca2+ handling. With each heartbeat, Ca2+ levels rise in systole, signaling sarcomeres to contract, and decline in diastole, causing sarcomeres to relax. These changes in cytoplasmic Ca2+ concentration are initiated by depolarization of the plasma membrane, which opens the L-type Ca2+ channel to allow a small amount of extracellular Ca2+ to enter the cell. This Ca2+ entry stimulates RYR2, located on the sarcoplasmic reticulum, to open and release much more Ca2+. This Ca2+-induced Ca2+ release rapidly increases cytosolic Ca2+, which coordinates sarcomere contraction. Time-dependent closure of the L-type Ca2+ channel and RYR2, along with active return of cytosolic Ca2+ to the sarcoplasmic reticulum by an ATP-dependent pump (SERCA2A), return Ca2+ concentrations to a low level in diastole. A small amount of Ca2+ returns to the extracellular space via the Na+/Ca2+ exchanger, NCX. CPVT mutations cause excessive diastolic Ca2+ release through RYR2. The elevated diastolic Ca2+ drives greater Na+/Ca2+ exchange. Since this exchange is electrogenic, elevated exchange results in membrane depolarization (after-depolarizations), which can result in another action potential (“triggered activity”) or create heterogeneity of repolarization that can cause arrhythmic impulse propagation (“re-entry”)6,7.
The mechanism of action of the therapeutic described herein is to limit the excessive activity of RYR2, which is central to the pathogenesis of CPVT. Importantly, dysfunction of RYR2 is a final common pathway of many types of heart disease, and therefore it is likely that the indications for this anti-arrhythmic therapy could be expanded to include other types of inherited or acquired cardiomyopathy, including atrial fibrillation8 (prevalence, 1% of population and 9% of patients over 80 years of age).
Patients with CPVT are imperfectly treated by current medical and surgical options5,9,10. The current medical options have substantial side effects and afford incomplete protection.
Our current medical option is exercise restriction. Exercise restriction is difficult in children and adolescents, and limiting exercise has lifelong psychosocial and medical implications. The long-term benefits of exercise are increasingly recognized and associated with cardiovascular, metabolic, and inflammatory disorders and the lifetime risk of breast, endometrial and colon malignancies.11-13
Another current option is utilizing high dose beta-blockers. High dose beta-blockade is frequently difficult to tolerate due to effects on overall energy level and mood. As a result, non-compliance with beta-blockers, or sub-therapeutic dosing, is common. In a recent study, treatment failure (syncope or cardiac arrest) occurred in 25% to 33% of patients managed primarily with beta-blockers5,14. Suboptimal dosing and non-adherence to prescribed therapy occurred in 41% and 48% of these treatment failures, respectively5.
Another current medical option is flecainide. The combination of beta-blocker plus flecainide, a sodium channel blocker, has been found to be effective for patients with CPVT15. In adult heart disease trials, flecainide had substantial pro-arrhythmic effects and increased mortality16. Whether or not flecainide increases long term survival in CPVT is not known. In acute exercise testing, 76% of patients responded to flecainide, and 24% did not17. In a retrospective study with limited follow-up (median 1.7 years), flecainide appeared promising, although 38% of patients had persistent symptoms5.
Yet another current medical option is left cardiac sympathetic denervation (LCSD). Surgical interruption of the left cervical sympathetic chain reduces adrenergic stimulation to the heart and has been beneficial to some CPVT patients who have breakthrough arrhythmias on medical management. LCSD should be performed at a specialized center, and surgical complications such as Homer's syndrome are not uncommon. LCSD reduced frequency of cardiac events, but in a median 37-month follow-up, 24% of patients had at least one recurrent cardiac event18.
Still another current medical option is implanted cardiac defibrillators (ICDs). In children and adolescents with CPVT, ICD complications were common and associated with a high burden of shocks10. ICDs were effective in terminating ventricular fibrillation but not ventricular tachycardia9. Furthermore, ICD discharge in an awake patient results in catecholamine release that can precipitate further arrhythmia, leading to potentially fatal “electrical storm”. Recent evidence shows no survival benefit from ICDs for patients who present with cardiac arrest secondary to CPVT. For these reasons, ICD placement for CPVT should be avoided whenever possible, although this leaves patients dependent on medication with the associated issues of compliance and breakthrough events19.
CPVT remains a major cause of morbidity and mortality in otherwise healthy, functional children with very significant societal and economic costs despite the relative rarity of the disease. Repeated hospital visits for clinical assessment and procedures expose the patient and institution to significant costs.
The present disclosure proposes compositions and methods for treating CPVT. The composition comprises AAV-CTDP, in which adeno-associated virus with a cardiomyocyte-selective promoter expresses a peptide, CTDP (MYBPC3 C-terminus-derived peptide), that reduces the aberrant activity of RYR2, the underlying cause of arrhythmia in CPVT and many other inherited and acquired arrhythmias.
The target population are all patients with CPVT, although patients who failed medical management (breakthrough arrhythmias on beta-blockers and flecainide) are started with. The gene therapy vector are delivered by intravenous infusion as single dose treatment. The gene therapy method described herein reduces mortality and breakthrough arrhythmias, reduce the need for LCSD and ICDs, reduces or eliminate the need for high dose beta-blockers, and permit some level of exercise. These changes would vastly improve quality of life for CPVT patients. Successful gene therapy would reduce the impact on patient outcome of medical compliance, which is a difficult issue with life or death consequences in these teenage and young adult patients. These benefits are expected based on the preliminary determination of efficacy in a CPVT mouse model and in human iPSC-derived cardiomyocytes harboring CPVT mutations.
It is anticipated that the compositions and methods described herein could extend to other arrhythmias that are more common than CPVT in which abnormal Ca2+ release from RYR2 is central to disease pathogenesis20. One likely expansion indication is atrial fibrillation, which affects 9% of patients 80 years of age and greater.
One potential alternative to AAV-mediated delivery of CTDP is delivery as a cell penetrating peptide. Compared to AAV gene therapy, peptide therapy has properties and cost more similar to a conventional pharmaceutical. However, peptide levels and cardiac specificity would likely be lower than for AAV gene therapy. Additionally, to be clinically effective, the product would need to be orally available, which could be a challenge for peptide therapy. For these reasons, the primary strategy is AAV gene therapy, with peptide-based therapy being a potential alternative that is contingent upon improvements in cell penetrating peptide technology.
Proximity proteomics were performed to identify proteins that localize to dyads, where RYR2 is localized. This identified peptides derived from the C-terminus of MYBPC3, a sarcomere protein (
To determine the functional significance of this interaction, AAV was developed to deliver portions of the MYBPC3 C-terminus to the mouse heart. MYBPC3 is composed of several immunoglobulin-like and fibronectin-like domains, labeled C1-C10 (
An important consideration for the feasibility of human gene therapy is the percent of cardiomyocytes that need to be transduced to achieve efficacy. A parallel question is whether partial myocardial transduction and resulting myocardial heterogeneity might be pro-arrhythmic. Although answers to these questions specifically with respect to AAV-MYBPC3 have yet to be determined, results from other gene therapy studies for CPVT are informative. In AAV gene replacement therapy for CPVT caused by CASQ2 deficiency (the autosomal recessive form of CPVT), Priori and colleagues reported therapeutic efficacy and no pro-arrhythmia in mice with ˜40% cardiomyocyte transduced24,25. Similarly, in the report of AAV-mediated CaMKII inhibition to treat CPVT caused by RYR2 mutation, therapeutic efficacy without pro-arrhythmia was observed in mice with 50% cardiomyocytes transduced21. Formal dose-response experiments are underway to determine the minimum transduction efficiency needed for efficacy; based on pilot experiments with low numbers of replicates, it is believed to be approximately 20%. The mechanism is likely based in a concept known as “source-sink mis-match”: Because cardiomyocytes are electrically connected to their neighbors, the activity of one cardiomyocyte is stabilized by its interactions with neighboring cells. For a cardiomyocyte to aberrantly depolarize, it needs to generate sufficient current to also depolarize neighboring cells. In this way, a low fraction of cardiomyocytes that are resistant to aberrant activity can stabilize a network of cells.
The effect of MYBPC3 on Ca2+ handling of human CPVT patient-derived iPSC-CMs was evaluated. MYBPC3 expression reduced the frequency of Ca2+ sparks in CPVT iPSC-CMs stimulated with isoproterenol, a beta-adrenergic agent (
Conducting dose response experiments with a therapeutic candidate vector without a reporter gene can make measurement of transduction efficiency difficult. However, this is a key parameter to scale dosing between species. To overcome this difficulty, RNA in situ hybridization methods were established in the laboratory. For example, for a separate project using AAV-TAZ to treat a mouse model of Barth syndrome, RNAscope RNA in situ hybridization was used to measure the fraction of cardiomyocytes that were transduced. This same technology are used here to measure transduction efficiency without relying on a reporter gene embedded in the therapeutic candidate vector.
Current standard of care has been effective at reducing the risk of cardiac arrest and death for CPVT patients. However, protection is incomplete and cardiac arrest and death continue to be a threat. Incomplete protection from current SOC is due to (1) intolerable side effects of current management, which result in non-compliance; and (2) failure to target the root cause of CPVT, dysfunction of RYR2. Exercise restriction, beta-blockers, and cardiac sympathetic denervation are designed to minimize pro-arrhythmic effects of beta-adrenergic signaling that trigger arrhythmia in CPVT patients. However, a recent retrospective study showed that about one fifth of cardiac events in CPVT were not provoked by an identifiable excitatory stimulus5, suggesting that removal of adrenergic signaling by itself may not be fully protective. The incomplete protection of many patients by exercise restriction, beta-blockers5,14, and even surgical sympathetic denervation indicate that targeting this signaling pathway alone is insufficient18. Likewise, flecainide is incompletely protective—in acute testing, 24% of patients did not respond, and in short term follow-up, 38% of patients continued to have significant events while on flecainide17.
It is demonstrated herein that AAV-CTDP improved outcomes by addressing both of these problems with current standard of care. Both RYR2 and MYBPC3 are cardiac specific proteins, and the AAV will selectively direct expression to the heart. Therefore, minimal effects outside of cardiomyocytes are expected. CTDP directly interacts with RYR2 and reduces spontaneous Ca2+ release through mutant RYR2 channels. This mechanism of action on the affected channel is more direct than current strategies of beta-blockade or flecainide. Importantly, these strategies are likely to be complementary, so that a multi-layered strategy might be envisioned to afford maximal protection while minimizing side effects. For example, administration of AAV-CTDP could directly reduce aberrant RYR2 activity. Additional protection could be afforded by beta-blocker, perhaps at lower doses that are more easily tolerated, or by flecainide. If the therapy was highly effective, some patients could return to some level of physical activity, guided by wearable heart rate monitors.
In summary, the AAV-CTDP described herein might supplant current standard of care and be sufficient as monotherapy. At the least, AAV-CTDP is able to synergize with current standard of care and permit lower level beta-blockade and less stringent exercise restriction, so that patients can be better protected from risk of sudden death while reducing side effects and thereby enhancing compliance.
Next the therapeutic candidate vector design is optimized. These optimization experiments are performed in human iPSC-CMs and CPVT mouse (RYR2-R176Q/+ and RYR2-R4650I/+) adult CMs. There are two parameters to consider.
The first parameter to consider is the RYR2 inhibitory peptide. Preliminary data suggests that the C-terminus of MYBPC3, is effective in reducing the aberrant activity of RYR2 containing a CPVT mutation. AAV that express different C-terminal peptides (C6-C10, C6-C8, C8-10, C9-C10, C10, C6-C9, C7-C9, C8-C9, C9) were constructed. Initial in vitro data indicated that peptides comprising the C6-C8 and C6-C9, and the C10 domain bind to the same sub-cellular location as RYR2 (
The fragments of MYPBC3 that interact with RYR2 were identified using a Biomolecular fluorescence complementation assay (BiFC) as outlined in
Results from the BiFC demonstrated that the C7 and C8 regions of MYBPC3 are the major contributor to the interaction between MYBPC3 and RYR2. Different fragments of MCBPC3 were test for binding to RYR2. Results from C9-C10, C10, C6-C10, C7-C10, and C8-C10 strongly suggested that the C7 and C8 regions both contribute to binding (
MYBPC3's established localization in cardiomyocytes is the A-band of sarcomeres. However, RYR2 is located in junctional SR/days, which are close to sarcomere Z-lines. Experiments were performed to determine if MYBCP3 fragments localize near the Z-line and therefore in the same region and RYR2. To do this, a MYBPC3 construct was made with a HA tag on the N-terminal and a Myc tag on the C-terminal (
To test this, cardiomyocyte lysates from wild type, wild-type+HA-MYBPC3-MYC, and MYBPC3 KO hearts were probed using HA or C10 (monoclonal Ab that recognizes the C-terminal most domain of MYBPC3) antibody (
To determine if the C7-C8 fragment localized to Z-line patterns in cardiomyocytes in vivo, mice were treated with AAV-cTnT-HA-C7C8-P2A-GFP (SEQ ID NO: 78). Heart sections were stained with HA and ACTN2 (a Z-line marker). Confocal images and signal intensity along a line parallel to the cardiomyocyte long axis show that HA stain had a striated pattern that co-localized with Z-lines showing that the C7-C8 fragment localizes to the same location as the RYR2 protein in vivo (
Response of Human CPVT iPSC-CMs to Overexpression of MYBPC3
It was further demonstrated the C6-C10 MYBC3 fragment suppresses abnormal calcium release in human iPSC-CMs with CPVT caused by a RYR2-5404 mutation (
RYR2 is a tetramer with higher order clustering that is important for normal Ca2+-induced Ca2+ release. This structural organization suggests the possibility that multimerizing the MYBPC3-derived interacting protein may increase potency or efficacy. Using the minimal region required for anti-arrhythmic effect in vivo identified above (e.g., C7-C8 or C7), concatemers are generated in which 2 or 3 copies are separated by a flexible linker. The efficacy of these constructs is compared using in vitro and in vivo assays. The effect on cardiac function is also examined by echocardiography. The optimized therapeutic construct is named C-terminus derived peptide, “CTDP”. The second parameter to consider when optimizing the therapeutic candidate vector is the promoter used to drive cardiomyocyte expression. Promoters and enhancers are tested to identify the combination with maximal level of expression and cardiomyocyte selectivity. A massively parallel reporter assay was previously developed to test thousands of candidate enhancers in parallel34, and this assay is currently being used to find the most potent and cardiac specific enhancers and promoters to drive expression from AAV.
These experiments are done with an AAV9 capsid because it is established as an efficient gene therapy vector in mice, and it has been used previously in an FDA-approved human product.
Next the therapeutic mechanism is evaluated. It is believed that the C-terminal region of MYBPC3 interacts with RYR2 and reduces diastolic Ca2+ flux. The effect of CTDP on RYR2WT and RYR2R176Q/+ diastolic Ca2+ flux is measured. RYR2-R176Q/+ and littermate control mice are treated with control AAV (AAV-GFP) or AAV-GFP-CTDP. 6-week old cardiomyocytes are isolated and diastolic sarcoplasmic reticulum Ca2+ leak are measured using an established protocol33.
To further test if MYBPC3 directly interacts with RYR2, a heterologous expression system and planar lipid bilayers is used. RYR2 wild-type or RYR2R176Q expression plasmid are transfected into HEK293 cells, and endoplasmic reticulum vesicles are purified. The vesicles are used to seed a planar lipid bilayer. Ca2+ current through the bilayer is measured after treatment with increasing concentration of recombinant CTDP. CTDP normalizes Ca2+ release by RYR2R176Q.
Next, dose-response and toxicity studies in the mouse CPVT model is performed. Using the optimized therapeutic candidate, dose-response experiments are performed in CPVT mice to determine the minimum percent of cardiomyocytes that must be transduced to suppress arrhythmia. In preliminary experiments, dose finding and biodistribution studies with AAV-CTDP are performed. 4-week old mice are injected intravenously with AAV-CTDP or control (AAV-GFP). At 8 weeks, mice are euthanized and tissues (heart, lung, spleen, liver, kidney, testes/ovaries, skeletal muscle, and brain) will be collected for histological and molecular studies. Cryosections are analyzed for GFP expression. Heart samples are analyzed by RNAscope in situ hybridization to directly measure the fraction of cardiomyocytes transduced by AAV-CTDP. Molecular studies measure RNA expression of GFP or CTDP, and viral genome copies per host genome.
Having established viral doses that yield 10%, 30%, and 50% cardiomyocyte transduction, dose-response studies are performed next. Two different mouse CPVT models are used, RYR2-R176Q/+ and RYR2-R4650I/+. These CPVT mutations occur in different mutation hotspot regions at opposite ends of the protein. Use of both genotypes help to show that the treatment is effective against multiple different CPVT-causing RYR2 mutations. Both CPVT models and littermate control mice are studied. The mice are treated at 4 weeks of age with these three doses of AAV-CTDP, or with AAV-GFP at a dose that transduces 50% of cardiomyocytes. After 4 weeks, mice undergo echocardiography and then an electrophysiology study. The electrophysiology study involves insertion of an octapolar pacing/recording catheter through the right carotid and into the right ventricle. Mice are treated with adrenergic stimulation (isoproterenol plus epinephrine) and with programmed ventricular stimulation as recently described21. Following the electrophysiology study, mice are euthanized and tissues preserved for histological and molecular assays. These studies are performed blinded to genotype and treatment group. There are 10 animals per group, 3 genotypes, and 3 doses, plus one dose of the control vector. This study requires dosing and an electrophysiology study of 120 mice.
Next the efficacy in a rabbit CPVT model is tested. Mouse cardiac physiology is significantly different from human. For example, mouse heart rate is 10 times faster than human, and the heart mass is 2000 times smaller. In contrast, rabbit cardiac physiology is more similar to human—the rabbit heart rate is about 2 times faster than human, and the mass is about 10 times lower. Heart rate and size have important implications for expression of cardiac ion channels and for susceptibility to arrhythmia. The closer alignment between rabbit and human cardiac electrophysiology indicates that demonstration of efficacy and safety in the rabbit model would significantly de-risk the therapeutic strategy. The rabbit model is expensive both in terms of rabbit breeding and housing, and production of sufficient AAV. Therefore, initial dose finding studies are performed in mouse models as described and then validated in rabbit models.
A rabbit CPVT model (R4650I/+) is being developed. Control and treated CPVT rabbits are compared for arrhythmic response to catecholamine stimulation or to programmed ventricular stimulation.
In initial dose-finding and biodistribution studies using AAV-GFP, several doses of the therapeutic vector are tested, and transduction of heart and other tissues are measured, as described in task two above for mice. Juvenile rabbits (8 weeks old) are treated intravenously with AAV-GFP. Four weeks later, transduction and expression are measured in heart, lung, spleen, liver, kidney, testes/ovaries, skeletal muscle, and brain. Rabbits are treated with AAV-CTDP at a comparable dose to confirm equivalent cardiac transduction efficiency, using RNAscope in situ hybridization.
CPVT and littermate control rabbits are treated with the dose of virus that transduces cardiomyocytes to the level that is found to be effective in mice as described in task two. A third cohort of CPVT rabbits are not treated. Four weeks after treatment, rabbits undergo echocardiography and then an electrophysiology study. An electrophysiology study consist of surface EKG and intracardiac recording during adrenergic stress (isoproterenol plus epinephrine) and programmed ventricular stimulation. There are a total of 10 rabbits per group in three groups for a total of 30 rabbits.
Next the efficacy in human iPSC-CMs across a range of CPVT genotypes is tested. AAV-CTDP on iPSC-CMs are tested from patients with several different CPVT genotypes that map to each of the 4 CPVT mutation hotspot regions. AAV2 capsid can be used to transfect cultured cells. The efficacy of the therapeutic candidate are measured across genotypes, using Ca2+ spark frequency as the primary readout.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.
Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
Where websites are provided, URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses. The actual web addresses do not contain the parentheses.
In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 63/049,398, filed Jul. 8, 2020, which is incorporated by reference herein in its entirety.
This invention was made with government support under Grant Nos. R01HL146634 and UG3HL141798 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63049398 | Jul 2020 | US |
