COMPOSITIONS AND METHODS FOR THE TREATMENT OF DOMINANTLY-INHERITED CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA

Abstract

Compositions and methods for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), particularly forms of the disease that are inherited in an autosomal dominant manner, by way of calsequestrin 2 (CASQ2) gene therapy can be used to treat CPVT caused by mutations in, for example, ryanodine receptor 2 and calmodulin, such as calmodulin 1 (CALM1) and CALM3. A variety of vectors are provided that can be used for the delivery of a CASQ2 transgene to a patient, such as a human patient suffering from autosomal dominant CPVT, including adeno-associated virus vectors, among others.

Claims

1. A method of treating dominant catecholaminergic polymorphic ventricular tachycardia (CPVT) in a human patient in need thereof, the method comprising the step of: administering to the patient a therapeutically effective amount of a composition comprising a transgene encoding calsequestrin 2 (CASQ2).

2.-8. (canceled)

9. The method according to claim 1, wherein the patient exhibits a gain-of-function mutation in an endogenous gene encoding ryanodine receptor 2 (RYR2).

10. The method according to claim 9, wherein the mutation in RYR2 results in an increase in cytosolic calcium release in a cardiac myocyte that is contacted with a RYR2 agonist relative to a cardiac myocyte that is not contacted with the RYR2 agonist.

11. The method according to claim 9, wherein the mutation in RYR2 is R4497C, N4104K, N4895D, R176Q, G178A, R420W, L433P, P1067S, S2246L, G2273R, F2307L, E2311D, L2344P, R2474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A4860G, I4867M, P4902S, and/or R4959Q.

12. The method according to claim 1, wherein the patient exhibits a mutation in an endogenous gene encoding a calmodulin.

13. The method according to claim 12, wherein the calmodulin is CALM1 or CALM3.

14. The method according to claim 13, wherein the mutation reduces CALM1 inhibition of calcium release from the sarcoplasmic reticulum of a cardiac myocyte.

15. The method according to claim 13, wherein the CALM1 or CALM3 harboring the mutation directly activates calcium release from the sarcoplasmic reticulum of a cardiac myocyte.

16. The method according to claim 13, wherein the mutation increases the stability of an open conformation of RYR2 in a CALM1-RYR2 complex present in an aqueous solution having a concentration of Ca.sup.2+ of from about 50 nM to about 5 M.

17. The method according to claim 13, wherein the mutation in CALM1 is N97S or N53I.

18.-19. (canceled)

20. The method according to claim 15, wherein the CALM3 mutation is A103V.

21. (canceled)

22. The method according to claim 1, wherein the CASQ2 transgene encodes a protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

23.-29. (canceled)

30. The method according to claim 1, wherein the composition is a vector.

31. The method according to claim 30, wherein the vector is a viral vector.

32.-33. (canceled)

34. The method according to claim 31, wherein the viral vector is an AAV.

35. The method according to claim 34, wherein the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74 serotype.

36. The method according to claim 31, wherein the viral vector is a pseudotyped AAV.

37.-38. (canceled)

39. The method according to claim 34, wherein the AAV comprises a recombinant capsid protein.

40. The method according to claim 1, wherein the composition is a liposome, vesicle, synthetic vesicle, exosome, synthetic exosome, dendrimer, or nanoparticle.

41. The method according to claim 1, wherein the transgene is operably linked to a promoter that induces expression of the CASQ2 in a cardiac myocyte.

42.-43. (canceled)

44. The method according to claim 1, wherein upon administration of the composition to the patient, expression of CASQ2 is increased in a cardiac cell in the patient.

45. The method according to claim 44, wherein the cardiac cell is a cardiac myocyte.

46. The method according to claim 44, wherein the expression of CASQ2 is assessed by measuring CASQ2 protein in the cell.

47. The method or the composition for use according to claim 44, wherein expression of CASQ2 is assessed by measuring CASQ2 mRNA in the cell.

48.-50. (canceled)

51. The method according to claim 1, wherein upon administration of the composition to the patient, expression of RYR2, triadin, and/or junctin is not substantially altered in a cardiac cell in the patient.

52. (canceled)

53. A kit comprising a composition comprising a transgene encoding CASQ2 and a package insert, wherein the package insert instructs a user of the kit to administer the composition to a patient suffering from dominant CPVT in accordance with the method according to claim 1.

54. The kit according to claim 53, wherein the composition is a vector.

55. The kit according to claim 54, wherein the vector is a viral vector.

56. (canceled)

57. The kit according to claim 5655, wherein the viral vector is an AAV.

58. The kit according to claim 57, wherein the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74 serotype.

59. The kit according to claim 55, wherein the viral vector is a pseudotyped AAV.

60.-61. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0089] FIG. 1 is a diagram of the pAAV2.1-CMV-CASQ2-IRES-eGP construct used in the murine CASQ2 gene therapy experiments described in Example 1, below. The vector contains, in the 5-to-3 direction, a 5 AAV2 inverted terminal repeat (ITR), cytomegalovirus (CMV) promoter, simian virus 40 (SV40) intron, wild-type murine calsequestrin 2 (CASQ2) transgene, internal ribosomal entry site (IRES), enhanced green fluorescent protein (eGFP) transgene, woodchuck posttranscriptional regulatory element (WPRE), bovine growth hormone (BGH) polyadenylation sequence, and 3 AAV2 ITR. The pAAV2.1-CMV-CASQ2-IRES-eGFP construct used in Example 1, below, was packaged into an AAV9 capsid, thereby generating a recombinant AAV2/9 vector.

[0090] FIG. 2 is a graph showing the effect of the pAAV2.1-CMV-CASQ2-IRES-eGFP vector on the percentage of arrhythmic events in R4496C/+ heterozygous knock in mice (Ryr2-Het), a model for autosomal dominant CPVT, upon exposure to epinephrine (2 mg/kg) and caffeine (120 mg/kg). The black bar (left) indicates that all untreated RyR2-Het mice (n=5) exhibited arrhythmias following epinephrine and caffeine administration. The results on the right of the graph demonstrate that none of the RyR2-Het mice infected with the pAAV2.1-CMV-CASQ2-IRES-eGFP vector (n=4) developed arrhythmias.

[0091] FIG. 3 is a graph showing mRNA expression of CASQ2, cardiac RYR2, triadin, and junctin in myocardial tissue of wild-type mice and RYR2 R4496C/+ mice that were either untreated or treated with the pAAV2.1-CMV-CASQ2-IRES-eGFP vector. Results are reported as fold expression of mRNA normalized to mRNA expression in wild type mice (WT, white bars). Black bars show fold expression of mRNA transcripts in untreated RyR2 R4496C/+ heterozygous knock in mice (Ryr2-Het) versus wild type (WT); grey bars show fold expression of mRNA transcripts in RyR2 R4496C/+ heterozygous knock in mice treated with the pAAV2.1-CMV-CASQ2-IRES-eGFP vector (Ryr2-Het AAV9-CASQ2) versus wild type (WT).

[0092] FIG. 4 is a diagram showing various components of another AAV vector found to exhibit an anti-arrhythmic effect in R4496C/+ heterozygous knock in mice. The vector contains a CASQ2 expression cassette flanked by 5 and 3 AAV2 ITRs. The expression cassette includes a transgene encoding wild-type CASQ2 operably linked to a desmin promoter.

[0093] FIG. 5 is a graph showing the effect of a recombinant, pseudotyped AAV2/8 containing the vector shown in FIG. 4 on the percentage of arrhythmic events in R4496C/+ heterozygous knock in mice (Ryr2-Het) upon exposure to epinephrine (2 mg/kg) and caffeine (120 mg/kg). The black bar (left) indicates that about 44% (4 out of 9) of untreated RyR2-Het mice (n=9) exhibited arrhythmias following epinephrine and caffeine administration. The results on the right of the graph demonstrate that none of the RyR2-Het mice infected with the rAAV2/8 vector encoding CASQ2 (n=9) developed arrhythmias.

DETAILED DESCRIPTION

[0094] Described herein are compositions and methods treating catecholaminergic polymorphic ventricular tachycardia (CPVT), and particularly autosomal dominant CPVT, in a patient, such as a human patient. Dominant CPVT is caused by mutations in one or more proteins that regulate the concentration of divalent calcium ions in cardiac myocytes. For example, dominant CPVT is associated with various mutations in the ryanodine receptor 2 (RYR2) protein, particularly gain-of-function mutations that upregulate RYR2-mediated release of divalent calcium ions from the sarcoplasmic reticulum. As described below, this leads to a cascade of signaling events and muscle contraction events that terminate in a heartbeat. Dominant CPVT is also associated with loss-of-function mutations in calmodulin proteins, such as calmodulin 1 (CALM1) and CALM3. These proteins assist in regulating cytosolic calcium by binding and sequestering divalent calcium ions, thereby preventing the propagation of an action potential. Excess calcium release from the sarcoplasmic reticulum during the diastolea period of cardiac relaxationengenders an extrasystolic heartbeat, which can trigger an arrhythmia.

[0095] The present disclosure is based, in part, on the discovery that dominant CPVT can be treated without the need to provide the patient with a functional RYR2, CALM1, or CALM3 protein or a transgene encoding the same. Using the compositions and methods described herein, a patient, such as a human patient, having dominant CPVT may be treated by administration of a transgene encoding a functional calsequestrin 2 (CASQ2) protein. Administration of the CASQ2, in turn, restores calcium homeostasis in the heart, leading to a reduction in arrhythmia events and the return of a normal heartbeat.

[0096] Using the compositions and methods described herein, a transgene encoding a functional CASQ2 protein may be delivered to a patient by way of viral gene therapy, among other approaches described below, such as the use of a liposome, vesicle, exosome, dendrimer, or nanoparticle. The sections that follow provide a summary of the pathophysiology that underlies dominant CPVT, as well as a description of exemplary vectors and other gene delivery vehicles that may be used for to provide patients with a functional CASQ2 transgene.

Dominant CPVT

[0097] CPVT is an inherited channelopathy characterized by high susceptibility to life threatening arrhythmias. Two forms of the disease have been described: the autosomal dominant and the autosomal recessive variants. The first is associated with mutations in the cardiac RYR2 gene, while the autosomal recessive variant is associated with mutations in the cardiac CASQ2 gene (Priori et al., Circulation 104 (Supplement 10:335 (2001), and Lahat et al., Am. J. Hum. Genet. 69:1378-1384 (2001)). Clinical observations have shown that patients having the dominant form of CPVT develop bidirectional and polymorphic ventricular tachycardia in response to sympathetic activation, whereas their resting electrocardiography profiles are unremarkable, and heart structure is preserved.

[0098] The pathology underlying dominant CPVT is linked to an abnormal function of the physiologic mechanism referred to as calcium-induced calcium release (CICR), which is fundamental for maintaining excitation-contraction coupling in the heart. The highly coordinated opening and closing of voltage-dependent ion channels located in the membrane of cardiac myocytes generates a cardiac action potential. During the plateau phase of the action potential, opening of voltage-dependent L-type Ca.sup.2+ channels allows the influx of Ca.sup.2+ in the plasmalemma. This process triggers the calcium transient and induces opening of sarcoplasmic reticulum Ca.sup.2+ release channel: RYR2 (Bers, Nature 415:198-205 (2002)). These local releases occur at specialized structures referred to as calcium-release units (CRUs). The CRUs are preferentially localized at the level of the transverse tubules (T-tubules), where the membrane of the sarcoplasmic reticulum is juxtaposed to the cellular membrane. One CRU is formed by clusters of RYR2, spanning the sarcoplasmic reticulum membrane, that are in close proximity to the L-type Ca.sup.2+ channels on the cell membrane (Franzini-Armstrong, et I., Ann. NY Acad. Sci. 1047:76-85 (2005)). The Ca.sup.2+ released from the sarcoplasmic reticulum binds to troponin C and induces a series of allosteric changes in myosin filaments leading to muscle fiber contraction. The subsequent removal of Ca.sup.2+ is mediated by the concomitant closing of the RYR2 and the action of sarcoplasmic reticulum Ca.sup.2+ ATPase that pumps Ca.sup.2+ back into the sarcoplasmic reticulum stores.

[0099] Another component of Ca.sup.2+-transient termination is the Na.sup.+-Ca.sup.2+ exchanger (NCX). The NCX extrudes one Ca.sup.2+ ion (two positive charges) for every three Na.sup.+ ions (three positive charges) taken into the cell. Thus, the NCX removes Ca.sup.2+ by generating a net inward depolarizing current: the transient inward current (I.sub.ti) (Pieske, et al., Circ. Res. 85:38-46 (1999)). The NCX becomes very important for the removal of Ca.sup.2+ in conditions characterized by calcium overload, for example, in case of RYR2 genetic mutations.

[0100] Arrhythmias in CPVT are elicited by Ca.sup.2+ release events that are not triggered by an action potential and are, therefore, referred to as spontaneous calcium releases (SCRs). SCR begins as a localized event involving a single CRU, but can also diffuse to neighboring CRUs triggering more Ca.sup.2+ release to produce a cell-wide calcium wave. The probability that SCR will lead to a calcium wave is influenced by the balance between sarcoplasmic reticulum Ca.sup.2+ content and the concentration of Ca.sup.2+ that induces Ca.sup.2+ release, the so-called sarcoplasmic reticulum calcium threshold. RYR2 function has a pivotal role in controlling this threshold. Several RYR2 mutations that are associated with CPVT have been suggested to act by decreasing the sarcoplasmic reticulum calcium threshold so that SCR is readily induced (Venetucci et al., Nat. Rev. Cardiol. 9:561-75 (2012)).

[0101] When abnormal Ca.sup.2+ leakage occurs, cytosolic Ca.sup.2+ concentration increases, and the cell must activate mechanisms to prevent disruption of Ca.sup.2+ homeostasis and re-establish the physiological diastolic level of Ca.sup.2+. It, current produces transient membrane depolarizations after completion of the action potential, known as a delayed after depolarization (DAD). When the amplitude of a DAD reaches the voltage threshold for Na.sup.+ channel activation, a triggered action potential is generated. Propagation of an action potential to the entire heart generates an extrasystolic beat. When this chain of events becomes repetitive and several DADs reach the threshold for the generation of propagating action potentials, triggered arrhythmic activity is elicited. In several studies, mutations of RYR2 have been shown to facilitate the occurrence of SCRs during -adrenergic stimulation and, in turn, elicit DADs and triggered activity leading to life-threatening arrhythmias (Liu et al., Circulation Research 99:292-298 (2006)).

[0102] The generation and characterization of a RYR2.sup.R4496C/+ knock-in mouse model for autosomal dominant CPVT (see, for example, Cerrone et al., M, Circulation Research 96:e77-e82 (2005), and U.S. Pat. No. 7,741,529) has provided great insight into the pathomechanism of this disease. RyR2.sup.R4496C/+ heterozygous mice recapitulate human CPVT and develop adrenergically-induced bidirectional and polymorphic ventricular arrhythmias. The R4496C mutation in mice, which corresponds to the R4497C mutation in humans, increases the sensitivity of the RYR2 channel to luminal calcium, thus facilitating the spontaneous release of calcium from the sarcoplasmic reticulum.

[0103] Exemplary mutations that may give rise to dominant CPVT are R4497C, N4104K, N4895D, R176Q, G178A, R420W, L433P, P1067S, S2246L, G2273R, F2307L, E2311D, L2344P, R2474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651 I, V4771 I, F4851L, A4860G, I4867M, P4902S, and R4959Q. Mutations in CALM1 that may lead to dominant CPVT include N97S and N53I, and an exemplary mutation in CALM3 that may lead to dominant CPVT is A103V.

[0104] It has presently been discovered that CPVT caused, e.g., by RYR2, CALM1, and/or CALM3 mutations that lead to arrhythmia, such as R4997C in humans, can be treated without the need to provide the patient with functional transgenes encoding these proteins. Rather, using the compositions and methods described herein, patients having dominant CPVT can be treated by administration of a delivery vehicle containing a CASQ2 transgene. Exemplary vehicles for the delivery of CASQ2 useful in conjunction with the compositions and methods described herein are described in detail below.

CPVT Treatment by CASQ2 Gene Therapy

CASQ2 Transgenes

[0105] Using the compositions and methods described herein, a transgene encoding CASQ2 can be provided to a patient, such as a human patient having dominant CPVT, so as to treat the pathology. The CASQ2 transgene may be delivered to the patient by way of a variety of vectors (e.g., viral vectors), among other compositions for gene therapy described herein. In some embodiments (e.g., in which a human patient is treated), the CASQ2 transgene encodes a protein having an amino acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of wild-type human CASQ2, which is provided in SEQ ID NO: 1, shown below. CASQ2 transgenes for use in conjunction with the compositions and methods described herein include those that encode a protein having one or more conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1. For example, the CASQ2 transgene may encode a protein having up to 50 conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 conservative amino acid substitutions) relative to the amino acid sequence of SEQ ID NO: 1. The CASQ2 transgene may, additionally or alternatively, have one or more nonconservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1. For example, the CASQ2 transgene may encode a protein having up to 50 nonconservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nonconservative amino acid substitutions) relative to the amino acid sequence of SEQ ID NO: 1.

[0106] In some embodiments, (e.g., in which a human patient is treated), the CASQ2 transgene has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of wild-type human CASQ2, which is provided in SEQ ID NO: 2, shown below.

[0107] Exemplary wild-type CASQ2 amino acid and nucleic acid sequences useful in conjunction with the compositions and methods described herein are described in Table 2, below.

TABLE-US-00002 TABLE2 ExemplaryCASQ2aminoacidandnucleicacidsequences SEQID NO. Description AminoAcid/NucleicAcidSequence 1 Wild-typeHumanCASQ2Protein MKRTHLFIVGIYFLSSCRAEEGLNFPTYDGK DRVVSLSEKNFKQVLKKYDLLCLYYHEPVS SDKVTQKQFQLKEIVLELVAQVLEHKAIGFV MVDAKKEAKLAKKLGFDEEGSLYILKGDRTI EFDGEFAADVLVEFLLDLIEDPVEIISSKLEV QAFERIEDYIKLIGFFKSEDSEYYKAFEEAAE HFQPYIKFFATFDKGVAKKLSLKMNEVDFYE PFMDEPIAIPNKPYTEEELVEFVKEHQRPTL RRLRPEEMFETWEDDLNGIHIVAFAEKSDP DGYEFLEILKQVARDNTDNPDLSILWIDPDD FPLLVAYWEKTFKIDLFRPQIGVVNVTDADS VWMEIPDDDDLPTAEELEDWIEDVLSGKINT EDDDEDDDDDDNSDEEDNDDSDDDDDE 2 Wild-typeHumanCASQ2cDNA ATGAAGAGAACTCACTTGTTTATTGTGGG (ExcludingUTRSequences) GATTTATTTTCTGTCCTCTTGCAGGGCAGA AGAGGGGCTTAATTTCCCCACATATGATG GGAAGGACCGAGTGGTAAGTCTTTCCGA GAAGAACTTCAAGCAGGTTTTAAAGAAATA TGACTTGCTTTGCCTCTACTACCATGAGC CGGTGTCTTCAGATAAGGTCACGCAAAAA CAGTTCCAACTGAAAGAAATCGTGCTTGA GCTTGTGGCCCAGGTCCTTGAACATAAAG CTATAGGCTTTGTGATGGTGGATGCCAAG AAAGAAGCCAAGCTTGCCAAGAAACTGGG TTTTGATGAAGAAGGAAGCCTGTATATTCT TAAGGGTGATCGCACAATAGAGTTTGATG GCGAGTTTGCAGCTGATGTCTTGGTGGAG TTCCTCTTGGATCTAATTGAAGACCCAGT GGAGATCATCAGCAGCAAACTGGAAGTCC AAGCCTTCGAACGCATTGAAGACTACATC AAACTCATTGGCTTTTTCAAGAGTGAGGA CTCAGAATACTACAAGGCTTTTGAAGAAG CAGCTGAACACTTCCAGCCTTACATCAAA TTCTTTGCCACCTTTGACAAAGGGGTTGC AAAGAAATTATCTTTGAAGATGAATGAGGT TGACTTCTATGAGCCATTTATGGATGAGC CCATTGCCATCCCCAACAAACCTTACACA GAAGAGGAGCTGGTGGAGTTTGTGAAGG AACACCAAAGACCCACTCTACGTCGCCTG CGCCCAGAAGAAATGTTTGAAACATGGGA AGATGATTTGAATGGGATCCACATTGTGG CCTTTGCAGAGAAGAGTGATCCAGATGGC TACGAATTCCTGGAGATCCTGAAACAGGT TGCCCGGGACAATACTGACAACCCCGATC TGAGCATCCTGTGGATCGACCCGGACGA CTTTCCTCTGCTCGTTGCCTACTGGGAGA AGACTTTCAAGATTGACCTATTCAGGCCA CAGATTGGGGTGGTGAATGTCACAGATGC TGACAGTGTCTGGATGGAGATTCCAGATG ATGACGATCTTCCAACTGCTGAGGAGCTG GAGGACTGGATTGAGGATGTGCTTTCTGG AAAGATAAACACTGAAGATGATGATGAAG ATGATGATGATGATGATAATTCTGATGAAG AGGATAATGATGACAGTGATGACGATGAT GATGAATAG 3 Wild-typeMurineCASQ2Protein MKRIYLLMVGVYLLSLSGAEEGLNFPTYDGK DRVVSLSEKNLKQMLKRYDLLCLYYHEPVS SDKVSQKQFQLKEIVLELVAQVLEHKNIGFV MVDSRKEAKLAKRLGFSEEGSLYVLKGDRT IEFDGEFAADVLVEFLLDLIEDPVEIVNNKL EVQAFERIEDQTKLLGFFKNEDSEYYKAFQEA AEHFQPYIKFFATFDKAVAKKLSLKMNEVGF YEPFMDEPNVIPNKPYTEEELVEFVKEHQR PTLRRLRPEDMFETWEDDLNGIHIVAFAEK SDPDGYEFLEILKQVARDNTDNPDLSILWID PDDFPLLVAYWEKTFKIDLFKPQIGVVNVTD ADSIWMEIPDDDDLPTAEELEDWIEDVLSGK INTEDDDNEDEDDDGDDNDDDDDDDDDND NSDEDNEDSDDDDDDDE 4 Wild-typeMurineCASQ2cDNA ATGAAGAGGATTTACC (ExcludingUTRSequences) TGCTCATGGTGGGGGTTTATCTGCTGTCC CTGAGCGGGGCAGAAGAGGGGCTGAACT TCCCCACGTACGATGGGAAAGACCGAGT GGTCAGCCTTTCTGAGAAGAACCTCAAGC AGATGTTGAAGAGATATGATTTGCTCTGTC TCTATTACCACGAACCTGTGTCTTCAGACA AGGTCTCACAAAAACAGTTCCAGCTGAAG GAGATTGTACTGGAGCTTGTGGCCCAGGT CCTGGAACATAAAAACATAGGCTTTGTGA TGGTGGATTCGAGGAAAGAGGCCAAGCTT GCTAAGAGGCTGGGATTCAGTGAAGAAG GAAGCCTGTATGTTCTGAAGGGTGACCGC ACGATTGAGTTTGACGGGGAGTTCGCAGC AGATGTCTTAGTGGAATTTCTCTTGGATCT CATTGAAGACCCAGTGGAGATCGTGAATA ACAAGCTGGAGGTCCAGGCCTTTGAGCG CATCGAGGACCAGACCAAGCTCCTTGGCT TCTTCAAGAATGAGGACTCAGAATATTACA AAGCATTCCAAGAGGCAGCTGAACACTTC CAGCCTTACATCAAGTTCTTTGCCACCTTT GACAAGGCGGTGGCAAAGAAGTTATCCTT GAAGATGAACGAAGTTGGCTTCTATGAGC CATTTATGGATGAGCCCAACGTCATCCCT AACAAACCGTACACAGAAGAGGAGCTTGT GGAGTTTGTGAAGGAACATCAAAGACCCA CCCTACGTCGCTTGCGCCCAGAGGACAT GTTTGAAACATGGGAAGACGACTTGAATG GGATCCACATCGTGGCCTTTGCGGAGAA GAGTGACCCAGATGGCTATGAGTTCCTAG AGATCCTGAAACAGGTTGCCCGGGACAAC ACTGACAATCCTGACTTGAGCATCTTGTG GATTGACCCAGATGACTTTCCACTGCTTG TTGCTTACTGGGAGAAGACTTTCAAGATT GACCTGTTCAAGCCACAGATTGGGGTGGT GAATGTAACCGATGCTGACAGTATCTGGA TGGAGATCCCAGATGATGACGACCTGCCC ACAGCTGAGGAGCTGGAAGACTGGATTG AAGATGTGCTTTCTGGAAAGATCAACACT GAAGATGATGACAATGAAGATGAAGATGA TGATGGTGATGACAACGATGACGATGATG ATGACGACGATGATAATGATAATTCTGATG AGGATAATGAAGACAGTGATGATGACGAT GATGACGATGAATAG

[0108] CASQ2 transgenes herein further include those that encode fragments of the foregoing proteins that retain the ability to polymerize and bind divalent calcium, for example, with an affinity that is within up to 25% (e.g., with an affinity that is within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) of the affinity of wild-type CASQ2 and/or with a stoichiometry that deviates from that of wild-type CASQ2 by no more than 25% (e.g., no more than 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less). Examples of assays that can be used to assess calcium binding affinity and stoichiometry are known in the art and described, for example, in Johnson et al. Methods Mol. Biol. 173:89-102 (2002), the disclosure of which is incorporated herein by reference in its entirety.

Transcription Regulatory Elements

[0109] CASQ2 transgenes as described herein may be incorporated into a delivery vehicle, such as a vector, plasmid, or other nucleic acid delivery vehicle described herein, such that the transgene is operably linked to a promoter. Exemplary promoters useful in conjunction with the compositions and methods described herein are those that induce transgene expression in a cardiac cell in a patient (e.g., a human patient), such as a cardiac myocyte. For example, promoters that may be used to induce expression of CASQ2 in the gene delivery vehicles described herein include, without limitation, a desmin promoter, cytomegalovirus promoter, myosin light chain-2 promoter, alpha actin promoter, troponin 1 promoter, Na.sup.+/Ca.sup.2+ exchanger promoter, dystrophin promoter, creatine kinase promoter, alpha7 integrin promoter, brain natriuretic peptide promoter, alpha B-crystallin/small heat shock protein promoter, alpha myosin heavy chain promoter, or atrial natriuretic factor promoter.

Vectors for Delivery of Exogenous Nucleic Acids to Target Cells

Viral Vectors for Nucleic Acid Delivery

[0110] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a gene of interest into the genome of a target cell in a patient (e.g., a mammalian cell, such as a human cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors that may be used in conjunction with the compositions and methods described herein are AAV, retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses that may be used in conjunction with the compositions and methods described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

AAV Vectors for Nucleic Acid Delivery

[0111] In some embodiments, nucleic acids of the compositions and methods described herein are incorporated into rAAV vectors and/or virions in order to facilitate their introduction into a cell, such as a target cardiac cell (e.g., cardiac myocyte) in a patient. rAAV vectors useful in the conjunction with the compositions and methods described herein include recombinant nucleic acid constructs that contain (1) a CASQ2 transgene and (2) viral nucleic acids that facilitate integration and expression of the heterologous genes. The viral nucleic acids may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors include those having one or more of the naturally-occurring AAV genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype (e.g., derived from serotype 2) suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000), and Monahan and Samulski, Gene Delivery 7:24-30 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0112] The nucleic acids and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for example, in U.S. Pat. Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0113] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8 and 9. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol. 72:2224-2232 (1998); Halbert et al., J. Virol. 74:1524-1532 (2000); Halbert et al., J. Virol. 75:6615-6624 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0114] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV2) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, among others). For example, a representative pseudotyped vector is an AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 8 or AAV serotype 9. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001).

[0115] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635-45 (2000). Other rAAV virions that can be used in methods of the disclosure include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001).

Additional Methods for the Delivery of Transgenes to Target Cells

Transfection Techniques

[0116] Techniques that can be used to introduce a CASQ2 transgene, such as a CASQ2 transgene operably linked to a transcription regulatory element described herein, into a target cell are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.

[0117] Additional techniques useful for the transfection of target cells include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0118] Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in US 2010/0227406, the disclosure of which is incorporated herein by reference.

[0119] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0120] Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein. For example, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

Incorporation of Target Genes by Gene Editing Techniques

[0121] In addition to the above, a variety of tools have been developed that can be used for the incorporation of a gene of interest into a target cell, such as a human cell. One such method that can be used for incorporating polynucleotides encoding target genes into target cells involves the use of transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by 5 and 3 excision sites. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some instances, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of similar excision sites that exist within the nuclear genome of the cell. This allows the gene of interest to be inserted into the cleaved nuclear DNA at the complementary excision sites, and subsequent covalent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the mammalian cell genome completes the incorporation process. In certain cases, the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail in, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail in, e.g., US 2005/0112764), the disclosures of each of which are incorporated herein by reference as they pertain to transposons for use in gene delivery to a cell of interest.

[0122] Another tool for the integration of target genes into the genome of a target cell is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site. In this manner, highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can design a CRISPR/Cas system to cleave any target DNA molecule of interest. This technique has been exploded in order to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an efficient means of site-specifically editing target cell genomes in order to cleave DNA prior to the incorporation of a gene encoding a target gene. The use of CRISPR/Cas to modulate gene expression has been described in, for example, U.S. Pat. No. 8,697,359, the disclosure of which is incorporated herein by reference as it pertains to the use of the CRISPR/Cas system for genome editing. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a gene of interest in a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al., Nature Reviews Genetics 11:636 (2010); and in Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosure of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.

[0123] Additional genome editing techniques that can be used to incorporate polynucleotides encoding target genes into the genome of a target cell include the use of ARCUS meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA. The use of these enzymes for the incorporation of genes encoding target genes into the genome of a mammalian cell is advantageous in view of the defined structure-activity relationships that have been established for such enzymes. Single chain meganucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a target gene into the nuclear DNA of a target cell. These single-chain nucleases have been described extensively in, for example, U.S. Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.

Methods of Measuring Transgene Expression

[0124] The expression level of a CASQ2 transgene expressed by a target cell in a patient (e.g., a cardiac myocyte) can be ascertained, for example, by evaluating the concentration or relative abundance of mRNA transcripts derived from transcription of the CASQ2 transgene. Additionally or alternatively, gene expression can be determined by evaluating the concentration or relative abundance of CASQ2 protein produced by transcription and translation of a CASQ2 transgene. Protein concentrations can also be assessed using functional assays, such as calcium abundance assays. The sections that follow describe exemplary techniques that can be used to measure the expression level of a CASQ2 transgene upon delivery to a patient, such as a patient having dominant CPVT as described herein. Transgene expression can be evaluated by a number of methodologies known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in-situ hybridization (e.g., fluorescence in-situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence activated cell sorting (FACS), northern analysis and/or PCR analysis of mRNAs.

Nucleic Acid Detection

[0125] Nucleic acid-based methods for determining transgene expression detection methods datasets suitable for analysis in conjunction with the compositions and methods described herein include imaging-based techniques (e.g., Northern blotting or Southern blotting), which may be used in conjunction with cells obtained from a patient following administration of the transgene. Northern blot analysis is a conventional technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, N.Y. 11803-2500. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).

[0126] Transgene detection techniques that may be used in conjunction with the compositions and methods described herein to evaluate CASQ2 expression further include microarray sequencing experiments (e.g., Sanger sequencing and next-generation sequencing methods, also known as high-throughput sequencing or deep sequencing). Exemplary next generation sequencing technologies include, without limitation, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLID sequencing, and nanopore sequencing platforms. Additional methods of sequencing known in the art can also be used. For instance, transgene expression at the mRNA level may be determined using RNA-Seq (e.g., as described in Mortazavi et al., Nat. Methods 5:621-628 (2008) the disclosure of which is incorporated herein by reference in their entirety). RNA-Seq is a robust technology for monitoring expression by direct sequencing the RNA molecules in a sample. Briefly, this methodology may involve fragmentation of RNA to an average length of 200 nucleotides, conversion to cDNA by random priming, and synthesis of double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology). Then, the cDNA is converted into a molecular library for sequencing by addition of sequence adapters for each library (e.g., from Illumina/Solexa), and the resulting 50-100 nucleotide reads are mapped onto the genome.

[0127] Transgene expression levels may be determined using microarray-based platforms (e.g., single-nucleotide polymorphism arrays), as microarray technology offers high resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No. 6,232,068 and Pollack et al., Nat. Genet. 23:41-46 (1999), the disclosures of each of which are incorporated herein by reference in their entirety. Using nucleic acid microarrays, mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then hybridize to one or more complementary nucleic acids arrayed and immobilized on a solid support. The array can be configured, for example, such that the sequence and position of each member of the array is known. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Expression level may be quantified according to the amount of signal detected from hybridized probe-sample complexes. A typical microarray experiment involves the following steps: 1) preparation of fluorescently labeled target from RNA isolated from the sample, 2) hybridization of the labeled target to the microarray, 3) washing, staining, and scanning of the array, 4) analysis of the scanned image and 5) generation of gene expression profiles. One example of a microarray processor is the Affymetrix GENECHIP system, which is commercially available and comprises arrays fabricated by direct synthesis of oligonucleotides on a glass surface. Other systems may be used as known to one skilled in the art.

[0128] Amplification-based assays also can be used to measure the expression level of a transgene in a target cell following delivery to a patient. In such assays, the nucleic acid sequences of the gene act as a template in an amplification reaction (for example, PCR, such as qPCR). In a quantitative amplification, the amount of amplification product is proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the expression level of the gene, corresponding to the specific probe used, according to the principles described herein. Methods of real-time qPCR using TaqMan probes are well known in the art. Detailed protocols for real-time qPCR are provided, for example, in Gibson et al., Genome Res. 6:995-1001 (1996), and in Heid et al., Genome Res. 6:986-994 (1996), the disclosures of each of which are incorporated herein by reference in their entirety. Levels of gene expression as described herein can be determined by RT-PCR technology. Probes used for PCR may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.

Protein Detection

[0129] Transgene expression can additionally be determined by measuring the concentration or relative abundance of a corresponding protein product (e.g., CASQ2) encoded by a gene of interest. Protein levels can be assessed using standard detection techniques known in the art. Protein expression assays suitable for use with the compositions and methods described herein include proteomics approaches, immunohistochemical and/or western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assay (ELIFA), mass spectrometry, mass spectrometric immunoassay, and biochemical enzymatic activity assays. In particular, proteomics methods can be used to generate large-scale protein expression datasets in multiplex. Proteomics methods may utilize mass spectrometry to detect and quantify polypeptides (e.g., proteins) and/or peptide microarrays utilizing capture reagents (e.g., antibodies) specific to a panel of target proteins to identify and measure expression levels of proteins expressed in a sample (e.g., a single cell sample or a multi-cell population).

[0130] Exemplary peptide microarrays have a substrate-bound plurality of polypeptides, the binding of an oligonucleotide, a peptide, or a protein to each of the plurality of bound polypeptides being separately detectable. Alternatively, the peptide microarray may include a plurality of binders, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins. Examples of peptide arrays may be found in U.S. Pat. Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference in their entirety.

[0131] Mass spectrometry (MS) may be used in conjunction with the methods described herein to identify and characterize transgene expression in a cell from a patient (e.g., a human patient) following delivery of the transgene. Any method of MS known in the art may be used to determine, detect, and/or measure a protein or peptide fragment of interest, e.g., LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, and the like. Mass spectrometers generally contain an ion source and optics, mass analyzer, and data processing electronics. Mass analyzers include scanning and ion-beam mass spectrometers, such as time-of-flight (TOF) and quadruple (Q), and trapping mass spectrometers, such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), may be used in the methods described herein. Details of various MS methods can be found in the literature. See, for example, Yates et al., Annu. Rev. Biomed. Eng. 11:49-79, 2009, the disclosure of which is incorporated herein by reference in its entirety.

[0132] Prior to MS analysis, proteins in a sample obtained from the patient can be first digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion. Complex peptide samples also benefit from the use of front-end separation techniques, e.g., 2D-PAGE, HPLC, RPLC, and affinity chromatography. The digested, and optionally separated, sample is then ionized using an ion source to create charged molecules for further analysis. Ionization of the sample may be performed, e.g., by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. Additional information relating to the choice of ionization method is known to those of skill in the art.

[0133] After ionization, digested peptides may then be fragmented to generate signature MS/MS spectra. Tandem MS, also known as MS/MS, may be particularly useful for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring in between the stages, which may be accomplished with individual mass spectrometer elements separated in space or using a single mass spectrometer with the MS steps separated in time. In spatially separated tandem MS, the elements are physically separated and distinct, with a physical connection between the elements to maintain high vacuum. In temporally separated tandem MS, separation is accomplished with ions trapped in the same place, with multiple separation steps taking place over time. Signature MS/MS spectra may then be compared against a peptide sequence database (e.g., SEQUEST). Post-translational modifications to peptides may also be determined, for example, by searching spectra against a database while allowing for specific peptide modifications.

Pharmaceutical Compositions

[0134] The CASQ2 transgenes described herein may be incorporated into a vehicle for administration into a patient, such as a human patient suffering from dominant CPVT, as described herein. Pharmaceutical compositions containing vectors, such as viral vectors, that contain the CASQ2 transgene can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

[0135] Mixtures of the nucleic acids and viral vectors described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. 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 may contain a preservative to prevent the growth of microorganisms. 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 (described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may 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.

[0136] For example, a solution containing a pharmaceutical composition described herein 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, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. 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. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

Routes of Administration and Dosing

[0137] Viral vectors, such as AAV vectors and others described herein, containing the transgene may be administered to a patient (e.g., a human patient) by a variety of routes of administration. The route of administration may vary, for example, with the onset and severity of disease, and may include, e.g., intravenous, intrathecal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. Intravascular administration includes delivery into the vasculature of a patient. In some embodiments, the administration is into a vessel considered to be a vein (intravenous), and in some administration, the administration is into a vessel considered to be an artery (intraarterial). Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and/or femoral vein. Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and/or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary.

[0138] Treatment regimens may vary, and often depend on disease severity and the age, weight, and sex of the patient. Treatment may include administration of vectors (e.g., viral vectors) or other agents described herein as useful for the introduction of a transgene into a target cell in various unit doses. Each unit dose will ordinarily contain a predetermined-quantity of the therapeutic composition.

[0139] In cases in which the transgene is delivered to the patient by way of viral gene therapy (e.g., by way of administration of an AAV vector described herein, such as an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AAV2/8, or AAV2/9, vector), the viral vector may be administered to the patient at a dose of, for example, from about 110.sup.6 genome copies (GC)/kg to about 110.sup.18 GC/kg (e.g., at a dose of about 110.sup.6 GC/kg, 210.sup.6 GC/kg, 310.sup.6 GC/kg, 410.sup.6 GC/kg, 510.sup.6 GC/kg, 610.sup.6 GC/kg, 710.sup.6 GC/kg, 810.sup.6 GC/kg, 910.sup.6 GC/kg, 110.sup.7 GC/kg, 210.sup.7 GC/kg, 310.sup.7 GC/kg, 410.sup.7 GC/kg, 510.sup.7 GC/kg, 610.sup.7 GC/kg, 710.sup.7 GC/kg, 810.sup.7 GC/kg, 910.sup.7 GC/kg, 110.sup.8 GC/kg, 210.sup.8 GC/kg, 310.sup.8 GC/kg, 410.sup.8 GC/kg, 510.sup.8 GC/kg, 610.sup.8 GC/kg, 710.sup.8 GC/kg, 810.sup.8 GC/kg, 910.sup.8 GC/kg, 110.sup.9 GC/kg, 210.sup.9 GC/kg, 310.sup.9 GC/kg, 410.sup.9 GC/kg, 510.sup.9 GC/kg, 610.sup.9 GC/kg, 710.sup.9 GC/kg, 810.sup.9 GC/kg, 910.sup.9 GC/kg, 110.sup.10 GC/kg, 210.sup.10 GC/kg, 310.sup.10 GC/kg, 410.sup.10 GC/kg, 510.sup.10 GC/kg, 610.sup.10 GC/kg, 710.sup.10 GC/kg, 810.sup.10 GC/kg, 910.sup.10 GC/kg, 110.sup.11 GC/kg, 210.sup.11 GC/kg, 310.sup.11 GC/kg, 410.sup.11 GC/kg, 510.sup.11 GC/kg, 610.sup.11 GC/kg, 710.sup.11 GC/kg, 810.sup.11 GC/kg, 910.sup.11 GC/kg, 110.sup.12 GC/kg, 210.sup.12 GC/kg, 310.sup.12 GC/kg, 410.sup.12 GC/kg, 510.sup.12 GC/kg, 610.sup.12 GC/kg, 710.sup.12 GC/kg, 810.sup.12 GC/kg, 910.sup.12 GC/kg, 110.sup.13 GC/kg, 210.sup.13 GC/kg, 310.sup.13 GC/kg, 410.sup.13 GC/kg, 510.sup.13 GC/kg, 610.sup.13 GC/kg, 710.sup.13 GC/kg, 810.sup.13 GC/kg, 910.sup.13 GC/kg, 110.sup.14 GC/kg, 210.sup.14 GC/kg, 310.sup.14 GC/kg, 410.sup.14 GC/kg, 510.sup.14 GC/kg, 610.sup.14 GC/kg, 710.sup.14 GC/kg, 810.sup.14 GC/kg, 910.sup.14 GC/kg, 110.sup.15 GC/kg, 210.sup.15 GC/kg, 310.sup.15 GC/kg, 410.sup.15 GC/kg, 510.sup.15 GC/kg, 610.sup.15 GC/kg, 710.sup.15 GC/kg, 810.sup.15 GC/kg, 910.sup.15 GC/kg, 110.sup.16 GC/kg, 210.sup.16 GC/kg, 310.sup.16 GC/kg, 410.sup.16 GC/kg, 510.sup.16 GC/kg, 610.sup.16 GC/kg, 710.sup.16 GC/kg, 810.sup.16 GC/kg, 910.sup.16 GC/kg, 110.sup.17 GC/kg, 210.sup.17 GC/kg, 310.sup.17 GC/kg, 410.sup.17 GC/kg, 510.sup.17 GC/kg, 610.sup.17 GC/kg, 710.sup.17 GC/kg, 810.sup.17 GC/kg, 910.sup.17 GC/kg, or 110.sup.18 GC/kg). In some embodiments, the vector is administered to the patient at a dose of from about 110.sup.8 GC/kg to about 110.sup.16 GC/kg (e.g., at a dose of about 110.sup.8 GC/kg, 210.sup.8 GC/kg, 310.sup.8 GC/kg, 410.sup.8 GC/kg, 510.sup.8 GC/kg, 610.sup.8 GC/kg, 710.sup.8 GC/kg, 810.sup.8 GC/kg, 910.sup.8 GC/kg, 110.sup.9 GC/kg, 210.sup.9 GC/kg, 310.sup.9 GC/kg, 410.sup.9 GC/kg, 510.sup.9 GC/kg, 610.sup.9 GC/kg, 710.sup.9 GC/kg, 810.sup.9 GC/kg, 910.sup.9 GC/kg, 110.sup.10 GC/kg, 210.sup.10 GC/kg, 310.sup.10 GC/kg, 410.sup.10 GC/kg, 510.sup.10 GC/kg, 610.sup.10 GC/kg, 710.sup.10 GC/kg, 810.sup.10 GC/kg, 910.sup.10 GC/kg, 110.sup.11 GC/kg, 210.sup.11 GC/kg, 310.sup.11 GC/kg, 410.sup.11 GC/kg, 510.sup.11 GC/kg, 610.sup.11 GC/kg, 710.sup.11 GC/kg, 810.sup.11 GC/kg, 910.sup.11 GC/kg, 110.sup.12 GC/kg, 210.sup.12 GC/kg, 310.sup.12 GC/kg, 410.sup.12 GC/kg, 510.sup.12 GC/kg, 610.sup.12 GC/kg, 710.sup.12 GC/kg, 810.sup.12 GC/kg, 910.sup.12 GC/kg, 110.sup.13 GC/kg, 210.sup.13 GC/kg, 310.sup.13 GC/kg, 410.sup.13 GC/kg, 510.sup.13 GC/kg, 610.sup.13 GC/kg, 7 142010.sup.13 GC/kg, 810.sup.13 GC/kg, 910.sup.13 GC/kg, 110.sup.14 GC/kg, 210.sup.14 GC/kg, 310.sup.14 GC/kg, 410.sup.14 GC/kg, 510.sup.14 GC/kg, 610.sup.14 GC/kg, 710.sup.14 GC/kg, 810.sup.14 GC/kg, 910.sup.14 GC/kg, 110.sup.15 GC/kg, 210.sup.15 GC/kg, 310.sup.15 GC/kg, 410.sup.15 GC/kg, 510.sup.15 GC/kg, 610.sup.15 GC/kg, 710.sup.15 GC/kg, 810.sup.15 GC/kg, 910.sup.15 GC/kg, or 110.sup.16 GC/kg). In some embodiments, the vector is administered to the patient at a dose of from about 110.sup.10 GC/kg to about 110.sup.14 GC/kg (e.g., at a dose of about 110.sup.10 GC/kg, 210.sup.10 GC/kg, 310.sup.10 GC/kg, 410.sup.10 GC/kg, 510.sup.10 GC/kg, 610.sup.10 GC/kg, 710.sup.10 GC/kg, 810.sup.10 GC/kg, 910.sup.10 GC/kg, 110.sup.11 GC/kg, 210.sup.11 GC/kg, 310.sup.11 GC/kg, 410.sup.11 GC/kg, 510.sup.11 GC/kg, 610.sup.11 GC/kg, 710.sup.11 GC/kg, 810.sup.11 GC/kg, 910.sup.11 GC/kg, 110.sup.12 GC/kg, 210.sup.12 GC/kg, 310.sup.12 GC/kg, 410.sup.12 GC/kg, 510.sup.12 GC/kg, 610.sup.12 GC/kg, 710.sup.12 GC/kg, 810.sup.12 GC/kg, 910.sup.12 GC/kg, 110.sup.13 GC/kg, 210.sup.13 GC/kg, 310.sup.13 GC/kg, 410.sup.13 GC/kg, 510.sup.13 GC/kg, 610.sup.13 GC/kg, 710.sup.13 GC/kg, 810.sup.13 GC/kg, 910.sup.13 GC/kg, or 110.sup.14 GC/kg).

EXAMPLES

[0140] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

Example 1. Delivery of CASQ2 to Heterozygous RYR 4496C/+ Mice Eliminates Arrhythmia in a Manner Independent of Expression of RYR2, Triadin, and Junctin

Materials and Methods

Animal Use

[0141] Animals were maintained and bred at the Charles River Laboratories in Calco, Italy, and transferred to the ICS Maugeri Spa-SB for characterization of the phenotype. Animals were maintained and studied according to the protocols approved by the committee for the animals' well-being by the University of Pavia.

Generation of Knock-in R4496C RYR2 Murine Model

[0142] The experiments described in this example utilized a heterozygous RYR2 R4496C/+ knock-in mouse model of dominant CPVT, which recapitulates the phenotype of the human RYR2 R4997C mutation. Details regarding the production of this mouse model are provided in U.S. Pat. No. 7,741,529, the disclosure of which is incorporated herein by reference as it pertains to the generation of the RYR2.sup.R4496C/+ knock-in model.

Vector Design and Production

[0143] A detailed description of the design of one of the vectors employed in these experiments is provided in U.S. Pat. No. 8,859,517. Briefly, a pseudotyped adeno-associated serotype 9 vector (AAV2/9) was generated containing the coding sequence, without UTR sequences, of wild-type murine CASQ2 (NCBI Reference Sequence No. NM_009814.2). Wild-type murine CASQ2 was cloned into pGEM-T-Easy vector (Promega). By enzymatic digestion using EcoRI, the insert corresponding to the CASQ2 transgene was excised from the pGEM vector and subcloned in bis-cistronic pIRES vector (BD Biosciences, Cat. No: 631605, Clontech Palo Alto Calif., USA). Subsequently, the fragment corresponding to the CASQ2 transgene followed by the IRES sequence was subcloned via PCR amplification using specific primers (Forward: 5-CACAGCGGCCGCACAATGAAGAGGATTTACCTGCTCATGG-3 (SEQ ID NO: 5) and Reverse 5-CGAAGCATTAACCCTCACTAAAGGG-3 (SEQ ID NO: 6) containing the NotI enzymatic site. The amplicon was inserted into the adenoviral backbone vector pAAV-2.1-eGFP, serotype 9 (containing a bovine growth hormone (BGH) polyA sequence and CMV promoter), provided by the Adeno-Associated Virus (AAV) vector Core facility (Tigem, Napoli, Italy), by way of enzymatic digestion with Not I. All plasmids were sequenced.

[0144] The AAV production was done in collaboration with the Tigem AAV Vector core facility.

[0145] The AAV vector was produced using a transient transfection of 3 plasmids in HEK 293 cells: pAd helper, pAAV rep-cap (packaging), pAAV Cis (including the CASQ2 insert, cloned in the pAAV2.1-CMV-eGFP plasmid MCS). Real time PCR and dot blot analysis was conducted to assess viral titer, and SDS PAGE followed by Coomassie staining was performed to evaluate the presence and purity of capsid proteins. Infectivity and sterility were also assessed. The service returned with a viral preparation in PBS with a total yield in excess of 110.sup.12 genome copies. All AAV2/9-WT-mCASQ2-IRES-eGFP stocks were frozen at 80 C. in single aliquots and thawed during the surgical procedure.

[0146] Similar recombinant AAV production methods were used to furnish a second vector investigated in these experiments. The second vector was a pseudotyped AAV2/8 vector containing a transgene encoding wild-type CASQ2 operably linked to a desmin promoter. Various components of this second vector are shown in FIG. 4.

Quantitative Real Time PCR

[0147] Total RNA from cardiac tissue of RyR2.sup.R4496C/+ mice and from RyR2.sup.R4496C/+ heterozygous knock-in mice infected with the AAV9-WT-mCASQ2-IRES-eGFP viral vector was purified with RNeasy mini kit (Qiagen). A total amount of 1 g template RNA per reaction was used for the RT-PCR assay performed with iScript cDNA Synthesis kit (Bio-Rad Laboratories, Inc., USA). Real Time quantification of mRNAs of various genes of interest (RyR2 CASQ2, Triadin, Junctin) and the reference gene (GAPDH) was performed in optical 96-well plates using CFX96 detection module (Bio-Rad Laboratories, Inc.). All samples were analyzed in triplicate with SsoFast EvaGreen Supermix using specific primer mix and 20 ng of cDNA template with primers reported previously (Denegri et al., Circulation 129:267381 (2014)). Real-time PCR was performed using the CFX96 Real-Time PCR Detection System and analyzed using the Bio-Rad CFX96 Manager Software (Version 1.5).

In Vivo Experiments

[0148] The AAV vector containing the construct shown in FIG. 1 was delivered via intraperitoneal injection of 100 l of purified virus at a dose of 6.410.sup.11 genome copies to n=4 neonatal mice with a 25 gauge syringe on the eighth postnatal day (P8). A total of n=5 littermates were not treated.

[0149] At 7 weeks of age, electrocardiography (ECG) and radiotelemetry monitors (Data Sciences International) were implanted subcutaneously under general anaesthesia (Isoflurane 1.5-3%) in all mice. After 72 hours of recovery from surgery, phenotype characterization was performed. Basal ECG was recorded for to assess for the presence of spontaneous arrhythmias. Immediately after, the susceptibility of mice to adrenergically-induced arrhythmias was tested by administering epinephrine (2 mg/kg) and caffeine (120 mg/kg) by intraperitoneal injection (Cerrone et al., M, Circulation Research 96:e77-e82 (2005), Liu et al., Circulation Research 99:292-298 (2006)). All animals were freely moving while ECG recordings were performed.

[0150] This experiment was then repeated using the AAV2/8 vector shown in FIG. 4 in order to investigate the anti-arrhythmic effects of this construct in a model organism for dominantly inherited CPVT. RyR2 R4496C/+ heterozygous mice (n=9) were administered the AAV2/8 vector at a dose of 610.sup.13 vector genomes (vg) per kg. Non-treated RyR2 R4496C/+ heterozygous mice (n=9) were used as a control. At two months post administration, mice were evaluated by electrophysiological analysis in vivo (ECG) under epinephrine and caffeine stimulation.

Results

[0151] As shown in FIG. 2, administration of an AAV vector encoding the CASQ2 transgene to RYR2.sup.R4996C/+ mice resulted in a complete suppression of caffeine- and epinephrine-induced arrhythmia. Following -adrenergic stimulation, none of the n=4 AAV CASQ2-treated mice exhibited arrhythmic activity, while all of the n=5 untreated mice demonstrated arrhythmia. Moreover, the therapeutic effects of CASQ2 delivery in RYR2.sup.R4996C/+ mice were not dependent upon the activity of RYR2 or the membrane-associated proteins, triadin and junctin. As shown in FIG. 3, administration of the AAV CASQ2 vector resulted in elevated CASQ2 expression in cardiac myocytes, but did not substantially alter RYR2, triadin, or junctin expression in such cells.

[0152] As shown in FIG. 5, similar results were obtained using the AAV2/8 construct. Particularly, administration of the AAV2/8-desmin-CASQ2 vector resulted in a complete suppression of caffeine- and epinephrine-induced arrhythmia in RYR2.sup.R4996C/+ mice, while 44% of the untreated mice demonstrated arrhythmia.

[0153] Collectively, these results demonstrate that CASQ2 transgene delivery in murine models of dominant CPVT fosters the correction of bidirectional and polymorphic arrhythmias. Surprisingly, CASQ2 gene transfer alone, without the need for RYR2 delivery, was found to prevent the physiological sinus rhythm that triggers arrhythmias due to adrenergic activation. CASQ2 gene therapy in murine dominant CPVT models resulted in augmented CASQ2 expression in cardiac myocytes and restored anti-arrhythmic cardiac contractile function.

[0154] In view of these results and the description provided herein, CASQ2 gene therapy can be used as a paradigm for the treatment of dominant CPVT, as illustrated in Example 2, below.

Example 2. Treatment of Dominant CPVT in a Human Patient by Administration of a Viral Vector Containing a CASQ2 Transgene

[0155] Using conventional molecular biology techniques known in the art, a gene encoding a CASQ2 protein, such as the wild-type human CASQ2 protein having the amino acid sequence of SEQ ID NO: 1 (or a variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1), can be incorporated into a viral vector and formulated for administration to a human patient. For example, a patient suffering from dominant CPVT, can be administered an AAV vector containing a CASQ2 transgene under the control of a transcriptional regulatory element that promotes CASQ2 expression in cardiac myocytes. The AAV vector may be a pseudotyped vector that incorporates the CASQ2 transgene between the 5 and 3 inverted terminal repeats of AAV2 and that is encapsidated by AAV8 or AAV9 capsid proteins (e.g., an AAV2/8 or AAV2/9 vector). The AAV vector can be administered to the subject by a variety of routes of administration, such as intravenously, intramuscularly, or subcutaneously, among others.

[0156] Following administration of the vector to a patient, a practitioner of skill in the art can monitor the expression of the CASQ2 gene, and the patient's improvement in response to the therapy, by a variety of methods. For example, a physician can monitor the patient's cardiac function by analyzing the quantity or frequency of arrhythmia events exhibited by the patient following administration of the transgene. A finding that the patient exhibits less or no arrhythmia activity in a specified time period following administration of the CASQ2 transgene relative to an equivalent time period assessed prior to administration of the transgene may indicate that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

[0157] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0158] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the disclosure that come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.