Compositions and methods for treatment of dominantly inherited catecholaminergic polymorphic ventricular tachycardia

Abstract

To provide 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.SOLUTION: Provided is a method of treating dominant catecholaminergic polymorphic ventricular tachycardia (CPVT) in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a transgene encoding calsequestrin 2 (CASQ2). The compositions and methods described herein can be used to treat CPVT caused by mutations in, for example, ryanodine receptor 2 and calmodulin, such as calmodulin 1 (CALM1) and CALM3. The disclosure provides a variety of vectors 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of nucleic acid biotechnology and relates to the use of nucleic acid fragments for the treatment of genetic inherited disorders. The present invention provides compositions and methods for: [Background technology]

[0002] Catecholaminergic polymorphic ventricular tachycardia Orphic ventricular tachycardia (CPVT) is a condition in which the heart It is characterized by dysregulation of calcium signaling in cells. Patients with dominant CPVT: Excessive or dysregulated calcium release from the sarcoplasmic reticulum of cardiomyocytes, especially during cardiac diastole. This release of calcium often indicates an action potential that can produce premature beats. This triggers a series of signaling events that ends with the If left untreated, arrhythmias can be fatal. Available strategies to improve this condition are scarce, and therapeutic approaches to cure the disease are needed. Summary of the Invention

[0003] Described herein are methods for treating dominantly inherited catecholamine-induced inflammatory bowel disorders in patients, such as human patients. Compositions and methods for the treatment of polymorphic ventricular tachycardia (CPVT). The method and methodology described herein involve the administration of functional calsequestrin 2 (CASQ) to patients with dominant CPVT. 2) Delivery of genes encoding proteins, e.g., the compositions and methods described herein. Using the methods and techniques described herein, a transgene encoding a functional CASQ2 protein is introduced into a viral vector. The transgene may be delivered to the patient via gene therapy. The compound may be delivered in a manner that promotes expression of CASQ2 in the patient's cells, such as The present disclosure provides a method for producing a functional CASQ2 protein by delivery of a gene encoding CASQ2. Instead, they are targeting one or more deleterious targets in other calcium-regulating proteins. The study is based in part on the discovery that it can improve CPVT in patients suffering from a genetic mutation. For example, Patients have a gain-of-function mutation in the gene encoding ryanodine receptor 2 (RYR2) and and / or calmodulin 1 (CALM1) and calmodulin 3 (CALM3) Various mutations that cause dominant CPVT, including mutations in the gene encoding calmodulin, have been reported. The compositions and methods described herein can be used to detect various Despite the presence of mutations in calcium-regulating proteins, Cardiac calciton in patients with dominant CPVT without the need to provide a functional version This may restore umbilical cord regulation.

[0004] In a first aspect, the present invention provides a therapeutic composition comprising a transgene encoding CASQ2. administering a therapeutically effective amount to a patient (e.g., a mammal, such as a human patient, in need thereof); The present invention features a method of treating or preventing dominant CPVT in a patient, comprising:

[0005] In another aspect, the present disclosure provides a method for the treatment of a composition comprising a transgene encoding CASQ2. A subject (e.g., a mammal, such as a human subject) diagnosed with dominant CPVT may be administered an effective amount of The present invention features a method for reducing arrhythmia in a patient by administering to the The disclosure provides a method for treating a CPVT infection in a subject, comprising administering a therapeutically effective amount of a composition comprising a transgene encoding CASQ2 to a subject. by administering to a patient (e.g., a mammal, such as a human patient) diagnosed with The present disclosure further features a method for reducing the amount of arrhythmic events in a patient. The present invention relates to a method for treating a patient diagnosed with dominant CPVT, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a transgene encoding 2. to a patient (e.g., a mammal, such as a human patient), Methods for reducing the frequency of arrhythmia events are featured.

[0006] In another aspect, the present disclosure provides a method for the treatment of a composition comprising a transgene encoding CASQ2. A subject (e.g., a mammal, such as a human subject) diagnosed with dominant CPVT may be administered an effective amount of and a method for restoring calcium homeostasis in a patient's heart by administering to the patient a do.

[0007] In a further aspect, the present disclosure provides a composition comprising a transgene encoding CASQ2. A therapeutically effective amount is administered to a patient (e.g., a mammal, such as a human patient) diagnosed with dominant CPVT. By administering the drug to the patient, spontaneous calcium release in the patient's cardiomyocytes is reduced. The present invention is characterized by a method for reducing

[0008] In a further aspect, the present disclosure provides a composition comprising a transgene encoding CASQ2. A therapeutically effective amount is administered to a patient (e.g., a mammal, such as a human patient) diagnosed with dominant CPVT. The present invention features a method for reducing delayed afterdepolarizations in a patient by administering to the patient a therapeutically effective amount of a vasopressin (vasopressin) to reduce delayed afterdepolarizations in the patient.

[0009] In some embodiments of any of the above aspects of the disclosure, the patient has RYR2 These are gain-of-function mutations in endogenous genes that code for RYR2. The mutations increased cardiomyocyte activity in response to RYR2 agonists compared with cardiomyocytes not in contact with RYR2 agonists. This can result in increased cytoplasmic calcium release in cardiomyocytes in contact with R The YR2 mutations were R4497C, N4104K, N4895D, R176Q, and G17 8A, R420W, L433P, P1067S, S2246L, G2273R, F230 7L, E2311D, L2344P, R2474S, T2504M, K3717R, L3 778F, G3926D, G3946S, Q4159P, V4319-K4324dup , R4651I, V4771I, F4851L, A4860G, I4867M, P490 2S, and / or R4959Q.

[0010] For example, a patient may have a combination of two or more of the above RYR2 mutations. In some embodiments, the patient has a R4497C mutation and a N4104K, N4 895D, R176Q, G178A, R420W, L433P, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 0G, I4867M, P4902S, or R4959Q mutation.

[0011] In some embodiments, the patient has a N4104K mutation and a R4497C, N4 895D, R176Q, G178A, R420W, L433P, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0012] In some embodiments, the patient has a N4895D mutation and a R4497C, N4 104K, R176Q, G178A, R420W, L433P, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0013] In some embodiments, the patient has a R176Q mutation and a R4497C, N41 04K, N4895D, G178A, R420W, L433P, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0014] In some embodiments, the patient has a G178A mutation and 04K, N4895D, R176Q, R420W, L433P, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0015] In some embodiments, the patient has a R420W mutation and a R4497C, N41 04K, N4895D, R176Q, G178A, L433P, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0016] In some embodiments, the patient has a G178A mutation and 04K, N4895D, R176Q, R420W, L433P, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0017] In some embodiments, the patient has a L433P mutation and 04K, N4895D, R176Q, R420W, G178A, P1067S, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0018] In some embodiments, the patient has a P1067S mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, S224 6L, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0019] In some embodiments, the patient has a S2246L mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, G2273R, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0020] In some embodiments, the patient has a G2273R mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, F2307L, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0021] In some embodiments, the patient has a F2307L mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, E2311D, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0022] In some embodiments, the patient has an E2311D mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, L2344P, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0023] In some embodiments, the patient has a L2344P mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, R2474S, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0024] In some embodiments, the patient has a R2474S mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, T2 504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0025] In some embodiments, the patient has a T2504M mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0026] In some embodiments, the patient has a K3717R mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0027] In some embodiments, the patient has a L3778F mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0028] In some embodiments, the patient has a G3926D mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0029] In some embodiments, the patient has a G3946S mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, Q4159P, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0030] In some embodiments, the patient has a Q4159P mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, V4319-K4324dup, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0031] In some embodiments, the patient has a V4319-K4324dup mutation and 4497C, N4104K, N4895D, R176Q, R420W, G178A, L4 33P, P1067S, S2246L, G2273R, F2307L, E2311D, L 2344P, R2474S, T2504M, K3717R, L3778F, G3926D , G3946S, Q4159P, R4651I, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0032] In some embodiments, the patient has a R4651I mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, V4771I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0033] In some embodiments, the patient has a V4771I mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, F4851L, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0034] In some embodiments, the patient has a F4851L mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, A486 and one or more of the following mutations: 0G, I4867M, P4902S, and R4959Q. Yes.

[0035] In some embodiments, the patient has an A4860G mutation and an R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F485 and one or more of the following mutations: 1L, I4867M, P4902S, and R4959Q. Yes.

[0036] In some embodiments, the patient has a I4867M mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F485 and one or more of the following mutations: 1L, A4860G, P4902S, and R4959Q. Yes.

[0037] In some embodiments, the patient has a P4902S mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F485 and one or more of the following mutations: 1L, A4860G, I4867M, and R4959Q. Yes.

[0038] In some embodiments, the patient has a R4959Q mutation and a R4497C, N4 104K, N4895D, R176Q, R420W, G178A, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F485 and one or more of the following mutations: 1L, A4860G, I4867M, and P4902S. Yes.

[0039] In some embodiments, the patient has a defect in an endogenous gene encoding calmodulin. For example, patients show a mutation in the CA1 gene that regulates calcium release from the sarcoplasmic reticulum of cardiomyocytes. This may indicate a mutation in CALM1, such as a mutation that reduces LM1 inhibition. In morphology, CALM1 harboring the mutation inhibits calcium release from the sarcoplasmic reticulum in cardiomyocytes. For example, the mutation directly activates 2+ Concentration (e.g. For example, about 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 35 0nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1μM, 2 μM, 3 μM, 4 μM, or 5 μM Ca 2+ C present in an aqueous solution having a concentration It may increase the stability of the open structure of RYR2 in the ALM1-RYR2 complex. In some embodiments, the CALM1 mutation is N97S or N53I.

[0040] In some embodiments, the patient is experiencing a decrease in calcium release from the sarcoplasmic reticulum of cardiomyocytes. In some embodiments, CALM3 mutations, such as mutations that result in activation of the CALM3 gene, are shown. In this case, the CALM3 mutation is A103V.

[0041] In some embodiments, RYR2 or calmodulin (e.g., CALM1 and The mutations in CALM1 and / or CALM3 are heterozygous.

[0042] In some embodiments (e.g., embodiments for treating human patients), a CASQ2-introduced The transgene is at least 85% identical to the amino acid sequence of SEQ ID NO:1 (e.g., SEQ ID NO: The amino acid sequence of No. 1 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, and 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 1 The CASQ2 transgene encodes a protein with an amino acid sequence that is 0.00% identical to the CASQ2 transgene. The protein has one or more conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO:1. For example, the CASQ2 transgene can encode a protein that corresponds to the amino acid sequence of SEQ ID NO:1. 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, 2 3, 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). In an embodiment, the CASQ2 transgene is up to 25 amino acids from the amino acid sequence of SEQ ID NO:1. 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, or 25 conservative amino acid substitutions). In this embodiment, the CASQ2 transgene has up to 10 amino acids with respect to the amino acid sequence of SEQ ID NO:1. Conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative In some embodiments, the C The ASQ2 transgene differs from the amino acid sequence of SEQ ID NO:1 only by conservative amino acid substitutions. The amino acid sequence of the polypeptide is

[0043] In some embodiments (e.g., embodiments for treating human patients), a CASQ2-introduced The transgene has one or more non-conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO:1. For example, the CASQ2 transgene encodes a protein having the amino acid sequence of SEQ ID NO:1. Up to 50 non-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, 3 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 The present invention can encode proteins having 1, 49, or 50 non-conservative amino acid substitutions. In some embodiments, the CASQ2 transgene has the amino acid sequence of SEQ ID NO:1. up to 25 non-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, 2 3, 24, or 25 conservative amino acid substitutions). In some embodiments, the CASQ2 transgene has the amino acid sequence of SEQ ID NO:1 Up to 10 non-conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or or 10 conservative amino acid substitutions).

[0044] In some embodiments, the CASQ2 transgene has the amino acid sequence of SEQ ID NO:3. At least 85% identical (e.g., 85%, 86%, 87%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 29 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 7%, 98%, 99%, 99.9%, or 100% identical) amino acid sequence The CASQ2 transgene encodes a protein having the amino acid sequence of SEQ ID NO:3. It may encode a protein having one or more conservative amino acid substitutions. For example, CASQ2 The transgene may contain up to 50 conservative amino acid substitutions (e.g. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 6, 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) In some embodiments, the CASQ2 transgene can encode a protein that , up to 25 conservative amino acid substitutions (e.g., 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, or 25 conservative amino acid substitutions) In some embodiments, the CASQ2 transgene encodes a protein that Up to 10 conservative amino acid substitutions (e.g., 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions) In some embodiments, the CASQ2 transgene encodes a conserved amino acid sequence. The amino acid sequence of SEQ ID NO:3 is encoded by a sequence different from that of SEQ ID NO:3 only by the amino acid sequence of SEQ ID NO:3. do.

[0045] In some embodiments, the CASQ2 transgene comprises the amino acid sequence of SEQ ID NO:3. For example, CA The SQ2 transgene may contain up to 50 non-conserved amino acids relative to the amino acid sequence of SEQ ID NO:3. 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, 2 8, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nonconserved amino acids In some embodiments, the CASQ2 derivative may encode a protein having a CASQ2 substitution. The transgene may contain up to 25 non-conservative amino acid substitutions (e.g. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 6, 17, 18, 19, 20, 21, 22, 23, 24, or 25 conserved amino acids In some embodiments, the CASQ2-introduced The gene may contain up to 10 non-conservative amino acid substitutions (e.g., For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions It encodes a protein that

[0046] In some embodiments (e.g., embodiments for treating human patients), a CASQ2-introduced The transgene is at least 85% identical to the nucleic acid sequence of SEQ ID NO:2 (e.g., SEQ ID NO:2 of nucleic acid sequences and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical The nucleic acid sequence is

[0047] In some embodiments, the CASQ2 transgene has at least the nucleic acid sequence of SEQ ID NO:4. 4. 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of the nucleic acid sequence.

[0048] In some embodiments, the composition comprising the CASQ2 transgene is a viral vector. Viral vectors include, for example, adeno-associated viruses (AAVs). , adenovirus, lentivirus, retrovirus, poxvirus, baculovirus herpes simplex virus, vaccinia virus, or synthetic viruses (e.g., chimeric viruses, virus, mosaic virus, or pseudotyped virus, and / or foreign protein The therapeutic agent may be a virus, including a material, a synthetic polymer, a nanoparticle, and / or a small molecule. In some embodiments, the viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV AV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74 blood The AAV vector may be a pseudotyped vector. In some embodiments, the pseudotyped AAV vector is AAV2 / 8 or AV2 / 9. do.

[0049] In some embodiments, the composition comprises a vector (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV31, AAV32, A AV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh7 4. AAV vectors such as those mentioned above, exemplified by AAV2 / 8 or AV2 / 9 vectors. viral vector), and the vector is approximately 1x10 6 Genome copies (GC) / kg ~ approx. 1x10 18 Dose of GC / kg (e.g., approximately 1x10 6 GC / kg, 2x10 6 GC / k g, 3x10 6 GC / kg, 4x10 6 GC / kg, 5x10 6 GC / kg, 6x10 6 GC / kg, 7x10 6 GC / kg, 8x10 6 GC / kg, 9x10 6 GC / kg, 1 x10 7 GC / kg, 2x10 7 GC / kg, 3x10 7 GC / kg, 4x10 7 GC / kg, 5x10 7 GC / kg, 6x10 7 GC / kg, 7x10 7 GC / kg, 8x10 7 GC / kg, 9x10 7 GC / kg, 1x10 8 GC / kg, 2x10 8 GC / kg, 3x10 8 GC / kg, 4x10 8 GC / kg, 5x10 8 GC / kg, 6x10 8 GC / kg, 7x10 8 GC / kg, 8x10 8 GC / kg, 9x10 8 GC / kg, 1x1 0 9 GC / kg, 2x10 9 GC / kg, 3x10 9 GC / kg, 4x10 9 GC / kg , 5x10 9GC / kg、6x10 9 GC / kg、7x10 9 GC / kg、8x10 9 G C / kg、9x10 9 GC / kg、1x10 10 GC / kg、2x10 10 GC / kg、 3x10 10 GC / kg、4x10 10 GC / kg、5x10 10 GC / kg、6x10 10 GC / kg、7x10 10 GC / kg、8x10 10 GC / kg、9x10 10 GC / kg、1x10 11 GC / kg、2x10 11 GC / kg、3x10 11 GC / kg、 4x10 11 GC / kg、5x10 11 GC / kg、6x10 11 GC / kg、7x10 11 GC / kg、8x10 11 GC / kg、9x10 11 GC / kg、1x10 12 GC / kg、2x10 12 GC / kg、3x10 12 GC / kg、4x10 12 GC / kg、 5x10 12 GC / kg、6x10 12 GC / kg、7x10 12 GC / kg、8x10 12 GC / kg、9x10 12 GC / kg、1x10 13 GC / kg、2x10 13 GC / kg、3x10 13 GC / kg、4x10 13 GC / kg、5x10 13 GC / kg、 6x10 13GC / kg、7x10 13 GC / kg、8x10 13 GC / kg、9x10 13 GC / kg、1x10 14 GC / kg、2x10 14 GC / kg、3x10 14 GC / kg、4x10 14 GC / kg、5x10 14 GC / kg、6x10 14 GC / kg、 7x10 14 GC / kg、8x10 14 GC / kg、9x10 14 GC / kg、1x10 15 GC / kg、2x10 15 GC / kg、3x10 15 GC / kg、4x10 15 GC / kg、5x10 15 GC / kg、6x10 15 GC / kg、7x10 15 GC / kg、 8x10 15 GC / kg、9x10 15 GC / kg、1x10 16 GC / kg、2x10 16 GC / kg、3x10 16 GC / kg、4x10 16 GC / kg、5x10 16 GC / kg、6x10 16 GC / kg、7x10 16 GC / kg、8x10 16 GC / kg、 9x10 16 GC / kg、1x10 17 GC / kg、2x10 17 GC / kg、3x10 17 GC / kg、4x10 17 GC / kg、5x10 17 GC / kg、6x10 17 GC / kg、7x10 17GC / kg, 8x10 17 GC / kg, 9x10 17 GC / kg, or 1x10 18 In some embodiments, the patient is administered a dose of 100 mg / kg of GC. The vector is about 1x10 8 GC / kg ~ approx. 1x10 16 Dose of GC / kg (e.g. Approx. 1x10 8 GC / kg, 2x10 8 GC / kg, 3x10 8 GC / kg, 4x10 8 G C / kg, 5x10 8 GC / kg, 6x10 8 GC / kg, 7x10 8 GC / kg, 8x 10 8 GC / kg, 9x10 8 GC / kg, 1x10 9 GC / kg, 2x10 9 GC / k g, 3x10 9 GC / kg, 4x10 9 GC / kg, 5x10 9 GC / kg, 6x10 9 GC / kg, 7x10 9 GC / kg, 8x10 9 GC / kg, 9x10 9 GC / kg, 1 x10 10 GC / kg, 2x10 10 GC / kg, 3x10 10 GC / kg, 4x10 1 0 GC / kg, 5x10 10 GC / kg, 6x10 10 GC / kg, 7x10 10 GC / kg, 8x10 10 GC / kg, 9x10 10 GC / kg, 1x10 11 GC / kg, 2 x10 11 GC / kg, 3x1011 GC / kg、4x10 11 GC / kg、5x10 1 1 GC / kg、6x10 11 GC / kg、7x10 11 GC / kg、8x10 11 GC / kg、9x10 11 GC / kg、1x10 12 GC / kg、2x10 12 GC / kg、3 x10 12 GC / kg、4x10 12 GC / kg、5x10 12 GC / kg、6x10 1 2 GC / kg、7x10 12 GC / kg、8x10 12 GC / kg、9x10 12 GC / kg、1x10 13 GC / kg、2x10 13 GC / kg、3x10 13 GC / kg、4 x10 13 GC / kg、5x10 13 GC / kg、6x10 13 GC / kg、7x10 1 3 GC / kg、8x10 13 GC / kg、9x10 13 GC / kg、1x10 14 GC / kg、2x10 14 GC / kg、3x10 14 GC / kg、4x10 14 GC / kg、5 x10 14 GC / kg、6x10 14 GC / kg、7x10 14 GC / kg、8x10 1 4 GC / kg、9x10 14 GC / kg、1x10 15 GC / kg、2x10 15 GC / kg, 3x10 15 GC / kg, 4x10 15 GC / kg, 5x10 15 GC / kg, 6 x10 15 GC / kg, 7x10 15 GC / kg, 8x10 15 GC / kg, 9x10 1 5 GC / kg, or 1x10 16 It is administered to patients at a dose of 100 mg / kg. In an embodiment, the vector is about 1x10 10 GC / kg ~ approx. 1x10 14 GC / kg A dose of (e.g., about 1x10 10 GC / kg, 2x10 10 GC / kg, 3x10 10 G C / kg, 4x10 10 GC / kg, 5x10 10 GC / kg, 6x10 10 GC / kg , 7x10 10 GC / kg, 8x10 10 GC / kg, 9x10 10 GC / kg, 1x1 0 11 GC / kg, 2x10 11 GC / kg, 3x10 11 GC / kg, 4x10 11 G C / kg, 5x10 11 GC / kg, 6x10 11 GC / kg, 7x10 11 GC / kg , 8x10 11 GC / kg, 9x10 11 GC / kg, 1x10 12 GC / kg, 2x1 0 12 GC / kg, 3x10 12 GC / kg, 4x10 12 GC / kg, 5x10 12 G C / kg, 6x10 12GC / kg, 7x10 12 GC / kg, 8x10 12 GC / kg , 9x10 12 GC / kg, 1x10 13 GC / kg, 2x10 13 GC / kg, 3x1 0 13 GC / kg, 4x10 13 GC / kg, 5x10 13 GC / kg, 6x10 13 G C / kg, 7x10 13 GC / kg, 8x10 13 GC / kg, 9x10 13 GC / kg , or 1x10 14 The drug is administered to patients at a dose of 100 mg / kg.

[0050] In some embodiments, the composition comprises a liposome, a vesicle, a synthetic vesicle, an exosome, , synthetic exosomes, dendrimers, or nanoparticles.

[0051] In some embodiments, the transgene encoding CASQ2 is expressed in cardiomyocytes. The CASQ2 gene is operably linked to a promoter that induces expression of the CASQ2 gene. For example, the promoter The promoters are the desmin promoter, the cytomegalovirus promoter, and the myosin light chain 2 promoter. promoter, alpha actin promoter, troponin 1 promoter, Na + / Ca 2+ Exchange promoter, dystrophin promoter, creatine kinase promoter -, alpha 7 integrin promoter, brain natriuretic peptide promoter, Alpha B-crystallin / small heat shock protein promoter, alpha myosin heavy chain promoter, or the atrial natriuretic factor promoter. In an embodiment, the promoter is the desmin promoter.

[0052] In some embodiments, the desmin promoter is an endogenous human desmin transcription start promoter. A region having a nucleic acid sequence from nucleotides -984 to +76 relative to the site, or At least 85% identical to the amino acid sequence (e.g., 85%, 86%, 87%, 88%, 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, or 100% identical to desmin. The promoter is located at nucleotides -973 to - 693 or a region having at least 85% identity to this nucleic acid sequence ( For example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) In some embodiments, the desmin promoter is an endogenous human desmin promoter. a region having the nucleic acid sequence from nucleotide -984 to -644 relative to the start site; or is at least 85% identical to this nucleic acid sequence (e.g., 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% identical) regions. The desmin promoter is located at nucleotide -269 relative to the endogenous human desmin transcription start site. A region having a nucleic acid sequence of 76 to 77 or a region having at least 85% identity to this nucleic acid sequence. (For example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) Includes.

[0053] In some embodiments, the desmin promoter is a promoter that encodes (i) an endogenous human desmin promoter. (ii) a nucleic acid sequence from nucleotide -973 to -693 relative to the transcription start site; or The nucleic acid sequence from nucleotides -973 to -693 relative to the transcription initiation site of human desmin At least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or The desmin promoter further comprises a first region having a sequence identity (or 100% sequence identity) between the desmin promoter and the desmin promoter. (i) nucleotides −269 to +76 relative to the endogenous human desmin transcription start site; or (ii) nucleotide -2 relative to the endogenous human desmin transcription start site. At least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), In some embodiments, the first region is operably linked to the second region. For example, a first region can be directly linked to a second region, with or without intervening nucleotides. In some embodiments, the first region is located 5' of the second region. (i.e., the 3' end of the first region is linked to the 5' end of the second region). In some embodiments, the first region is located 3' of the second region (i.e., the first the 5' end of one region is linked to the 3' end of a second region).

[0054] In some embodiments, the desmin promoter is a promoter that encodes (i) an endogenous human desmin promoter. or (ii) a nucleic acid sequence from nucleotide -984 to -644 relative to the transcription start site; The nucleic acid sequence from nucleotides -984 to -644 relative to the transcription initiation site of human desmin At least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or The desmin promoter further comprises a first region having a sequence identity (or 100% sequence identity) between the desmin promoter and the desmin promoter. (i) nucleotides −269 to +76 relative to the endogenous human desmin transcription start site; or (ii) nucleotide -2 relative to the endogenous human desmin transcription start site. At least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity), In some embodiments, the first region is operably linked to the second region. For example, a first region may be directly adjacent to a second region, with or without intervening nucleotides. In some embodiments, the first region may be linked 5' to the second region. (i.e., the 3' end of the first region is linked to the 5' end of the second region). In some embodiments, the first region is located 3' to the second region (i.e., The 5' end of one region is linked to the 3' end of the second region.

[0055] An exemplary desmin promoter that may be used in conjunction with the compositions and methods of the present disclosure is G ao et al., Journal of Biological Chemist 273:6402-6409 (1998), the disclosure of which is incorporated by reference. No. 6,399,433, which is incorporated herein in its entirety.

[0056] In some embodiments, administration of the composition to a patient results in proliferation of the patient's cardiac cells (e.g., For example, the expression of CASQ2 is increased in the myocardial cells. CASQ can be assayed using gene expression assays known in the art and described herein. This can be assessed by measuring CASQ2 protein or CASQ2 mRNA. Upon administration of the composition to a patient, CASQ2 expression in cardiac cells is increased, for example, by about 1.1-fold to about 1.2-fold. About 10 times, or more (e.g., about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1. 5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.1x, 2.2x, 2.3x , 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3 .2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 5.6 times, 5.7x, 5.8x, 5.9x, 6x, 6.1x, 6.2x, 6.3x, 6.4x, 6. 5x, 6.6x, 6.7x, 6.8x, 6.9x, 7x, 7.1x, 7.2x, 7.3x , 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8x, 8.1x, 8 .2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9 times, 9.1 times, 9.2 times, 9.3 times, 9.4 times, 9.5 times, 9.6 times, 9.7 times, 9.8 For example, CASQ2 expression may be increased in the heart. In cells, about 1.5-fold to about 3.5-fold (e.g., about 0.5-fold, 1.6-fold, 1.7-fold, 1.8-fold) times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, and In some embodiments, CASQ2 expression may be increased by 3.5-fold in cells. Approximately 2 to 3 times (for example, approximately 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times) 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3x) increase in In terms of morphology, CASQ2 expression is increased by about 2.5-fold to about 3-fold (e.g., 2.5-fold) in cells. , 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, or 3-fold) increase.

[0057] In some embodiments, administration of the composition to a patient increases RYR2, triazines, and and / or junctional expression can be assessed, for example, by evaluating protein and / or mRNA expression. The antibody is then administered to the patient in a manner that is consistent with the antibody's intended use, as determined by measuring the activity of the antibody in the patient's cardiac cells (e.g., cardiomyocytes). For example, when the composition is administered to a patient, RYR2, triazine, and / or Junctin expression is less than 10% (e.g., 9.9%, 9.8%, 9.7%, 9.6%, 9.5%, 9.4%, 9.3%, 9.2%, 9.1%, 9%, 8.9%, 8. 8%, 8.7%, 8.6%, 8.5%, 8.4%, 8.3%, 8.2%, 8.1%, 8% , 7.9%, 7.8%, 7.7%, 7.6%, 7.5%, 7.4%, 7.3%, 7.2% ,7.1%,7%,6.9%,6.8%,6.7%,6.6%,6.5%,6.4%,6 .3%, 6.2%, 6.1%, 6%, 5.9%, 5.8%, 5.7%, 5.6%, 5.5 %, 5.4%, 5.3%, 5.2%, 5.1%, 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3. 8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3% , 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2% ,2.1%,2%,1.9%,1.8%,1.7%,1.6%,1.5%,1.4%,1 .3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5 %, 0.4%, 0.3%, 0.2%, 0.1%, 0.01%, 0.001%, or any (below) increase and / or decrease.

[0058] In some embodiments, the composition is administered intravenously, intrathecally, intramuscularly, intradermally, transdermally (transdermally). ansdermal, parenteral, intranasal, subcutaneous, percutaneous, By intratracheal, intraperitoneal, intra-arterial, intravascular, inhalation, perfusion, lavage, and / or oral administration It is administered to the patient.

[0059] In another embodiment, the invention includes a composition comprising a transgene encoding CASQ2. The kit further comprises any of the above aspects of the invention and / or According to any of the above embodiments, a dominant CP of a patient (e.g., a human patient) is identified. The user of the kit is instructed to administer the composition to a patient to treat VT or a symptom thereof. Instructions may be included in the package insert.

[0060] In some embodiments of the foregoing aspects, the CASQ2 transgene comprises the sequence of SEQ ID NO:1. The amino acid sequence of the nucleic acid sequence is at least 85% identical to that of SEQ ID NO:1 (e.g., 85% identical to the amino acid sequence of SEQ ID NO:1). , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.9%, or 100% identical It encodes a protein having the sequence.

[0061] In some embodiments of the foregoing aspects, the CASQ2 transgene comprises the nucleic acid sequence of SEQ ID NO:2. The nucleic acid sequence is at least 85% identical to the nucleic acid sequence of SEQ ID NO:2 (e.g., 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, 99.9%, or 100% identical) Code things.

[0062] In some embodiments of the foregoing aspects, the composition comprising the CASQ2 transgene is Viral vectors include, for example, adeno-associated viruses. AAV, adenovirus, lentivirus, retrovirus, poxvirus, It may be a baculovirus, a herpes simplex virus, or a vaccinia virus. In some embodiments, the viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV AV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74 blood The AAV vector may be a pseudotyped vector. In some embodiments, the pseudotyped AAV vector is AAV2 / 8 or AV2 / 9. do.

[0063] In some embodiments of the aforementioned aspects, the composition comprises a liposome, a vesicle, a synthetic vesicle, The exosome is an exosome, a synthetic exosome, a dendrimer, or a nanoparticle.

[0064] In some embodiments of the foregoing aspects, the transgene encoding CASQ2 is The CASQ2 gene is operably linked to a promoter that induces expression of CASQ2 in muscle cells. For example, the promoter may be the desmin promoter, the myosin light chain 2 promoter, the alpha a promoter, Cutin promoter, Troponin 1 promoter, Na + / Ca 2+ Exchanger Promoter , dystrophin promoter, creatine kinase promoter, alpha 7 inte Glycine promoter, brain natriuretic peptide promoter, alpha B-crystal The phospho / small heat shock protein promoter, the alpha myosin heavy chain promoter, and may be an atrial natriuretic factor promoter.

[0065] As used herein, the term "about" refers to a value within plus or minus 10% of the stated value. vinegar.

[0066] As used herein, the terms "calmodulin 1" ("CALM1") and "calmodulin Calm3 (CALM3) is a second messenger protein that functions as a second messenger protein in muscle cells (e.g., cardiomyocytes). CALM stands for calmodulin calcium-binding protein, which is expressed as a sensor. 1 and CALM3 help regulate the cytoplasmic concentration of divalent calcium ions. An exemplary amino acid sequence of ALM1 can be found in NCBI reference sequence NP_008819.1. Similarly, an exemplary amino acid sequence of wild-type CALM3 is set forth in NCBI Reference SEQ ID NO: Found in P_001316851.1.

[0067] As used herein, the terms "calsequestrin 2" and "CASQ2" refer to, for example, Calcium ions are involved in the regulation of the cytoplasmic concentration of divalent calcium in muscle tissues such as cardiac myocytes. CASQ2 is a naturally occurring gene encoding chromosome 1 in humans. It is located on the chromosome and is also known as "PDIB2". "CASQ2" and "PDIB2" are used interchangeably herein and refer to wild-type CASQ2 as well as variants of the wild-type CASQ2 protein and nucleic acids encoding same The amino acid sequence of wild-type human CASQ2 is provided herein as SEQ ID NO:1. and characterized in the art under NCBI reference sequence number NP_001223.2. Additionally, the nucleic acid sequence of wild-type human CASQ2 is provided herein as SEQ ID NO:2. and characterized in the art under NCBI reference sequence NM_001232.3. CASQ2 transfection genes that may be used in conjunction with the compositions and methods described herein are The gene is at least 85% identical to the amino acid sequence of SEQ ID NO:1 (e.g., SEQ ID NO:1 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100 a transgene encoding a protein having an amino acid sequence that is 100% identical to the amino acid sequence of the sequence One or more conservative amino acid substitutions (e.g., up to 5, up to 1 0, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or Up to 50 conservative amino acid substitutions) and / or one or more non-conservative amino acid substitutions (e.g. For example, max 5, max 10, max 15, max 20, max 25, max 30, max 35, max 4 0, up to 45, or up to 50 non-conservative amino acid substitutions) Exemplary transgenes that may be used in conjunction with the compositions and methods described herein include The CASQ2 transgene is at least 85% identical to the amino acid sequence of SEQ ID NO:2 ( For example, the amino acid sequence of SEQ ID NO: 2 and 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 the CA The SQ2 transgene may furthermore exhibit an affinity that is within, for example, up to 25% of the affinity of wild-type CASQ2. Gender (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%, or 25% affinity) and / or wild-type CASQ Within 25% or less of the stoichiometry of 2 (e.g., 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 (within the range of 0.01 to 0.50) and retain the ability to polymerize and bind divalent calcium. These include those encoding fragments of the aforementioned proteins. Examples of assays that can be used to assess stoichiometry are known in the art. See, e.g., Johnson et al. Methods Mol. Biol. 173:89-102 (2002), the disclosure of which is incorporated by reference in its entirety. is incorporated herein.

[0068] As used herein, the terms "catecholamine-induced polymorphic ventricular tachycardia" and "CPVT" refer to Refers to an inherited channelopathy characterized by a high susceptibility of patients to fatal arrhythmias. Two forms of the disease have been described: somatic dominant and autosomal recessive variants. The autosomal dominant form of rheumatoid arthritis is associated with mutations in the cardiac ryanodine receptor type 2 (RYR2) gene. Autosomal recessive variants are associated with mutations in the CASQ2 gene (e.g., Priori et al., Circulation 104(Supplement t II):335(2001), and Lahat et al., Am. J. See Hum. Genet. 69:1378-1384 (2001). The disclosures herein are incorporated by reference in their entireties. Patients with dominantly inherited disorders called "autosomal dominant CPVT" and "autosomal dominant CPVT" Typically, patients respond to sympathetic activation with bidirectional and polymorphic ventricular tachycardia. Patients with CPVT have unremarkable resting ECG profiles and preserved cardiac structure The arrhythmia of CPVT is referred to herein as "spontaneous calcium release" or "SCR." These are triggered by calcium release events that are not triggered by an action potential. SCR begins as a localized event involving a single calcium release unit (CRU); It may also diffuse to adjacent CRUs, resulting in more calcium release. This causes calcium waves throughout the cell. Abnormal calcium release from the sarcoplasmic reticulum occurs. The subsequent action potential can then propagate throughout the heart, causing a premature beat. If this sequence of events is repeated, triggered arrhythmic activity is induced. Dominant CPVT has been shown to promote the occurrence of SCR during β-adrenergic stimulation This is often caused by a mutation in the calcium receptor 2 (RYR2). Arrhythmias resulting from dysregulated activity can lead to fatal arrhythmias ( For example, Liu et al., Circulation Research 99: 292-298 (2006), the disclosure of which is incorporated herein by reference in its entirety. Mutations in RYR2 associated with dominant CPVT include R4497C, N4 104K, N4895D, R176Q, G178A, R420W, L433P, P106 7S, S2246L, G2273R, F2307L, E2311D, L2344P, R2 474S, T2504M, K3717R, L3778F, G3926D, G3946S, Q4159P, V4319-K4324dup, R4651I, V4771I, F485 These include 1L, A4860G, I4867M, P4902S, and R4959Q.

[0069] As used herein, protein mutations are expressed using the form "AA1-N-AA2." where AA1 represents the amino acid occurring at position N of the wild-type form of the protein of interest, and A A2 represents the amino acid occurring at position N in the mutant form of the protein. For example, the RYR2 mutant The variant "R4497C" is a mutant in which the arginine residue at position 4497 ​​of the wild-type RYR2 protein is replaced by a seq. This refers to a mutant form of RYR2 in which a stearate residue has been replaced by a stearate residue. 319-K4324dup" is a deletion that occupies amino acid residues V4319 to K432 of wild-type RYR2. The nucleotides coding for 4 are duplicated, resulting in 18 nucleotides coding for these amino acids. Insertion of additional instances of (e.g., nucleotide 12955 of the wild-type RYR2 gene) Insertion of residues GTCGAAGGTGCTAAAAAG (SEQ ID NO: 7) between 12956 and 12956 This refers to a mutant form of the RYR2 gene that results in

[0070] As used herein, the terms "conservative mutation," "conservative substitution," or "conservative amino acid substitution" are used interchangeably. "Acid substitution" refers to one or more compounds that exhibit similar physicochemical properties such as polarity, electrostatic charge, and steric volume. These characteristics are described in the table below. 1 is summarized for each of the 20 naturally occurring amino acids.

[0071] [Table 1]

[0072] From this table, conserved amino acid families include, for example, (i) G, A, V, L, I, P (ii) D and E; (iii) C, S, and T; (iv) H, K, and (v) N and Q; and (vi) F, Y, and W. Thus, conservative mutations or substitutions are used to group members of the same amino acid family into one amino acid. amino acid substitutions (e.g., Thr with Ser, or Arg with L ys).

[0073] As used herein, the term "desmin promoter" refers to (i) the human desmin transcription initiation site Endogenous human desmin promoter defined by nucleotides -984 to +76 (ii) a polynucleotide having the nucleic acid sequence of the vector, and (iii) variants and functional portions thereof. For example, the desmin promoter may be used in conjunction with the compositions and methods of the present disclosure. - includes the nucleic acid sequence from nucleotides -984 to +76 relative to the human desmin transcription start site. At least 85% identical (e.g., 85%, 86%, e.g., 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99%, or 99.9% or more identical) and the transgene is When operably linked to a promoter, the cell (e.g., a mammalian cell, a human cell, or These include those that promote the expression of CASQ2 transgenes in eukaryotic cells (e.g., human cardiomyocytes). Desmin promoters that may be used in conjunction with the compositions and methods described herein Further examples are given in Gao et al., Journal of Biologica Chemistry 273:6402-6409 (1998), The disclosure of which is incorporated herein by reference in its entirety. When used in the context of a transcriptional regulatory element, such as a transcription factor, the term "functional portion" refers to a transcription factor that is involved in the transcription of a target cell. It refers to a portion of a larger nucleic acid that retains the ability to stimulate transcription of a gene of interest in a cell. For example, the endogenous human desmin promoter is nucleoside relative to the desmin transcription start site. It contains residues -984 to +76 and is capable of retaining the transcription activation properties of larger nucleic acids. An example of a functional portion of this locus is nucleotide -9, relative to the desmin transcription start site. Another example is the region including nucleotides 73 to -693 relative to the desmin transcription start site. This is the region including positions -269 to +76.

[0074] Similarly, as used herein, the terms "cytomegalovirus promoter," "myosin "Light chain 2 promoter", "Alpha actin promoter", "Troponin 1 promoter" -," "Na + / Ca 2+ exchanger promoter," "dystrophin promoter," Creatine kinase promoter, alpha 7 integrin promoter, brain Natriuretic peptide promoter, alpha B crystallin / small heat shock protein "Protein promoter," "alpha myosin heavy chain promoter," and "atrial natriuretic The “human diuretic factor promoter” and “human cytomegalovirus promoter” are the wild-type endogenous human cytomegalovirus promoters, respectively. promoter, myosin light chain 2 promoter, alpha actin promoter, troponin 1 promoter Roboter, Na + / Ca 2+ Exchange promoter, dystrophin promoter, Cre Attenuated kinase promoter, alpha7 integrin promoter, brain sodium utilization Urinary peptide promoter, alpha B crystallin / small heat shock protein promoter -, alpha myosin heavy chain promoter, and atrial natriuretic factor promoter , as well as variants and functional portions thereof.

[0075] As used herein in reference to a mutation, the term "heterozygous" refers to a patient has one mutant allele of a particular gene, i.e., the mutant maternal allele or the mutant paternal allele. This refers to a case in which a person has either one of the two alleles.

[0076] As used herein in reference to a mutation, the term "homozygous" refers to a mutation in which the patient Two mutant alleles of a particular gene, namely the mutant maternal allele and the mutant paternal allele This refers to the case where a person has an allele.

[0077] As used herein, the term "gain-of-function mutation" refers to a mutation in one or more amino acids in a protein. Mutations that enhance protein function through amino acid substitutions, insertions, and / or deletions For example, the ryanodine receptor 2 (RYR2) protein is transported from the sarcoplasmic reticulum of cardiomyocytes to A channel protein that mediates the release of divalent calcium ions into the cytosol. An exemplary gain-of-function mutation in the case of R2 is RYR2-mediated calcitonin transporter (CALC) transporter from the sarcoplasmic reticulum. Gain-of-function mutations in RYR2 include R449 7C, N4104K, N4895D, R176Q, G178A, R420W, L433P , P1067S, S2246L, G2273R, F2307L, E2311D, L234 4P, R2474S, T2504M, K3717R, L3778F, G3926D, G3 946S, Q4159P, V4319-K4324dup, R4651I, V4771I , F4851L, A4860G, I4867M, P4902S, and / or R495 This includes 9Q, etc.

[0078] As used herein, the term "operably linked" refers to a second molecule (e.g., a second nucleic acid molecule) that is It refers to the binding of a first molecule (e.g., a first nucleic acid) to a first nucleic acid, which molecules The molecules are arranged so that they affect the function of the second molecule. The two molecules are connected in a single adjacent They may or may not be part of the same molecule and may or may not be adjacent to each other. For example, a promoter may regulate the transcription of a transcribable polynucleotide molecule of interest in a cell. In this case, the promoter is operably linked to the transcribable polynucleotide molecule. The two parts of a transcriptional regulatory element are such that the transcriptional activation function of one part depends on the presence of the other part. Therefore, when they are joined together, they are operably linked to each other. The two transcriptional regulatory elements are connected via a linker nucleic acid (e.g., an intervening non-coding nucleic acid). or can be operably linked to each other without intervening nucleotides. It can be linked to the function.

[0079] "Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence" " aligns sequences and introduces gaps (if necessary) to find the maximum percent After achieving sequence identity, the nucleic acid in the reference polynucleotide or polypeptide sequence or as the percentage of nucleic acids or amino acids in the candidate sequence that are identical to the amino acid For purposes of determining percent nucleic acid or amino acid sequence identity, Examples of such software include BLAST, BLAST-2, or Megalign software. Using publicly available computer software, various The method can be carried out by the method described above. Those skilled in the art can carry out the method of maximal alignment over the entire length of the sequences to be compared. For sequence alignment, including any algorithms required to achieve this alignment Appropriate parameters can be determined. For example, the percent sequence identity value can be calculated by The comparison computer program BLAST may be used to generate the BLAST sequence. Percent sequence identity of a given nucleic acid or amino acid sequence A to nucleic acid or amino acid sequence B identity (in other words, a particular percent sequence identity to a given nucleic acid or amino acid sequence B) For a given nucleic acid or amino acid sequence A) having the following properties, the following is calculated: 100×(fraction X / Y) where X is the sequence that is matched by a sequence alignment program (such as BLAST). Nucleotides scored as perfect matches in the alignment of sequences A and B or where Y is the number of amino acids in B, and Y is the total number of nucleic acids in B. If the length is not equal to the length of nucleic acid or amino acid sequence B, the percent alignment of A to B is Sequence identity is not equal to the percent sequence identity of B to A.

[0080] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical composition that is administered to a subject, such as a mammal, for example a human. Prevent, treat, or treat a specific disease or condition that is affecting or may affect The therapeutic agent administered to a subject to control, optionally in combination with one or more pharma- ceutical acceptable excipients. "A" refers to a mixture containing the active ingredient in combination with a diluent and / or carrier.

[0081] As used herein, the term "pharmacologically acceptable" means a compound that is compatible with a reasonable benefit-risk ratio. without commensurate undue toxicity, irritation, allergic reactions, and other problems in mammals (e.g. Compounds, materials, compositions suitable for contact with tissue of a subject, e.g., a human, and / or Refers to the dosage form.

[0082] As used herein, the terms "ryanodine receptor 2" and "RYR2" refer to, for example, myocardial cells. RYR2 is a calcium channel protein located outside the sarcoplasmic reticulum in the endoplasmic reticulum. It mediates the release of divalent calcium from the sarcoplasmic reticulum, thereby initiating the activity that leads to cardiac contraction. An exemplary amino acid sequence of wild-type human RYR2 is set forth in NCBI Reference Sequence No. See No. NP_001026.2.

[0083] As used herein, the terms "ryanodine receptor 2 agonist" and "RYR2 agonist" are used interchangeably. "RyR2 channel" refers to an agent that stimulates the calcium-releasing properties of the RYR2 channel protein. Potential RYR2 agonists include caffeine, epinephrine, trifluoroperazine, and and maurocalcine, but are not limited to these.

[0084] As used herein, the term "sample" refers to a specimen (e.g., blood, plasma, etc.) isolated from a subject. Fluid components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissues (e.g., placenta, A subject may be, for example, a genetically modified organism, a human or human skin sample, a pancreatic fluid, a chorionic villus sample, or cells. The present invention relates to a method for treating cardiac disorders, such as tachycardia, including dominant catecholaminergic polymorphic ventricular tachycardia. The patient may be suffering from a disease described in the document.

[0085] As used herein, the expressions "specifically bind" and "bind" refer to specifically bind to a Ions, salts, etc. that are recognized by ligands or receptors, such as calcium-binding proteins , divalent calcium ions, etc., in heterogeneous populations of small molecules, and / or proteins. Refers to a binding reaction that determines the presence of a particular molecule or ion. A specific binding ligand (e.g., a calcium binding protein) may be present at, for example, less than 1 mM. K of Mitsu D For example, a ligand that specifically binds to a species can bind to a species at up to 100 μM (e.g., between 1 pM and 100 μM) D can bind to a species with another molecule or ion Ligands that do not exhibit specific binding to a particular molecule or ion are concentrated to 1 mM (e.g. K greater than 1 μM, 100 μM, 500 μM, 1 mM, or more DIt can be shown that A variety of assay formats are available to determine the affinity of a ligand for a particular protein. For example, a solid-phase ELISA assay is used to detect a specific antibody that binds to a target protein. These are routinely used to identify ligands that bind to specific proteins. For a description of assay formats and conditions available for ow & Lane, Antibodies, A Laboratory Manu al, Cold Spring Harbor Press, New York(1 988), and Harlow & Lane, Using Antibodies , A Laboratory Manual, Cold Spring Harbo See, for example, IEEE Press, New York (1999). Exemplary methods available for determining the affinity of a ligand for divalent calcium ions (Ca2+, divalent calcium ions) are Such assays are described, for example, in Johnson et al. Methods Mol. B iol. 173:89-102 (2002), the disclosure of which is incorporated by reference. It is incorporated herein in its entirety.

[0086] As used herein, the terms "subject" and "patient" refer to a subject who is suffering from a particular disease or condition described herein. Treatment of conditions (e.g., inherited cardiac disorders such as autosomal dominant catecholaminergic polymorphic ventricular tachycardia) Examples of subjects and patients include those receiving treatment for a disease or condition described herein. This includes mammals, such as humans, who are receiving

[0087] As used herein, the term "tachycardia" refers to a cardiac arrest or tachycardia that occurs in a patient (eg, a young adult) that exceeds the normal resting heart rate of a given species. For example, a human patient with dominant CPVT. In medical treatment, tachycardia includes when a patient's heart rate exceeds 100 beats per minute. Methods for monitoring are known in the art and are described, for example, in Example 1 below. do.

[0088] As used herein, the term "transcriptional regulatory element" refers to an element that regulates the transcription of a gene of interest at least A transcriptional regulatory element refers to a nucleic acid that controls or partially controls gene transcription. Promoters, enhancers, and other nucleic acids that help control Examples of transcriptional regulatory elements include, for example, ene Expression Technology: Methods in En zymology 185(Academic Press, San Diego, CA, 1990).

[0089] As used herein, the terms "treat" or "treatment" refer to therapeutic treatment, the purpose of which is to Unfavorable physiology, such as the progression of inherited cardiac disorders such as CPVT, especially dominant CPVT In the case of CPVT treatment, the beneficial effects are Desired clinical outcomes include alleviation of symptoms, improvement of disease, whether detectable or undetectable. a decrease in the severity of the disease, a stable (i.e., not worsening) state of the disease, a delay in the progression of the disease, or These include, but are not limited to, slowing, improvement or alleviation of symptoms, and remission (partial or total). Treatment of patients with CPVT (e.g., dominant CPVT) can be achieved by administering the methods described herein to patients without the need for any of the following: Arrhythmic events (e.g., tachycardial events) exhibited by a patient during a specified period following administration of the therapeutic agent described herein. ) (e.g., a reduction in the amount or frequency of Arrhythmic events exhibited by a patient following administration of a vector, such as a viral vector (e.g., This may manifest as one or more detectable changes, such as a decrease in the amount or frequency of tachycardial events. Indications for successful treatment of patients with dominant CPVT using the compositions and methods described herein include: , an arrhythmic event exhibited by the patient during a period (e.g., a period of the same length) prior to administration of the therapeutic agent. The amount and / or frequency of tachycardial events (e.g., tachycardia events) in a patient's The amount or frequency of arrhythmia events (e.g., tachycardia events) exhibited by subjects was 1%, 2%, 3%, or %, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, This includes findings of an 85%, 90%, 95%, or 100% reduction in the efficacy and safety of CPVT patients. Another clinical indication of therapeutic success includes the development of a therapeutic agent in the patient's cardiomyocytes (e.g., cardiac myocytes) following administration of the therapeutic agent to the patient. Cytosolic calcium ([Ca 2+ ]) is, for example, 1% , 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% Further indications of treatment success include the finding that the patient's (e.g. For example, the expression of CASQ2 in the patient's cardiac cells (e.g., cardiomyocytes) increases after administration of a therapeutic drug. The expression of CASQ2 was found to be increased at 1%, 2%, 3%, 4%, 5%, , 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x The concentration of CASQ2 protein may be increased by, for example, 50 to 100 μg / ml, or more. using protein detection assays known in the art, including ELISA assays such as The concentration of nucleic acid encoding CASQ2 may be determined ex vivo. Using the nucleic acid detection assays described herein (e.g., RNASeq assays), It may be determined by ivo.

[0090] As used herein, the term "vector" refers to a vector or vectors that are used for any purpose, such as replication and / or expression. as a vehicle for delivering genes to cells (e.g., mammalian cells, such as human cells). The term "nucleic acid" refers to a nucleic acid, such as DNA or RNA, that can function in a specific manner. Exemplary vectors useful in conjunction with the replicon, or other suitable replicon (e.g., viral vector). To deliver a polynucleotide encoding a protein to a prokaryotic or eukaryotic cell, Various vectors have been developed. Examples of such expression vectors are described in, for example, WO19 94 / 11026, the disclosure of which is incorporated herein by reference. The expression vectors described herein can be used to express polynucleotide sequences, as well as to express, e.g., proteins. Expression of the polypeptide and / or integration of these polynucleotide sequences into the genome of mammalian cells. The sequence elements included herein are useful for expression of the transgenes described herein. The specific vectors available contain promoter and enhancer regions that direct gene transcription. Other useful vectors for the expression of transgenes include plasmids containing regulatory sequences such as the ribosomal region. The activators either increase the rate of translation of these genes or increase the mRNA levels resulting from gene transcription. These sequence elements include polynucleotide sequences that improve the stability or nuclear export of the These include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and and polyadenylation signals to direct efficient transcription of genes carried by the expression vector. The expression vectors described herein also include a transcription factor receptor site. The polynucleotide may include a polynucleotide encoding a marker for the selection of the cell. Examples include ampicillin, chloramphenicol, kanamycin, or nourseothricin. These include genes that code for resistance to antibiotics such as rifampin. [Brief description of the drawings]

[0091] [Figure 1] 1 is a diagram of the pAAV2.1-CMV-CASQ2-IRES-eGP construct used in the mouse CASQ2 gene therapy experiment described in Example 1 below. The vector contains, from 5' to 3', a 5'AAV2 inverted terminal repeat (ITR), a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) intron, a wild-type mouse calsequestrin 2 (CASQ2) transgene, an internal ribosome entry site (IRES), an enhanced green fluorescent protein (eGFP) transgene, a woodchuck posttranscriptional regulatory element (WPRE), a bovine growth hormone (BGH) polyadenylation sequence, and a 3'AAV2 ITR. The pAAV2.1-CMV-CASQ2-IRES-eGFP construct used in Example 1 below was packaged into an AAV9 capsid, thus generating a recombinant AAV2 / 9 vector. [Diagram 2] Graph showing the effect of pAAV2.1-CMV-CASQ2-IRES-eGFP vector on the percentage of arrhythmic events in R4496C / + heterozygous knock-in mice (Ryr2-Het), a model of autosomal dominant CPVT, upon exposure to epinephrine (2 mg / kg) and caffeine (120 mg / kg). The black bars (left) show that all untreated RyR2-Het mice (n=5) showed arrhythmias after administration of epinephrine and caffeine. The results on the right side of the graph show that none of the RyR2-Het mice infected with the pAAV2.1-CMV-CASQ2-IRES-eGFP vector (n=4) developed arrhythmias. [Diagram 3] Graph showing mRNA expression of CASQ2, cardiac RYR2, triadin, and junctin in myocardial tissues of wild-type mice and RYR2R4496C / + mice untreated or treated with 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 RYR2R4496C / + heterozygous knock-in mice (Ryr2-Het) relative to wild-type (WT). Grey bars show fold expression of mRNA transcripts in RYR2R4496C / + heterozygous knock-in mice treated with pAAV2.1-CMV-CASQ2-IRES-eGFP vector (Ryr2-Het AAV9-CASQ2) relative to wild-type (WT). [Figure 4] 1 shows various components of another AAV vector that was found to have antiarrhythmic effects in R4496C / + heterozygous knock-in mice. The vector contains a CASQ2 expression cassette flanked by 5' and 3' AAV2 ITRs. The expression cassette contains a transgene encoding wild-type CASQ2 operably linked to the desmin promoter. [Diagram 5]Graph showing the effect of recombinant pseudotyped AAV2 / 8 containing the vectors shown in Figure 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 bars (left) show that approximately 44% (4 of 9) of untreated RyR2-Het mice (n=9) exhibited arrhythmias after administration of epinephrine and caffeine. The results on the right side of the graph show that none of the RyR2-Het mice (n=9) infected with the rAAV2 / 8 vector encoding CASQ2 developed arrhythmias. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] Provided herein is a method for treating catecholaminergic polymorphic ventricular tachycardia (CPV) in a patient, such as a human patient. This invention describes compositions and methods for treating pulmonary venous thrombocytopenic purpura (PVT), particularly autosomal dominant CPVT. mutations in one or more proteins that regulate the concentration of divalent calcium ions in cardiomyocytes For example, dominant CPVT is caused by ryanodine receptor 2 (RYR2) transcription factor. Various mutations in the protein, particularly in the sarcoplasmic reticulum, have been implicated in the secretion of divalent calcium ions via RYR2. This is associated with gain-of-function mutations that upregulate the release of This leads to a cascade of signaling and muscle contraction events that culminates in the heartbeat. also express calmodulin proteins such as calmodulin 1 (CALM1) and CALM3. These proteins are also associated with loss-of-function mutations in Helps regulate cytoplasmic calcium by binding and sequestering ions, thereby During diastole, i.e. the period of relaxation of the heart, excess calcium from the sarcoplasmic reticulum is released. Sium release produces premature heart beats that can lead to arrhythmias.

[0093] The present disclosure provides a method for treating a dominant CPVT disorder comprising administering to a subject an in vivo or in vivo treatment with a genetic disorder comprising administering to a subject ... This means that the drugs can be used to treat patients without the need to provide them with the gene or transgenes that encode them. The present invention is based in part on the discovery that the compositions and methods described herein can be used to induce dominant CPVT. Patients, such as human patients with pulmonary edema, express functional calsequestrin 2 (CASQ2) protein. This can be treated by administering a transgene that encodes CASQ2, which induces cardiac cardiogenesis. Sodium homeostasis is restored, arrhythmic events are reduced, and normal heart rate returns.

[0094] Using the compositions and methods described herein, a gene encoding a functional CASQ2 protein can be produced. The transgene can be delivered using liposomes, vesicles, exosomes, dendrimers, or nanoparticles. Among other approaches described below, the use of The following sections provide an overview of the pathophysiology underlying dominant CPVT, Exemplary vectors and methods available for providing a functional CASQ2 transgene to a patient include: and other gene delivery vehicles are described.

[0095] Dominant CPVT CPVT is an inherited channelopathy characterized by high susceptibility to life-threatening arrhythmias Two forms of the disease have been reported: an autosomal dominant and an autosomal recessive variant. Autosomal dominant is associated with mutations in the cardiac RYR2 gene, while autosomal recessive variants are It is associated with mutations in the cardiac CASQ2 gene (Priori et al., Circulation 104(Supplement II):335(2001) and Lahat et al., Am. J. Hum. Genet. 69 :1378-1384(2001)). Clinical observations have shown that patients with a dominant form of CPVT Patients develop bidirectional and polymorphic ventricular tachycardia in response to sympathetic activation but not at rest. The electrocardiogram profile was unremarkable, indicating that cardiac structure was preserved.

[0096] The pathology underlying dominant CPVT is the loss of CALCIUM, a fundamental component of cardiac excitation-contraction coupling. The abnormal functioning of a physiological mechanism called calcium-induced calcium release (CICR) It is related to the highly coordinated opening and closing of voltage-dependent ion channels in the membranes of cardiac myocytes. This generates the cardiac action potential. During the plateau phase of the action potential, voltage-dependent L-type Ca 2+ blood The opening of the Ca channel in the plasma membrane 2+ This process allows the inflow of Ca2+ transients and sarcoplasmic reticulum Ca 2+ Release channel: RYR2 opening (Bers, Nature 415:198-205(2002)). Their local release occurs from specialized structures called calcium release units (CRUs). CRU preferentially localizes to the level of transverse tubules (T-tubules), where the membrane of the sarcoplasmic reticulum is apposed to the plasma membrane. One CRU is localized to L-type Ca2+ receptors on the cell membrane. 2+ In the sarcoplasmic reticulum membrane, close to the channel It is formed by a cluster of RYR2 spanning ng, et l., Ann. NY Acad. Sci. 1047:76-85 (2005)). Ca released from the sarcoplasmic reticulum 2+ Binds to troponin C and myosin It induces a series of allosteric changes in the filaments, causing the muscle fiber to contract. Ca 2+ Removal of Ca results in the simultaneous closure of RYR2 and 2+ back into the sarcoplasmic reticulum store Sarcoplasmic reticulum Ca 2+ It is mediated by the action of ATPase.

[0097] Ca 2+ Another component of the transient termination is Na + -Ca 2+ Exchanger (NCX) NCX is the 3 Na+ + ion (three positive charges) for every Ca 2+ ions (two positive charges) are pushed out. Thus, NCX produces a net inward depolarizing current , i.e., the transient inward current (I ti ) to generate Ca 2+ Remove (Pieske, et al., Circ. Res. 85:38-46(199 9) NCX is characterized by calcium overload, for example in the case of inherited mutations in RYR2. Under conditions where Ca 2+ is very important for the removal of

[0098] The arrhythmia of CPVT is caused by Ca2+ deficiencies that are not triggered by action potentials. 2+ Release events (spontaneous The SCR is triggered by a single CRU-associated calcium release (SCR). It starts as a localized event that affects the CRUs but can spread to neighboring CRUs, resulting in More Ca 2+ This triggers the release of calcium, generating calcium waves throughout the cell. The probability of generating calcium waves is 2+ Content and sarcoplasmic reticulum calcium threshold Called Ca 2+Ca release trigger 2+ It is influenced by the balance between the concentration of RYR2 Function plays an important role in controlling this threshold. Several R YR2 mutations lower the sarcoplasmic reticulum calcium threshold so that SCR is readily induced It has been suggested that the mechanism of action of the α-amyloid complex is through the α-amyloid complex (Venetucci et al., Nat. Rev. Cardiol. 9:561-75(2012)).

[0099] Abnormal Ca 2+ When leakage occurs, cytoplasmic Ca 2+ As the concentration increases, the cells 2+ permanent Prevents the breakdown of normality and Ca 2+ Activates mechanisms that re-establish physiological diastolic levels of I need to ti The current produces a transient membrane depolarization after the completion of the action potential, which occurs after a delay The amplitude of the DAD is known as the depolarization (DAD). + Voltage threshold for channel activation When the heart reaches the triggered action potential, a triggered action potential is generated. As the action potential propagates throughout the heart, a premature ventricular contraction occurs. This sequence of events is repeated until some DADs reach the threshold for generating a propagating action potential. When this threshold is reached, triggered arrhythmic activity is induced. promotes the occurrence of SCR during β-adrenergic stimulation, which in turn induces DAD and leads to lethal It has been shown that vasopressin induces arrhythmia-related activity (Liu et al., 2013). irculation Research 99:292-298(2006)).

[0100] RYR2 for autosomal dominant CPVT R4496C / + Liver knock-in mouse model Construction and characterization (e.g., Cerrone et al., M, Circulat ion Research 96:e77-e82(2005) and US7,74 1,529) provides excellent insight into the pathomechanism of this disease. R2 R4496C / + Heterozygous mice recapitulate human CPVT and show adrenergic Human R4497C induces bidirectional and polymorphic ventricular arrhythmias. The corresponding mouse R4496C mutation inhibits RYR2 channel response to luminal calcium. It increases the sensitivity of the nerve endings and promotes the spontaneous release of calcium from the sarcoplasmic reticulum.

[0101] Examples of mutations that can cause dominant CPVT are R4497C, N4104K , N4895D, R176Q, G178A, R420W, L433P, P1067S, S 2246L, G2273R, F2307L, E2311D, L2344P, R2474S , T2504M, K3717R, L3778F, G3926D, G3946S, Q415 9P, V4319-K4324dup, R4651I, V4771I, F4851L, A 4860G, I4867M, P4902S, and R4959Q. CALM1 mutations that may lead to idiopathic pulmonary edema include N97S and N53I, with the majority An example of a CALM3 mutation that can lead to progressive CPVT is A103V.

[0102] Arrhythmogenic RYR2, CALM1, and / or CPVT caused by CALM3 mutations is a functional gene encoding these proteins. It has been discovered that treatment can be achieved without the need to provide a transgene to the patient. Using the compositions and methods described herein, patients with dominant CPVT can be treated with a CASQ2 transgene. The treatment can be achieved by administration of a delivery vehicle comprising the compositions described herein and Exemplary vehicles for delivery of CASQ2 useful in conjunction with the methods are described in detail below. .

[0103] Treatment of CPVT with CASQ2 gene therapy CASQ2 transgene Using the compositions and methods described herein, a transgene encoding CASQ2 can be introduced into The method can be provided to a patient, such as a human patient with dominant CPVT, to treat the pathology. The CASQ2 transgene can be used in, among other compositions for gene therapy described herein. , can be delivered to a patient via a variety of vectors (e.g., viral vectors). In embodiments (e.g., embodiments for treating a human patient), the CASQ2 transgene is and at least the amino acid sequence of wild-type human CASQ2 set forth in SEQ ID NO:1 below. 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or or 100% identical) amino acid sequence to the polypeptide described herein. The CASQ2 transgene for use in conjunction with the compositions and methods described herein includes the CASQ2 transgene of SEQ ID NO:1. A protein encoding a protein having one or more conservative amino acid substitutions relative to the amino acid sequence. For example, the CASQ2 transgene may have the following amino acid sequence: The 50 largest 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, 2 4, 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). The child may additionally or alternatively have one or more non-conservative amino acid residues relative to the amino acid sequence of SEQ ID NO:1. For example, the CASQ2 transgene may have an amino acid substitution in the amino acid sequence of SEQ ID NO:1. up to 50 non-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, 4 The polypeptide may encode proteins having 9, 10, 20, 30, 40, 50, or 60 non-conservative amino acid substitutions.

[0104] In some embodiments (e.g., embodiments for treating human patients), a CASQ2-introduced The transgene has at least the nucleic acid sequence of wild-type human CASQ2, as set forth in SEQ ID NO:2, shown below. 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) nucleic acid sequences. Exemplary Wild-Type CASQ2 Amino Acids Useful in Conjunction with the Compositions and Methods Described Herein and the nucleic acid sequences are set forth in Table 2 below.

[0105] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0106] The CASQ2 transgene herein may further be used to enhance, for example, the affinity of wild-type CASQ2. Affinity within up to 25% of the target (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% ,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%, (within 19%, 20%, 21%, 22%, 23%, 24%, or 25% affinity) and and / or within 25% or less of the stoichiometry of wild-type CASQ2 (e.g., 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) of stoichiometry. These include those encoding fragments of the aforementioned proteins that retain the ability to bind to the Examples of assays that can be used to assess calcium binding affinity and stoichiometry are Methods of ion exchange polymerization are known in the art and are described, for example, in Johnson et al. Mol. Biol. 173:89-102 (2002), and the The disclosure is incorporated herein by reference in its entirety.

[0107] Transcriptional Regulatory Elements The CASQ2 transgenes described herein comprise a transgene operably linked to a promoter. As discussed above, the vector, plasmid, or other nucleic acid delivery vehicle described herein may be In conjunction with the compositions and methods described herein, Exemplary useful promoters are those that are expressed in cardiac cells, such as cardiomyocytes, of a patient (e.g., a human patient). For example, the gene delivery vectors described herein induce expression of the transgene in the host. Promoters that can be used to induce expression of CASQ2 in the vehicle include desmopressin, desmopressin, and desmopressin. Min promoter, cytomegalovirus promoter, myosin light chain 2 promoter, Alpha actin promoter, troponin 1 promoter, Na + / Ca 2+ Exchanger P promoter, dystrophin promoter, creatine kinase promoter, alpha 7 integrin promoter, brain natriuretic peptide promoter, alpha B- Crystallin / small heat shock protein promoter, alpha myosin heavy chain promoter or atrial natriuretic factor promoter, but are not limited to .

[0108] Vectors for delivering exogenous nucleic acids to target cells Viral Vectors for Nucleic Acid Delivery The viral genome is transferred to the genome of a target cell of a patient (e.g., a mammalian cell, such as a human cell). This provides a plentiful source of vectors available for efficient delivery of genes of interest to viruses. A genome is a set of polynucleotides that are typically either generalized or specialized. It is integrated into the genome of target cells by controlled transduction, making it a unique tool for gene delivery. These processes are part of the natural viral replication cycle and are useful vectors for This occurs naturally and does not require the addition of any proteins or reagents to induce gene integration. Examples of viral vectors that can be used in conjunction with the compositions and methods described herein include AAV , retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, Orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), etc. Negative-stranded RNA viruses, positive-stranded RNA viruses such as picornaviruses and alphaviruses and adenoviruses, herpes viruses (e.g., herpes simplex virus 1 type 2, Epstein-Barr virus, cytomegalovirus, and poxvirus Viruses (e.g., vaccinia, modified vaccinia Ankara (MVA), chicken pox, and canary pox) The double-stranded DNA viruses that may be used in conjunction with the compositions and methods described herein include Other viruses that may be used include, for example, Norwalk virus, Togavirus, Flavivirus, These include viruses such as rabies, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B and D viruses. These include viruses, HTLV-BLV group, lentiviruses, and spumaviruses (Cof fin, JM, Retroviridae: The viruses an d their replication, In Fundamental Viro logy, Third Edition, BN Fields, et al ., Eds., Lippincott-Raven Publishers, Ph. iladelphia, 1996). Other examples include murine leukemia virus, murine Sarcoma virus, Mouse mammary tumor virus, Bovine leukemia virus, Feline leukemia virus, Nephroblastoma virus, Avian sarcoma virus, Avian leukosis virus, Human T-cell leukemia virus, Sheep endogenous virus s, Gibbon Ape Leukemia Virus, Mason Pfizer Monkey Virus, Simian Immunodeficiency Viruses include simian sarcoma virus, rous sarcoma virus, and lentiviruses. Other examples of vectors are described, for example, in US Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference. As it relates to viral vectors for use in gene therapy, the disclosure of which is incorporated herein by reference. do.

[0109] AAV vectors for nucleic acid delivery In some embodiments, the nucleic acids of the compositions and methods described herein are derived from patient samples. Delivery of rAAV vectors and / or virions to cells, such as targeted cardiac cells (e.g., cardiomyocytes). To facilitate transduction of the gene, the gene may be incorporated into the rAAV vector and / or virion. rAAV vectors useful in conjunction with the compositions and methods described herein include: (1) CA SQ2 transgene, and (2) a viral core that facilitates integration and expression of heterologous genes. The viral nucleic acid is the nucleic acid that is responsible for replicating DNA into virions. and AAV proteins required in cis for packaging (e.g., functional ITRs). Such rAAV vectors may contain marker or reporter genes. Useful rAAV vectors may also contain the entirety of one or more naturally occurring AAV genes. These include those that have been deleted in whole or in part but retain functional flanking ITR sequences. AAVITR may be derived from any serotype (e.g., 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 Monah an and Samulski, Gene Delivery 7:24-30(2 000), whose disclosures relate to AAV vectors for gene delivery. and is hereby incorporated by reference.

[0110] The nucleic acids and vectors described herein facilitate the introduction of the nucleic acid or vector into a cell. To make it more efficient, the capsid protein of AAV can be incorporated into rAAV virions. The protein constitutes the external, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins required for virion assembly. The rAAV virion is made up of, for example, US5,173,414, US5,139,941, US5,863,541, US5,86 9,305, 6,057,152, and 6,376,237, as well as Rabi nowitz et al., J. Virol. 76:791-801(2002 ) and Bowles et al., J. Virol. 77:423-432( 2003), whose disclosures relate to AAV vectors for gene delivery. and is hereby incorporated by reference.

[0111] rAAV virions useful in conjunction with the compositions and methods described herein include AAV1, These include those derived from various AAV serotypes, including 2, 3, 4, 5, 6, 7, 8, and 9. The construction and use of AAV vectors and AAV proteins of different serotypes is described in, e.g. , 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 e. t al., J. Virol. 75:6615-6624(2001), and A Uricchio et al., Hum. Molec. Genet. 10:3 No. 075-3081 (2001), the disclosures of which are incorporated herein by reference for purposes of gene delivery. As it relates to AAV vectors, it is incorporated herein by reference.

[0112] Useful in conjunction with the compositions and methods described herein include pseudotyped rAAV vectors. Pseudotyped vectors can be vectors of a given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9) It contains an AAV vector of a given serotype (e.g., AAV2) pseudotyped with the capsid gene. For example, representative pseudotyped vectors are derived from AAV serotype 8 or AAV serotype 9. AAV2 vectors encoding therapeutic proteins pseudotyped with the capsid gene of interest Techniques involving rAAV virions and uses of pseudotyped rAAV virions are known in the art. It is known in the field, e.g., Duan et al., J. Virol. 75:76 62-7671(2001), Halbert et al., J. Virol. 74:1524-1532(2000), Zolotukhin et al., Me. thods, 28:158-167(2002), and Auricchio et al. al., Hum. Molec. Genet., 10:3075-3081(20 01).

[0113] AAV virions with mutations in the virion capsid are similar to the unmutated capsid. can be used to infect certain cell types more efficiently than virions. For example, AAV mutants have been developed to enhance the expression of ligands that facilitate targeting of AAV to specific cell types. Insertion mutants, alanine screening mutants, and Construction and characterization of AAV capsid mutants, including epitope-tagged mutants In Wu et al., J. Virol. 74:8635-45 (2000) Other rAAV virions that can be used in the methods of the invention include Capsid hybrids generated by molecular breeding and exon shuffling of . For example, Soong et al., Nat. Genet., 25 :436-439(2000), and Kolman and Stemmer, Na See, e.g., T. Biotechnol. 19:423-428(2001).

[0114] Additional methods for transgene delivery to target cells Transfection technology A CASQ2 transgene operably linked to the transcriptional regulatory elements described herein. The techniques available for introducing the CASQ2 transgene into target cells are well known in the art. For example, electroporation involves applying an electrostatic potential to cells of interest. used to permeabilize mammalian cells (e.g., human target cells) by adding Mammalian cells, such as human cells, exposed to an external electric field in this manner can They are then susceptible to uptake of exogenous nucleic acids. For example, see Chu et al., Nucleic Acids Research. h 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection, is incorporated herein by reference. TM (Nucleic acid gene injection The technique involves stimulating the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. , utilizing an applied electric field. TM and to implement this technology. Useful protocols can be found, for example, in Distler et al., Experimental al Dermatology 14:315(2005), and US2010 / 03 17114, the disclosures of which are incorporated herein by reference. do.

[0115] An additional technique useful for transfection of target cells is squeezeporation. This technique involves the transfer of exogenous D through membrane pores that form in response to an applied stress. To stimulate NA uptake, rapid mechanical deformation of cells is induced. This technique is The advantage of this approach is that it does not require a vector to deliver the nucleic acid into a cell, such as a target cell. The queesporation is described, for example, in Sharei et al., Journal of f Visualized Experiments 81:e50980(2013) No. 6,399,433, the disclosure of which is incorporated herein by reference.

[0116] Lipofection is another technique useful for transfection of target cells. The method includes loading a nucleic acid into a liposome, which is directed toward the exterior of the liposome. They often exhibit cationic functional groups such as diamines or protonated amines. The anionic nature of the cell membrane promotes electrostatic interactions between the liposomes and the cells, ultimately , for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection results in the uptake of exogenous nucleic acids. 6, the disclosure of which is incorporated herein by reference. A similar technique that uses ionic interactions to induce the uptake of foreign nucleic acids is the cathodopsin technique. The method includes contacting the nucleic acid complex with an isotopic polymer-nucleic acid complex that is favorable for interaction with the cell membrane. Exemplary cationic molecules that bind to polynucleotides to impart a positive charge include activated deoxyribonucleic acid (ADCA) and activated deoxyribonucleic acid (ADCA). ndrimer (e.g., Dennig, Topics in Current Chem is described in US Pat. No. 6,393,311, the disclosure of which is incorporated herein by reference. (which are incorporated herein by reference in their entirety), and diethylaminoethyl (DEAE) dextran. Their use as transfection agents is described, for example, in Gulick et al. l., Current Protocols in Molecular Biolo gy 40:I:9.2:9.2.1 (1997), the disclosure of which is The magnetic beads are capable of directing the delivery of target cells in a gentle and efficient manner. is another tool that can be used to transfect cells. This method This technique is used in, for example, US2 No. 6,227,406, the disclosure of which is incorporated herein by reference. Be absorbed.

[0117] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is the laser. fection, which gently permeabilizes the cells and allows the polynucleotide to bind to the cell membrane. A technique that involves exposing cells to specific wavelengths of electromagnetic radiation in order to make them permeable. This technique is described, for example, in Rhodes et al., Methods in C The disclosure of which is described in detail in E. L. Biology 82:309 (2007). is incorporated herein by reference.

[0118] The microvesicles can be used to modify the genome of a target cell according to the methods described herein. These represent other potential vehicles for the fusion of the glycoprotein VSV-G and nucleases, etc. Using microvesicles induced by co-overexpression of genomic modified proteins, The protein is efficiently delivered into cells and then transduced into the target polynucleotide, such as a gene or regulatory sequence. Endogenous polynucleotides are synthesized to prepare the genome of cells for covalent incorporation of oligonucleotides. It catalyzes the site-specific cleavage of DNA sequences. It is also known as Gesicles, a method for genetic modification of eukaryotic cells. The use of such vesicles is described, for example, in Quinn et al., Genetic Modification of Target Cells by Direct D elivery of Active Protein [abstract]. : Methylation changes in early embryonic genes in cancer [abstract], in: Proceed ings of the 18th Annual Meeting of the A American Society of Gene and Cell Therapy ; 2015 May 13, Abstract No. 122 do.

[0119] Targeted gene integration using gene editing technology In addition to the above, various methods are available for introducing genes of interest into target cells, such as human cells. Various tools have been developed. One available method for incorporating genomic DNA involves the use of transposons. , a polynucleotide encoding a transposase enzyme, and 5' and 3' excision sites The transposon delivers the desired polynucleotide sequence or gene to the cell. Once this is reached, expression of the transposase gene begins, resulting in the transfection of the gene of interest. This activity is the transposase-mediated transfection of the sposon. This is mediated by site-specific recognition of poson excision sites. In some cases, these excision sites The position may be a terminal repeat or an inverted terminal repeat. The target gene is expressed via transposase-catalyzed cleavage of a similar excision site present in the nuclear genome of the cell. This allows the gene of interest to be integrated into the genome of a mammalian cell by can be inserted into cleaved nuclear DNA at specific excision sites and subsequently into the mammalian cell genome Integration by covalent phosphodiester linkages that connect the gene of interest to DNA The process is complete. In some cases, the transposon also The offspring are first transcribed into RNA products, which are then converted to DNA before being integrated into the mammalian cell genome. The transposon may be a retrotransposon that is reverse transcribed into A. The system may be a piggyBac transposon (described in detail in, for example, WO2010 / 085699). (see references) and Sleeping Beauty transposons (e.g., US2 005 / 0112764), the disclosures of which are incorporated herein by reference in their entirety. The present disclosure is incorporated by reference as it relates to transposons used for gene delivery to Can be enjoyed.

[0120] Another tool for integrating target genes into the genome of target cells is clustered and ordered Clustered regularly interleaved short palindromic repeats rspaced short palindromic repeats (CRISPR )) / Cas system, which was originally developed to protect bacteria and archaea against viral infections. It is a system that evolved as an adaptive defense mechanism. The CRISPR / Cas system has The palindromic repeat sequence in the plasmid DNA and the associated Cas9 nuclease This ensemble of DNA and proteins is what initially binds the foreign DNA to the CRISPR receptor. By integrating into the gene locus, they direct site-specific DNA cleavage of the target sequence. Polynucleotides containing the foreign sequence and the repeat spacer element of the CRISPR locus The guide RNA is then transcribed by the host cell to create a guide RNA, which is then annealed to the target sequence. By using the cDNA clone, the Cas9 nuclease can be localized to this site. The interaction that brings as9 into close proximity to the target DNA molecule is an RNA:DNA hybridization The cas9-mediated DNA cleavage is highly site-specific because it is governed by the Thus, any target DNA molecule of interest can be generated by the It is possible to design CRISPR / Cas systems that can open the genome of eukaryotic organisms. (Hwang et al., Nature Biol. Echnology 31:227(2013)), a set of genes encoding the target genes An efficient means to site-specifically edit the genome of target cells to cleave DNA prior to insertion The use of CRISPR / Cas to regulate gene expression has been demonstrated, for example, in For example, see US 8,697,359, the disclosure of which describes CR for genome editing. As it relates to the use of ISPR / Cas systems, it is incorporated herein by reference. An alternative for site-specific cleavage of genomic DNA prior to integration of a gene of interest into target cells Methods include the use of zinc finger nucleases (ZFNs) and transcription activator-like effectors. This involves the use of TALENs, which are different from the CRISPR / Cas system. These enzymes contain guide polynucleotides that allow them to localize to specific target sequences. Instead, target specificity is controlled by the DNA-binding domain within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov e t al., Nature Reviews Genetics 11:636(20 10) and Joung et al., Nature Reviews Molec This is described in ular Cell Biology 14:49 (2013), The disclosure of which is incorporated herein by reference for its disclosure regarding compositions and methods for genome editing. To be incorporated.

[0121] Can be used to integrate a polynucleotide encoding a target gene into the genome of a target cell Another genome editing technique that can be used is the rational design of genes that cleave genomic DNA in a site-specific manner. ARCUS can TM The use of meganucleases is included. The use of these enzymes to integrate nucleotides into the genome of mammalian cells has led to the development of novel methods for the synthesis of such enzymes. This is advantageous in view of the defined structure-activity relationships established in the past. , a nuclease that selectively cleaves DNA at desired positions by modifying specific amino acid positions This generates a plasmid that allows site-specific integration of the target gene into the nuclear DNA of the target cell. The single-stranded nucleases described by et al. are, for example, in US Pat. No. 8,021,867 and US Pat. No. 8,445,233. 51, the disclosures of which are directed to compositions and methods for genome editing. For purposes of this disclosure, the disclosure is hereby incorporated by reference.

[0122] Methods for measuring transgene expression Expression of the CASQ2 transgene expressed by the patient's target cells (e.g., cardiomyocytes) The level may be, for example, the concentration of mRNA transcripts resulting from transcription of the CASQ2 transgene or This can be confirmed by assessing relative abundance. Gene expression is determined by the transcription and translation of the CASQ2 transgene. This can be determined by assessing the concentration or relative abundance of a protein. Protein concentration can also be assessed using functional assays such as calcium abundance assays. The following sections provide information regarding the efficacy and safety of the CPVT inhibitors when delivered to patients, such as those with dominant CPVT, as described herein. Exemplary techniques available for measuring CASQ2 transgene expression levels Transgene expression can be analyzed by nucleic acid sequencing, microarray analysis, proteomics, and i In-situ hybridization (e.g., fluorescent in-situ hybridization) FISH), amplification-based assays, in situ hybridization, and fluorescent in situ hybridization. Photoactivated cell sorting (FACS), Northern analysis, and / or mRNA PCR This can be assessed by a number of methods known in the art, including, but not limited to, R analysis. It is Noh.

[0123] Nucleic Acid Detection Transgene expression detection methods suitable for analysis in conjunction with the compositions and methods described herein. Nucleic acid-based methods for determining the marker set include imaging-based techniques (e.g., Northern or Southern blotting, which allows Northern blot analysis may be used with cells obtained from the patient after administration. These techniques are well known in the art and are described, for example, in Molecular Cloning, a aboratory manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainvie 11803-2503. A typical protocol for evaluating the effect of β-glucose on the cellular metabolism is described, for example, in Ausubel et al., ed. S., 1995, Current Protocols In Molecular Biology, Units 2(Northern Blotting), 4( Southern Blotting), 15(Immunoblotting) a nd 18 (PCR Analysis).

[0124] Can be used in conjunction with the compositions and methods described herein to assess CASQ2 expression Further transgene detection techniques include microarray sequencing experiments (e.g., high-throughput sequencing). Sanger sequencing and next-generation sequencing, also known as batch sequencing or deep sequencing, Exemplary next-generation sequencing technologies include Illumina sequencing, Ionto sequencing, and Rent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms Other sequencing methods known in the art include, but are not limited to, the following: For example, expression of the transgene at the mRNA level can be achieved using RNA-Se q can be determined using (e.g., Mortazavi et al., Nat. Methods 5:621-628 (2008), the disclosure of which is incorporated by reference. (The entire contents of this document are incorporated herein by reference.) RNA-Seq is a method for directly identifying RNA molecules in a sample. It is a robust technique for monitoring expression by sequencing. This method involves fragmenting RNA to an average length of 200 nucleotides and random priming. This can involve conversion of the cDNA to a cDNA fragment containing the desired sequence, and synthesis of double-stranded cDNA (e.g., Agilent Technologies, Inc.). Technology's Just cDNA DoubleStranded cDN The cDNA was then extracted from each library using the A Synthesis Kit. By adding sequence adaptors (e.g., Illlumina® / Solexa) The resulting 50-100 nucleotides are then converted into a molecular library for sequencing. The leution reads are mapped to the genome.

[0125] Since microarray technology provides high resolution, the expression level of the transgene can be measured by microarray analysis. It may be determined using an array-based platform (such as a single nucleotide polymorphism array). Details of various microarray methods are described in the literature. 68 and Pollack et al., Nat. Genet. 23:41-4 6 (1999), the disclosures of which are incorporated herein by reference in their entireties. Nucleic acid microarrays are used to reverse transcribe and label mRNA samples for cDNA synthesis. The probe is then attached to one or more arrays of immobilized NAs on a solid support. Each member of the array can hybridize to a complementary nucleic acid. The sequences and positions of the labeled probes and the identified Hybridization to the array members of a probe indicates that the sample from which the probe was derived is a genetic The expression level was determined by the hybridized probe-sample ratio. The complex may be quantified according to the amount of signal detected. The experiment involves the following steps: 1) isolation of fluorescently labeled RNA from samples; 2) hybridization of the labeled targets to the microarray; 3) hybridization of the labeled targets to the array. 4) washing, staining, and scanning the cells; 5) analyzing the scanned images; and 6) analyzing gene expression profiles. An example of a microarray processor is the GENE The CHIP® system, which is commercially available, delivers oligonucleotides onto a glass surface. Other systems known to those skilled in the art include arrays fabricated by direct synthesis of It may be used.

[0126] Amplification-based assays measure the expression levels of transgenes in target cells after delivery to patients. In such assays, the nucleic acid sequence of a gene is amplified and In quantitative amplification, the nucleotide sequence of the nucleotides in the nucleotides serve as a template for a PCR reaction (e.g., PCR, such as qPCR). The amount of amplified product is proportional to the amount of template in the original sample. According to the principles described herein, the expression of the gene corresponding to the particular probe used is A measure of the level of IgG is provided. The methods are well known in the art. Detailed protocols for real-time qPCR can be found, for example, in Gibson et al., Genome Res. 6:995-1001(19 96) and Heid et al., Genome Res. 6:986-994 (1996), the disclosures of which are incorporated herein by reference in their entireties. The levels of gene expression described herein are determined by RT-PCR techniques. The probes used in PCR can be, for example, radioisotopes, fluorescent compounds, etc. a detectable compound, such as a marker, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme The antibody may be labeled with a marker.

[0127] Protein detection Expression of the transgene further results in the production of the corresponding protein encoded by the gene of interest. This can be determined by measuring the concentration or relative abundance of a substance (e.g., CASQ2). Protein levels can be assessed using standard detection techniques known in the art. Protein expression vectors suitable for use in the compositions and methods described herein can be used. Assays include proteomic methods, immunohistochemical and / or western blot analysis. precipitation, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assay (ELISA) ), mass spectrometry, mass spectrometric immunoassays, and biochemical enzyme activity assays. In addition, proteomics methods are being used to collect large-scale, multiplexed protein expression data. Proteomics methods use mass spectrometry to generate a set of polypeptides. (e.g., protein) and / or capture reagents specific for a panel of target proteins (e.g., The peptide microarrays are used to detect and quantify the amount of the peptides in the sample (e.g., antibodies). For example, to simultaneously measure the expression levels of proteins expressed in a single cell sample or multiple cell populations It can be determined and measured.

[0128] An exemplary peptide microarray has a plurality of polypeptides bound to a substrate, Each of a plurality of linked polypeptides, such as oligonucleotides, peptides, or proteins Alternatively, the peptide microarray may be a monoclonal antibody. Monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid The antibody may include multiple binders, including but not limited to, aptamers. These specifically detect specific oligonucleotide, peptide, or protein binding. Examples of peptide arrays can be found in US Pat. No. 6,060,513, and No. 5,143,854, the disclosures of which are incorporated herein by reference. The entirety of which is incorporated herein.

[0129] Mass spectrometry (MS) can be used to characterize the expression of genomic DNA in cells from a patient (e.g., a human patient) following delivery of a transgene. In combination with the methods described herein, Any MS method known in the art can be used to identify the protein or proteins of interest. or peptide fragments can be determined, detected, and / or measured, e.g. , LC-MS, ESI-MS, ESI-MS / MS, MALDI-TOF-MS, MAL DI-TOF / TOF-MS, tandem MS, etc. can be used. Typically, it contains an ion source and optics, a mass spectrometer, and data processing electronics. These include scanning and ion beam mass spectrometers such as time-of-flight (TOF) and quadruple (Q) The Ion Trap (IT), Orbitrap, and Fourier Transform Ion Cyclotron are all A trapping mass spectrometer such as a FT-ICR may be used in the methods described herein. Details of the various MS methods are described in the literature, e.g., Yates et al. ., Annu. Rev. Biomed. Eng. 11:49-79, 200 9, the disclosure of which is incorporated herein by reference in its entirety.

[0130] Prior to MS analysis, proteins in samples obtained from patients are first chemically characterized (e.g. , via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion. Complex peptide samples can be digested into small peptides using 2D-PAGE, HPLC, Also the use of front-end separation techniques such as RPLC and affinity chromatography The digested and optionally separated sample is then ionized using an ion source. The ionization of a sample can be achieved by, for example, electrochemical analysis. electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, Child ionization, fast atom bombardment (FAB) / liquid secondary ionization (LSIMS), matrix Assisted Laser Desorption / Ionization (MALDI), Field Ionization, Field Desorption, -This is done by ionization with phospray / plasmaspray and particle beam ionization. Additional information regarding the selection of an ionization method is known to those of skill in the art.

[0131] After ionization, the digested peptides are fragmented to perform signature MS / MS analysis. Tandem MS, also known as MS / MS, can produce spectra of complex mixtures. Tandem MS can be particularly useful for the analysis of compounds. Between stages some form of ion fragmentation occurs, which is spatial separation. Separate individual mass analyzer elements or a single mass analyzer with the MS steps separated in time In spatially separated tandem MS, the elements are They are temporally separate and distinct, with the high vacuum maintained by physical connections between the elements. In tandem MS, separation is achieved with ions trapped in the same location, Multiple separation steps are performed over time, and then signature MS / MS spectra are generated. Comparison with peptide sequence databases (e.g., SEQUEST) may be used. Post-modifications can also be performed, for example, by searching for specific peptide modifications against a database. The vector can be determined by searching the

[0132] Pharmaceutical Compositions The CASQ2 transgene described herein can be used to treat a dominant CPV as described herein. The composition may be incorporated into a vehicle for administration to a patient, such as a human patient, suffering from T. A pharmaceutical composition comprising a vector, such as a viral vector, containing an ASQ2 transgene is The compositions can be prepared using methods known in the art. For example, the compositions can be prepared in a physiologically acceptable form. Acceptable carriers, excipients, or stabilizers (Remington's Pharmaceuticals tical Sciences 16th edition, Osol, A. Ed. (1980), incorporated herein by reference, to prepare a lyophilized formulation or It can be prepared in a desired form, such as an aqueous solution.

[0133] The mixture of nucleic acids and viral vectors described herein may be mixed with one or more excipients, carriers, The dispersion can also be prepared in water suitably mixed with a diluent. It may also be prepared in liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Suitable pharmaceutical forms for injectable use include sterile injectable solutions or dispersions. These include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of liquids (US5, 466,468, the disclosure of which is incorporated herein by reference. In any event, the formulation may be sterile and may be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and is protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and their appropriate mixtures The appropriate fluidity may be a solvent or dispersion medium, including, for example, a water-soluble polymer, a water-soluble polymeric ... For example, the use of a coating such as lecithin can help maintain the required particle size in the case of dispersion. Prevention of microbial action can be achieved by the use of surfactants and by the use of microbial agents. Various antibacterial agents such as rabenes, chlorobutanol, phenol, sorbic acid, and thimerosal and antifungal agents. Often, isotonic agents such as sugars or sodium chloride are used. Prolonged absorption of the injectable compositions is preferably achieved by including aluminum monostearate. This can be achieved by the use in the compositions of agents that delay absorption, such as polysaccharides and gelatin.

[0134] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered, if necessary; The liquid diluent is first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Suitable sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. The dose is dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous solution. Depending on the condition of the subject being treated, However, some variation in dosage will inevitably occur. The person responsible for administration should, in any event, determine the appropriate dosage for the individual subject. For human administration, the preparations are also approved by the Food and Drug Administration for use in the manufacture of pharmaceuticals and pharmaceutical products. Required by FDA Office of Biologics standards It may meet sterility, pyrogenicity, and general safety and purity standards.

[0135] Route of administration and dosing Viruses, such as the AAV vectors and other vectors described herein, that contain a transgene. The vector can be administered to a patient (e.g., a human patient) by a variety of routes of administration. The route will vary depending on, for example, the onset and severity of the disease, and may be, for example, intravenous, intrathecal, or intradermal. intramuscular, intranasal, subcutaneous, transdermal, parenteral cutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, irrigation, lavage, and transfusion Intravascular administration includes delivery into the vascular system of a patient. In the study, administration was into a blood vessel that is considered to be a vein (intravenous), and some In administration, administration is into a blood vessel that is considered to be an artery (intra-arterial). The internal jugular vein, peripheral veins, coronary veins, hepatic veins, portal vein, great saphenous vein, pulmonary veins, superior vena cava, inferior vena cava, Vena cava, gastric vein, splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and / or femoral vein Arteries include, but are not limited to, the coronary arteries, pulmonary arteries, brachial arteries, and internal jugular arteries. These may include, but are not limited to, the aorta, aortic arch, femoral artery, peripheral artery, and / or ciliary artery. Delivery can be via or to an arteriole or capillary. It is believed that the following could be carried out.

[0136] Treatment plans vary and often depend on the severity of the disease and the age, weight, and sex of the patient. Therapeutics are useful for the introduction of transgenes into target cells at various unit doses. A vector (e.g., a viral vector) or other agent as described herein. Each unit dose typically contains a predetermined amount of the therapeutic composition.

[0137] The transgene is delivered by viral gene therapy (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, AA For administration of an AAV vector as described herein, such as an AAV2 / 8 or AAV2 / 9 vector, Thus, when delivered to a patient, the viral vector may be, for example, about 1 x 10 6 Genome Copy -(GC) / kg ~ approx. 1x10 18 At a dose of GC / kg (e.g., approximately 1x10 6 GC / k g, 2x10 6 GC / kg, 3x10 6 GC / kg, 4x10 6 GC / kg, 5x10 6 GC / kg, 6x10 6 GC / kg, 7x10 6 GC / kg, 8x10 6 GC / kg, 9 x10 6 GC / kg, 1x10 7 GC / kg, 2x10 7 GC / kg, 3x10 7 GC / kg, 4x10 7 GC / kg, 5x10 7 GC / kg, 6x10 7 GC / kg, 7x10 7 GC / kg, 8x10 7GC / kg、9x10 7 GC / kg、1x10 8 GC / kg、 2x10 8 GC / kg、3x10 8 GC / kg、4x10 8 GC / kg、5x10 8 GC / kg、6x10 8 GC / kg、7x10 8 GC / kg、8x10 8 GC / kg、9x1 0 8 GC / kg、1x10 9 GC / kg、2x10 9 GC / kg、3x10 9 GC / kg 、4x10 9 GC / kg、5x10 9 GC / kg、6x10 9 GC / kg、7x10 9 G C / kg、8x10 9 GC / kg、9x10 9 GC / kg、1x10 10 GC / kg、2 x10 10 GC / kg、3x10 10 GC / kg、4x10 10 GC / kg、5x10 1 0 GC / kg、6x10 10 GC / kg、7x10 10 GC / kg、8x10 10 GC / kg、9x10 10 GC / kg、1x10 11 GC / kg、2x10 11 GC / kg、3 x10 11 GC / kg、4x10 11 GC / kg、5x10 11 GC / kg、6x10 1 1 GC / kg、7x10 11 GC / kg、8x10 11 GC / kg、9x1011 GC / kg、1x10 12 GC / kg、2x10 12 GC / kg、3x10 12 GC / kg、4 x10 12 GC / kg、5x10 12 GC / kg、6x10 12 GC / kg、7x10 1 2 GC / kg、8x10 12 GC / kg、9x10 12 GC / kg、1x10 13 GC / kg、2x10 13 GC / kg、3x10 13 GC / kg、4x10 13 GC / kg、5 x10 13 GC / kg、6x10 13 GC / kg、7x10 13 GC / kg、8x10 1 3 GC / kg、9x10 13 GC / kg、1x10 14 GC / kg、2x10 14 GC / kg、3x10 14 GC / kg、4x10 14 GC / kg、5x10 14 GC / kg、6 x10 14 GC / kg、7x10 14 GC / kg、8x10 14 GC / kg、9x10 1 4 GC / kg、1x10 15 GC / kg、2x10 15 GC / kg、3x10 15 GC / kg、4x10 15 GC / kg、5x10 15 GC / kg、6x10 15 GC / kg、7 x10 15 GC / kg、8x10 15GC / kg, 9x10 15 GC / kg, 1x10 1 6 GC / kg, 2x10 16 GC / kg, 3x10 16 GC / kg, 4x10 16 GC / kg, 5x10 16 GC / kg, 6x10 16 GC / kg, 7x10 16 GC / kg, 8 x10 16 GC / kg, 9x10 16 GC / kg, 1x10 17 GC / kg, 2x10 1 7 GC / kg, 3x10 17 GC / kg, 4x10 17 GC / kg, 5x10 17 GC / kg, 6x10 17 GC / kg, 7x10 17 GC / kg, 8x10 17 GC / kg, 9 x10 17 GC / kg, or 1x10 18 GC / kg) to the patient. In some embodiments, the vector is about 1x10 8 GC / kg ~ approx. 1x10 16 G At a dose of C / kg (e.g., approximately 1x10 8 GC / kg, 2x10 8 GC / kg, 3x10 8 GC / kg, 4x10 8 GC / kg, 5x10 8 GC / kg, 6x10 8 GC / kg, 7x10 8 GC / kg, 8x10 8 GC / kg, 9x10 8 GC / kg, 1x10 9 GC / kg, 2x10 9 GC / kg, 3x10 9GC / kg、4x10 9 GC / kg、5x1 0 9 GC / kg、6x10 9 GC / kg、7x10 9 GC / kg、8x10 9 GC / kg 、9x10 9 GC / kg、1x10 10 GC / kg、2x10 10 GC / kg、3x10 10 GC / kg、4x10 10 GC / kg、5x10 10 GC / kg、6x10 10 GC / kg、7x10 10 GC / kg、8x10 10 GC / kg、9x10 10 GC / kg、 1x10 11 GC / kg、2x10 11 GC / kg、3x10 11 GC / kg、4x10 11 GC / kg、5x10 11 GC / kg、6x10 11 GC / kg、7x10 11 GC / kg、8x10 11 GC / kg、9x10 11 GC / kg、1x10 12 GC / kg、 2x10 12 GC / kg、3x10 12 GC / kg、4x10 12 GC / kg、5x10 12 GC / kg、6x10 12 GC / kg、7x10 12 GC / kg、8x10 12 GC / kg、9x10 12 GC / kg、1x10 13 GC / kg、2x10 13 GC / kg、 3x10 13 GC / kg、4x10 13GC / kg, 5x10 13 GC / kg, 6x10 13 GC / kg, 7x10 13 GC / kg, 8x10 13 GC / kg, 9x10 13 GC / kg, 1x10 14 GC / kg, 2x10 14 GC / kg, 3x10 14 GC / kg, 4x10 14 GC / kg, 5x10 14 GC / kg, 6x10 14 GC / kg, 7x10 14 GC / kg, 8x10 14 GC / kg, 9x10 14 GC / kg, 1x10 15 GC / kg, 2x10 15 GC / kg, 3x10 15 GC / kg, 4x10 15 GC / kg, 5x10 15 GC / kg, 6x10 15 GC / kg, 7x10 15 GC / kg, 8x10 15 GC / kg, 9x10 15 GC / kg, or 1x10 16 GC / kg) In some embodiments, the vector is administered to a subject at a dose of about 1 x 10 10 GC / kg ~ approx. 1x10 14 At a dose of GC / kg (e.g., approximately 1x10 10 GC / kg, 2x10 1 0 GC / kg, 3x10 10 GC / kg, 4x10 10 GC / kg, 5x10 10 GC / kg, 6x10 10 GC / kg, 7x10 10 GC / kg, 8x10 10 GC / kg, 9 x10 10 GC / kg, 1x10 11 GC / kg, 2x10 11 GC / kg, 3x10 1 1 GC / kg, 4x10 11 GC / kg, 5x10 11 GC / kg, 6x10 11 GC / kg, 7x10 11 GC / kg, 8x10 11 GC / kg, 9x10 11 GC / kg, 1 x10 12 GC / kg, 2x10 12 GC / kg, 3x10 12 GC / kg, 4x10 1 2 GC / kg, 5x10 12 GC / kg, 6x10 12 GC / kg, 7x10 12 GC / kg, 8x10 12 GC / kg, 9x10 12 GC / kg, 1x10 13 GC / kg, 2 x10 13 GC / kg, 3x10 13 GC / kg, 4x10 13 GC / kg, 5x10 1 3 GC / kg, 6x10 13 GC / kg, 7x10 13 GC / kg, 8x10 13 GC / kg, 9x10 13 GC / kg, or 1x10 14 GC / kg) was administered to the patient. can be. EXAMPLES

[0138] How the compositions and methods described herein can be used, made, and evaluated The following examples are provided to provide a person skilled in the art with an understanding of the present invention. It is intended to be merely illustrative of the invention and is not intended to limit the scope of what the inventors perceive as their invention. It is not intended to

[0139] Example 1: Delivery of CASQ2 into heterozygous RYR4496C / + mice enhances RYR2 , triadin, and junctionin expression to eliminate arrhythmias. material and method Animal Use Animals were cultured at the Charles River Laboratorio, Calco, Italy. The mice were maintained and bred at ICS MaugeriSpa-S for phenotypic characterization. The animals were kept in a laboratory at the University of Pavia, Italy, in accordance with a protocol approved by the Animal Welfare Committee. The animals were maintained and studied according to the protocol.

[0140] Generation of knock-in R4496CRYR2 mouse model The experiments described in this example recapitulate the phenotype of the human RYR2R4997C mutation. Heterozygous RYR2 for dominant CPVT R4496C / + Using knock-in mouse models Details regarding the production of this mouse model are described in US 7,741,529. ,The disclosure is RYR2 R4496C / + As it relates to generating knock-in models, see and is incorporated herein by reference.

[0141] Vector design and production A detailed description of the design of one of the vectors used in these experiments is provided in US8,859 Briefly, wild-type mouse CAS without UTR sequences is provided in A pseudotyped adenovirus containing the coding sequence of Q2 (NCBI reference sequence NM_009814.2) We generated an AAV2 / 9 vector. Wild-type mouse CASQ2 was fused to pGE The DNA was cloned into the MT-Easy vector (Promega) using EcoRI. The insert corresponding to the CASQ2 transgene was extracted from the pGEM vector by enzymatic digestion. The biscistronic pIRES vector (BD Biosciences, Ca t. No:631605, Clontech, Palo Alto, CA, USA) The fragment corresponding to the CASQ2 transgene was then subcloned in and the subsequent IRES sequence were subcloned by PCR amplification using specific primers. The forward 5'CACAGCGGCCGCACA ATGAAGAGGATTTACCTGCTCATGG-3' (SEQ ID NO: 5) and 5'-CGAAGCATTAACCCTCACTAAAGGG-3' (SEQ ID NO: 6) The amplicon was isolated from the adeno-associated virus (AAV) by enzymatic digestion with NotI. Adenou provided by the Vector Core Facility (Tigem, Naples, Italy). Virus backbone vector pAAV-2.1-eGFP, serotype 9 (bovine growth hormone (BGH) containing polyA sequence and CMV promoter. The mid sequence was determined.

[0142] AAV production was performed in collaboration with the Tigem AAV Vector Core Facility. Transient transfection of the following three plasmids in HEK293 cells: pAd helper, pAAV rep-cap (packaging pAAVCis (containing the CASQ2 insert, pAAV2.1-CMV -eGFP plasmid cloned into MCS) to assess viral titers. Real-time PCR and dot blot analysis were performed to confirm the presence and purity of capsid protein. To evaluate the Infectivity and sterility were also evaluated. 12 Total yield exceeds genome copies All AAV2 / 9-WT-mCASQ virus preparations were reconstituted in PBS at 100 μl. Freeze the 2-IRES-eGFP stock in single aliquots at -80 °C for the duration of the surgical procedure. Unzip it to .

[0143] A similar recombinant AAV production method was used to produce the second vector investigated in these experiments. The second vector contained a wild-type CA operably linked to the desmin promoter. The second vector was a pseudotyped AAV2 / 8 vector containing a transgene encoding SQ2. The various components of the vector are shown in FIG.

[0144] Quantitative real-time PCR RYR2 R4496C / + and AAV9-WT-mCASQ2 from mouse cardiac tissue RYR2 infected with IRES-eGFP viral vector R4496C / + Heterozygosity Total RNA from knock-in mice was purified using the RNeasy mini kit (Qiagen). A total of 1 μg of template RNA was used per reaction using the iScript cDNA synthesis kit. The R was performed using a RT-PCR kit (Bio-Rad Laboratories, Inc., USA). Various genes of interest (RYR2CASQ2, Triazine , Junctin) and reference gene (GAPDH) mRNA quantification in real time. CFX96 detection module (Bio-Rad Laboratories, Inc.) All samples were run in optical 96-well plates using a specific primer mix. and the previously reported primers (Denegri et al., Circulation n 129:267381(2014)) was used as a 20ng cDNA template. The samples were analyzed in triplicate with SsoFast EvaGreen Supermix. Immuno-PCR was performed using a CFX96 real-time PCR detection system and Bio-Ray d Analyzed using CFX96 Manager software (version 1.5).

[0145] In vivo experiments AAV vectors containing the constructs shown in Figure 1 were inoculated into the oocytes at postnatal day 8 (P8) using a 25-gauge syringe. 6.4x10 in n=4 neonatal mice 11 100 μl of purified virus at a dose of 1 genome copy A total of n=5 litters were not treated.

[0146] At 7 weeks of age, electrocardiograms (ECGs) and radiotelemetry monitors (Data Scie All mice were anesthetized with isoflurane (1 L) and 100 µg / mL. After 72 hours of recovery from surgery, phenotypic characterization was performed. A baseline electrocardiogram was recorded to assess for the presence of spontaneous arrhythmias. The susceptibility of mice to agonist-induced arrhythmias was assessed by administering epinephrine (2 The mice were tested by administering 100 mg / kg of caffeine and 120 mg / kg of caffeine. (Cerrone et al., M, Circulation Research 96:e77-e82(2005), Liu et al., Circulatio (N Research 99:292-298(2006)). ECG recording was performed. All animals were free to move during the study.

[0147] The antiarrhythmic effects of the construct were then investigated in model organisms for dominantly inherited CPVT. To investigate this, the experiment was repeated using the AAV2 / 8 vector shown in Figure 4. YR2R4496C / + heterozygous mice (n=9) were transfected with AAV2 / 8 vectors at 1 kg Per 6x10 13 The vector genome (vg) was administered at the dose of 100 mg / kg / day. 96C / + heterozygous mice (n=9) were used as controls. In this study, we performed in vivo electrophysiological studies on mice under epinephrine and caffeine stimulation. It was assessed by analysis (ECG).

[0148] result As shown in Figure 2. RYR2 of the AAV vector encoding the CASQ2 transgene R4 996C / + Administration to mice completely abolished caffeine- and epinephrine-induced arrhythmias. After β-adrenergic stimulation, n=4 AAVCASQ2-treated mice None of the mice showed arrhythmic activity, whereas all untreated mice (n=5) showed arrhythmias. RYR2 R4996C / + The therapeutic effect of CASQ2 delivery in mice was The effect was not dependent on the activity of the membrane-associated proteins triadin and junctionin. As shown in 3, administration of the AAVCASQ2 vector resulted in the expression of CASQ2 in cardiomyocytes. Expression was increased, but the expression of RYR2, triadin, or junctionin in the cells was not There was virtually no change.

[0149] Similar results were obtained using the AAV2 / 8 construct, as shown in Figure 5. Administration of the V2 / 8-desmin-CASQ2 vector resulted in RYR2 R4996C / + Mau Caffeine- and epinephrine-induced arrhythmias were completely suppressed in patients with 44% of the mice exhibited arrhythmias.

[0150] Taken together, these results support the notion that CASQ2 transgene expression in a mouse model of dominant CPVT. We demonstrate that gene delivery promotes the correction of bidirectional and polymorphic arrhythmias. Interestingly, CASQ2 gene transfer alone suppressed adrenaline action without the need for RYR2 delivery. Dynamic activation was found to prevent physiological sinus rhythm, which can lead to arrhythmias. CASQ2 gene therapy in a dominant CPVT model results in increased CASQ2 expression in cardiomyocytes The addition of 100 mg / kg / day restored antiarrhythmic cardiac contractile function.

[0151] In light of these results and the explanations presented herein, CASQ2 gene therapy may be As shown in Example 2 above, it can be used as a paradigm for the treatment of dominant CPVT. It is possible.

[0152] Example 2: Dominant C in human patients by administration of a viral vector containing the CASQ2 transgene Treatment of PVT Using conventional molecular biology techniques known in the art, the amino acid sequence of SEQ ID NO:1 can be identified. A wild-type human CASQ2 protein having the amino acid sequence of SEQ ID NO:1 (or at least the amino acid sequence of SEQ ID NO:1) Genes encoding CASQ2 proteins, such as CASQ2A and its variants, which have 85% sequence identity with each other, are also included. The gene can be incorporated into a viral vector and formulated for administration to a human patient. For example, in patients with dominant CPVT, promoting CASQ2 expression in cardiomyocytes By administering an AAV vector containing a CASQ2 transgene under the control of a transcriptional regulatory element, AAV vectors contain a CAS sequence between the 5' and 3' inverted terminal repeats of AAV2. Incorporating the Q2 transgene, AAV8 or AAV9 capsid protein (AAV2 / 8 or AAV2 / 9 vectors) AAV vectors can be administered via a variety of routes, including intravenously, intramuscularly, or subcutaneously, among others. It can be administered to a subject by any route.

[0153] After administration of the vector to the patient, the skilled artisan may determine whether the CAS Expression of the Q2 gene and improvement of the patient in response to treatment can be monitored. For example, a physician may analyze the amount or frequency of arrhythmic events a patient experiences following administration of a transgene. The cardiac function of the patient can be monitored by the Patients showed no arrhythmic activity during a specific period following administration of the CASQ2 transgene, compared with a comparable period. Little or no evidence indicates that the patient is responding favorably to treatment. Subsequent doses can be determined and administered as needed.

[0154] Other embodiments All publications, patents, and patent applications mentioned herein are hereby incorporated by reference as if each was individually incorporated by reference. Each individual publication or patent application is specifically and individually indicated to be incorporated by reference. to the same extent as if each of the preceding claims were incorporated by reference herein.

[0155] While this invention has been described in connection with specific embodiments thereof, it is capable of further modifications. This application generally describes the principles of the present invention and the art to which the present invention pertains. to the extent that it is known or practiced in the art and applies to the essential features set forth above and the present invention, including such developments from the present invention according to the claims. It will be understood that any modifications, uses, or adaptations are intended to be covered.

[0156] Other embodiments are within the scope of the claims.

Claims

1. 1. A pharmaceutical composition for the treatment of dominant catecholaminergic polymorphic ventricular tachycardia (CPVT) in a human patient in need thereof, comprising a transgene encoding calsequestrin 2 (CASQ2), The patient exhibits a mutation in the endogenous gene encoding ryanodine receptor 2 (RYR2). Pharmaceutical compositions.

2. 1. A pharmaceutical composition for reducing arrhythmia in a human patient diagnosed with dominant CPVT, comprising a transgene encoding calsequestrin 2 (CASQ2), The patient exhibits a mutation in the endogenous gene encoding ryanodine receptor 2 (RYR2). Pharmaceutical compositions.

3. 1. A pharmaceutical composition for restoring calcium homeostasis in a human patient diagnosed with dominant CPVT, comprising a transgene encoding calsequestrin 2 (CASQ2), The patient exhibits a mutation in the endogenous gene encoding ryanodine receptor 2 (RYR2). Pharmaceutical compositions.

4. 1. A pharmaceutical composition for reducing delayed afterdepolarization events in a human patient diagnosed with dominant CPVT, comprising a transgene encoding calsequestrin 2 (CASQ2), The patient exhibits a mutation in the endogenous gene encoding ryanodine receptor 2 (RYR2). Pharmaceutical compositions.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the CASQ2 transgene encodes a protein having an amino acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, to the amino acid sequence of SEQ ID NO:1, and / or the CASQ2 transgene has a nucleic acid sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, to the nucleic acid sequence of SEQ ID NO:

2.

6. The pharmaceutical composition of claim 5 , wherein the CASQ2 transgene encodes a protein having the amino acid sequence of SEQ ID NO:1 and / or comprises the nucleic acid sequence of SEQ ID NO:

2.

7. The pharmaceutical composition according to any one of claims 1 to 6, comprising a vector.

8. 8. The pharmaceutical composition of claim 7, wherein the vector comprises a viral vector, preferably the viral vector comprises an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus.

9. 9. The pharmaceutical composition of claim 8, wherein the synthetic virus is a chimeric, mosaic, or pseudotyped virus and / or comprises a foreign protein, synthetic polymer, nanoparticle, or small molecule.

10. 9. The pharmaceutical composition of claim 8, wherein the viral vector is AAV, preferably the AAV comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74 serotype.

11. 9. The pharmaceutical composition of claim 8, wherein the viral vector is a pseudotyped AAV, preferably the pseudotyped AAV comprises AAV2 / 8 or AAV2 / 9.

12. The pharmaceutical composition of claim 10 , wherein the AAV comprises a recombinant capsid protein.

13. The pharmaceutical composition of any one of claims 1 to 6, wherein the pharmaceutical composition comprises a liposome, a vesicle, a synthetic vesicle, an exosome, a synthetic exosome, a dendrimer, or a nanoparticle.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the transgene is operably linked to a promoter capable of inducing expression of the CASQ2 in cardiomyocytes.

15. The promoters include the desmin promoter, the cytomegalovirus promoter, the myosin light chain 2 promoter, the alpha actin promoter, the troponin 1 promoter, the Na ＋ / Ca ２＋ 15. The pharmaceutical composition according to any of claims 1 to 14, wherein the promoter is a desmin promoter, exchanger promoter, dystrophin promoter, creatine kinase promoter, alpha 7 integrin promoter, brain natriuretic peptide promoter, alpha B crystallin / small heat shock protein promoter, alpha myosin heavy chain promoter, or atrial natriuretic factor promoter, preferably a desmin promoter.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the expression of CASQ2 is increased in cardiac cells, preferably cardiomyocytes, of the patient upon administration of the pharmaceutical composition to the patient.

17. The pharmaceutical composition of claim 16, wherein the expression of CASQ2 is assessed by measuring CASQ2 protein in the cells or CASQ2 mRNA in the cells, preferably by measuring CASQ2 protein or CASQ2 mRNA in a patient after administration of the pharmaceutical composition.

18. The pharmaceutical composition of claim 17, wherein the expression of CASQ2 mRNA is increased by about 1.5-fold to about 3.5-fold in the cell, preferably by about 2-fold to about 3-fold in the cell, more preferably by about 2.5-fold to about 3-fold in the cell.

19. The pharmaceutical composition of any of claims 1 to 18, wherein administration of said pharmaceutical composition to said patient does not substantially alter expression of RYR2, triadin, and / or junctionin in cardiac cells of said patient.

20. 20. The pharmaceutical composition according to any of claims 1 to 19, wherein the pharmaceutical composition is administered to the patient by intravenous, intrathecal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, irrigation, lavage, and / or oral administration.

21. The pharmaceutical composition of claim 1, wherein the mutation of RYR2 results in increased cytoplasmic calcium release in cardiomyocytes contacted with the RYR2 agonist compared to cardiomyocytes not contacted with the RYR2 agonist.

22. 22. The pharmaceutical composition of claim 1 or 21, wherein the RYR2 mutation 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.

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