JP-2025517257-A5 -

Dates

Publication Date

20260511

Application Date

20230425

Description

Those skilled in the art will be able to recognize or confirm, using experiments not exceeding the conventional scope, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims. Another aspect of the present invention may be as follows: [1] A method for achieving remission of dilated cardiomyopathy (DCM) in a subject requiring remission of DCM, To deliver a therapeutically effective amount of a composition to target tissue in a subject, which can introduce a transcription activator to a site specific to a regulatory sequence that controls or affects TTN gene expression. A method comprising increasing the expression of a functional TTN gene product in the target cardiac tissue or skeletal muscle tissue. [2] The method according to [1], wherein the DCM in the subject is triggered by a single TTN allele in the subject's genomic DNA that encodes a TTN gene that produces a dysfunctional titin protein gene product. [3] The method according to [2], wherein the TTN allele encodes a truncated variant of the TTN gene. [4] The method according to [2], wherein the TTN allele encodes a nonsense mutation of the TTN gene. [5] The method according to [2], wherein the TTN allele encodes a frameshift mutation of the TTN gene. [6] The method according to [2], wherein the TTN allele encodes a splicing variant mutation of the TTN gene. [7] The method according to [2], wherein the TTN allele codes for a variant that reduces the TTN expression level. [8] The method according to [1], wherein the target tissue is cardiac tissue. [9] The method according to [1], wherein the target tissue is skeletal muscle tissue. [10] The method according to [1], wherein the composition delivered to the target tissue is a CRISPR-Cas9 complex comprising a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific to a regulatory sequence that controls or affects TTN gene expression. [11] The method according to [10], wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VPR. [12] The method according to [10], wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VP64. [13] The method according to [10], wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-SunTag. [14] The method according to [10], wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-SAM. [15] The method according to any one of the above [1] to [14], wherein the regulatory sequence for TTN gene expression is located within the TTN gene promoter region. [16] The method according to [15], wherein the guide RNA that targets the TTN gene promoter region is an sgRNA having a sequence identified by any one of SEQ ID NOs: 9, SEQ ID NOs: 13-21, SEQ ID NOs: 23-25, SEQ ID NOs: 27-32, and SEQ ID NOs: 38-41. [17] The method according to [15], wherein the guide RNA targeting the TTN gene promoter region is an sgRNA having a sequence identified by any one of SEQ ID NOs: 13, 14, 15, 21, 24, 25, 27, 28, 30, 31, and 38-41. [18] The method according to any one of the above [1] to [14], wherein the regulatory sequence for TTN gene expression is located within the TTN gene enhancer region. [19] The method according to [18], wherein the guide RNA that targets the TTN gene enhancer region is an sgRNA having a sequence identified by any one of SEQ ID NOs. 33 and SEQ ID NOs. 35-37. [20] The method according to [18], wherein the guide RNA is an sgRNA having a sequence identified by sequence number 33. [21] The method according to any one of the claims [10] to [14], wherein the CRISPR-Cas9 complex delivered to the target tissue in the subject is delivered by one or more expression constructs encoding the nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific to a regulatory sequence related to TTN gene expression. [22] The method according to [21], wherein the CRISPR-Cas9 complex or one or more expression constructs are constructed for delivery to cardiac tissue or skeletal muscle tissue. [23] An sgRNA molecule identified by any of the following: SEQ ID NOs: 9, 13-21, 23-25, 27-32, 33, 35-37, and 38-41. [24] A pharmaceutical composition comprising a CRISPR-Cas9 complex containing a nuclease-inactive Cas9 protein linked to an activator protein, and a guide RNA specific to a regulatory sequence that controls or affects TTN gene expression, and a pharmaceutically acceptable carrier. [25] The pharmaceutical composition according to [24], wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VPR. [26] The pharmaceutical composition according to [24], wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VP64. [27] The pharmaceutical composition according to [24], wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-SunTag. [28] The pharmaceutical composition