Method for treating dilated cardiomyopathy and pharmaceutical composition therefor

Abstract

The present invention relates to a genetic method for altering gene expression in humans or animals. Specifically, the present invention relates to increasing the expression of the TTN gene in animal or human cells or tissues, where at least one allele of said gene carries a mutation that results in a truncated titin protein product. Therapeutic treatments, pharmaceutical compositions, and methods of treatment utilizing such pharmaceutical compositions are also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 334,539, filed Apr. 25, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference of Electronically - Provided Sequence Listing This application contains a sequence listing submitted as an electronic text file named "22 - 0135 - WO.xml", created on Apr. 24, 2023, having a size of 105 kb in byte - units. The information contained in this electronic file is hereby incorporated by reference in its entirety. The present invention relates to genetic methods for altering gene expression in humans or animals. Specifically, the present invention relates to increasing the expression of the TTN gene in animal or human cells or tissues, where at least one allele of said gene carries a mutation that results in a dysfunctional protein product, specifically a truncated titin protein product. Pharmaceutical compositions and methods of therapeutic treatment using said pharmaceutical compositions are also provided.

Background Art

[0003] Dilated cardiomyopathy (DCM) is a common condition that occurs at a rate of 1 in 200 people and is associated with high morbidity and mortality despite current therapeutic agents and heart transplantation (Hershberger et al., 2013, Nat Rev Cadiol 10: 531-547). DCM is diagnosed by a decrease in ejection fraction and left ventricular dilation (Mestroni et al., 1999, Eur. Heart J. 20: 93-102) and may have acquired or genetic causes or a combination of both factors (Japp et al., 2016, J Amer Coll Cardiol. 67: 2996-3010). Facilitated by the rapid expansion of clinical genetic testing, pathogenic variants in DCM-related genes can be identified in 17-26% of DCM individuals (Mazzaratto et al., 2020, Circulation 141: 387-398).

[0004] Among DCM-related variants, titin (TTN) gene variants (TTNtv) that result in premature protein truncation, such as nonsense, frameshift, or splicing variants, are the most frequently identified genetic lesions found in DCM individuals (Roberts et al., 2015, Sci Trnsl Med 7: 270). In addition, according to gene-environment interaction studies, TTNtv is also involved in acquired heart failure such as peripartum cardiomyopathy (Ware et al., 2016, N. Engl. J. Med. 374: 233-241) and alcoholic cardiomyopathy (Ware et al., 2018, J. Am. Coll. Cardiol. 71: 2293-2302), and the co-inheritance of the TTNtv allele along with environmental stress synergistically increases the risk of DCM. The TTN locus has also been associated with an increased risk of DCM with common genetic variants near TTN (Tadros et al., 2021, Nat. Genet. 53: 128-134), as well as in genome-wide association studies (GWAS) regarding DCM-like changes in heart structure (e.g., ventricular dilation) and function (e.g., decreased ejection fraction) in healthy population studies (Pirruccello et al., 2020, Nat. Commun. 11: 2254), playing a role as a modifier in DCM. Collectively, these human genetic studies strongly demonstrate that the inheritance of both rare and common variants that disrupt TTN function are major risk factors for DCM, but at present, there are no clinically available treatments to restore TTN function. The fact that there are no treatments targeting TTN indicates, in part, that it is not fully understood how TTN variants cause DCM. Historically, studying TTN function has been difficult in the cardiac context because of the large size of TTN (e.g., the TTN N2BA isoform is 34,350 residues (https: / / varsome.com / transcript / hg19 / ENST00000591111.1)), which is further compounded by the lack of a robust TTNtv animal model similar to human cardiac physiology. In recent years, a biomimetic three-dimensional TTNtv microtissue model composed of human induced pluripotent stem cell-derived cardiomyocytes (“CMs”) that exhibits a robust DCM phenotype, including reduced contractile function and myofibrillar content similar to features observed in human DCM hearts, has been developed (Hinson et al., 2015, Science 349: 982-986). Cardiac microtissue models offer additional translational advantages compared to animal models, such as the use of human cells expressing human sarcomere components and human gene sequences that enable the efficient development and human application of therapeutics targeting human TTN. As proof of principle, recent studies have demonstrated that DCM-related TTNtv can be functionally restored using clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 technology (Romano et al., 2022, Circulation 145: 194-205). This method functions by treating heterozygous TTNtv cardiac microtissues with Cas9 derived from Streptococcus pyogenes and a custom guide RNA (gRNA) programmed to bind and cleave only the TTNtv allele. These results demonstrated that CMs can repair DNA double-strand breaks using an endogenous repair process that results in the restoration of the TTN reading frame and normalization of TTN protein levels and function in cardiac microtissue function assays.This approach has achieved success in the functional restoration of a single TTNtv identified in the DCM family. However, following the extremely rare occurrence of individual TTNtv in the DCM population, this specific CRISPR treatment is considered not to be generalizable to other DCM individuals with different TTNtv.

[0005] Therefore, in this technical field, there remains a need for therapeutic interventions, methods, and pharmaceutical compositions aimed at restoring TTN gene function in DCM patients.

Summary of the Invention

[0006] The present invention provides a treatment method and a pharmaceutical composition aimed at restoring TTN gene function in DCM patients, which may be generalizable to most individuals with dilated cardiomyopathy (DCM).

[0007] The present invention provides a method for remitting dilated cardiomyopathy (DCM) in a subject who can be a human or an animal in need of remission of DCM. In certain embodiments, these methods involve delivering a therapeutically effective amount of a composition capable of introducing a transcriptional activator to a site specific for a regulatory sequence that controls or affects TTN gene expression to a target tissue in a human or an animal, wherein the expression of a functional TTN gene product is increased in human or animal heart tissue or skeletal muscle tissue. Specifically, these methods can be used in a subject in which DCM is the result of one TTN allele in the genomic DNA of the subject encoding the TTN gene that produces a dysfunctional titin protein product. In certain embodiments, the TTN allele encodes a truncated mutation, nonsense mutation, frameshift mutation, or splice variant mutation of the TTN gene. In certain embodiments, the TTN allele encodes a gene variant that reduces TTN expression levels. In certain embodiments, the target tissue is heart tissue or skeletal muscle tissue. In the methods of the present invention, the composition is preferably delivered to the target tissue as a CRISPR-Cas9 complex. In these embodiments, the CRISPR-Cas9 complex comprises a Cas9 protein with reduced or abolished nuclease activity (referred to herein as a "nuclease-inactive Cas9 protein"). Further, in these embodiments, the nuclease-inactive Cas9 protein is linked to a eukaryotic transcriptional activator protein and is associated with a guide RNA specific for a regulatory sequence that controls or affects TTN gene expression. In these embodiments, the guide RNA molecule is identified by any of SEQ ID NO: 9, SEQ ID NOs: 13-21, SEQ ID NOs: 23-25, SEQ ID NOs: 27-32, SEQ ID NO: 33, SEQ ID NOs: 35-37, and SEQ ID NOs: 38-41. In a specific embodiment, the activator protein is VPR (referred to herein as dCas9-VPR). In an alternative specific embodiment, the activator protein is VP64R (referred to herein as dCas9-VP64).In a further specific embodiment, the activator protein is SunTag (referred to herein as dCas9-SunTag). In a further specific embodiment, the activator protein is SAM (referred to herein as dCas9-SAM). In a specific embodiment, the regulatory sequence that controls or affects TTN gene expression is located within the TTN gene promoter region. In certain embodiments, the guide RNA targeting the TTN gene promoter region is an sgRNA having a sequence identified by any one of SEQ ID NO: 9, SEQ ID NOs: 13-21, SEQ ID NOs: 23-25, SEQ ID NOs: 27-32, and SEQ ID NOs: 38-41. In certain advantageous embodiments, 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. In a specific embodiment, the regulatory sequence regarding TTN gene expression is located within the TTN gene enhancer region. In certain embodiments, the guide RNA targeting the TTN gene enhancer region is an sgRNA having a sequence identified by any one of SEQ ID NO: 33 and SEQ ID NOs: 35-37. In certain advantageous embodiments, the guide RNA targeting the TTN gene enhancer region is an sgRNA having a sequence identified by SEQ ID NO: 33.

[0008] In certain embodiments, the CRISPR-Cas9 complex delivered to cardiac or skeletal muscle tissue in a subject is delivered by one or more expression constructs encoding a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific for a regulatory sequence regarding TTN gene expression.

[0009] The present invention also provides a composition, specifically a therapeutic composition, for restoring TTN gene function in DCM patients, which may be generalizable to most DCM individuals. In certain embodiments, the composition is a pharmaceutical composition. In specific embodiments, the present invention provides a therapeutic composition and a pharmaceutical composition comprising a CRISPR-Cas9 complex. In these embodiments, the CRISPR-Cas9 complex comprises a nuclease-inactive Cas9 protein. Further, in these embodiments, the nuclease-inactive Cas9 protein is linked to a eukaryotic transcriptional activator protein and is associated with a guide RNA specific to a regulatory sequence that controls or affects TTN gene expression. In these embodiments, the CRISPR-Cas9 complex comprises a Cas9 protein with reduced or abolished nuclease activity (referred to herein as "nuclease-inactive Cas9 protein"). Further, in these embodiments, the nuclease-inactive Cas9 protein is linked to a eukaryotic transcriptional activator protein and is associated with a guide RNA specific to a regulatory sequence that controls or affects TTN gene expression. In these embodiments, the pharmaceutical composition further comprises a guide RNA molecule identified by any one of SEQ ID NO: 9, SEQ ID NO: 13-21, SEQ ID NO: 23-25, SEQ ID NO: 27-32, SEQ ID NO: 33, SEQ ID NO: 35-37, and SEQ ID NO: 38-41. In a specific embodiment, the activator protein is VPR (referred to herein as dCas9-VPR). In an alternative specific embodiment, the activator protein is VP64R (referred to herein as dCas9-VP64). In a further specific embodiment, the activator protein is SunTag (referred to herein as dCas9-SunTag). In a further specific embodiment, the activator protein is SAM (referred to herein as dCas9-SAM). In a specific embodiment, the CRISPR-Cas9 complex comprises dCas9-VPR.In certain embodiments, the regulatory sequences that control or affect TTN gene expression are located within the TTN gene promoter region. In certain embodiments, the guide RNA targeting the TTN gene promoter region is an sgRNA having a sequence identified by any one of SEQ ID NO: 9, SEQ ID NOs: 13-21, SEQ ID NOs: 23-25, SEQ ID NOs: 27-32, and SEQ ID NOs: 38-41. In certain advantageous embodiments, 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. In certain embodiments, the regulatory sequences related to TTN gene expression are located within the TTN gene enhancer region. In certain embodiments, the guide RNA targeting the TTN gene enhancer region is an sgRNA having a sequence identified by any one of SEQ ID NO: 33 and SEQ ID NOs: 35-37. In certain advantageous embodiments, the guide RNA targeting the TTN gene enhancer region is an sgRNA having a sequence identified by SEQ ID NO: 33.

[0010] In certain embodiments, the CRISPR-Cas9 complex delivered to cardiac or skeletal muscle tissue in a subject is delivered by one or more expression constructs encoding a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific for a regulatory sequence related to TTN gene expression.

[0011] The present invention provides a method for remitting dilated cardiomyopathy (DCM) in a subject who can be a human or an animal in need of remission of DCM. In certain embodiments, these methods involve introducing a transcription activator in a therapeutically effective amount of a composition capable of being delivered to a target tissue in a human or an animal to a site specific to a regulatory sequence that controls or affects a wild-type TTN allele that produces a functional titin protein gene product, wherein the expression of the functional titin protein is specifically increased in the cardiac or skeletal muscle tissue of the subject. Specifically, these methods can be used in a subject in which DCM is the result of a mutant TTN allele in the genomic DNA of an individual encoding a TTN gene that produces a dysfunctional titin protein gene product. In certain embodiments, the TTN allele encodes a truncated mutation, nonsense mutation, frameshift mutation, or splice variant mutation of the TTN gene. In certain embodiments, the TTN allele encodes a genetic variant that reduces the TTN expression level. In certain embodiments, the target tissue is cardiac or skeletal muscle tissue. In the methods of the present invention, the composition is preferably delivered to the target tissue as a CRISPR-Cas9 complex. In these embodiments, the CRISPR-Cas9 complex comprises a Cas9 protein with reduced or abolished nuclease activity (referred to herein as a "nuclease-inactive Cas9 protein"). Further, in these embodiments, the nuclease-inactive Cas9 protein is linked to a eukaryotic transcription activator protein and is associated with a guide RNA specific to a regulatory sequence that controls or affects wild-type TTN allele gene expression. In a specific embodiment, the activator protein is VPR (referred to herein as dCas9-VPR). In an alternative specific embodiment, the activator protein is VP64R (referred to herein as dCas9-VP64). In a further specific embodiment, the activator protein is SunTag (referred to herein as dCas9-SunTag).In a further specific embodiment, the activating factor protein is SAM (referred to herein as dCas9-SAM). In a specific embodiment, the regulatory sequence that controls or affects TTN gene expression is located within the wild-type TTN allele gene promoter region or the wild-type TTN allele gene enhancer region. In certain embodiments, the guide RNA targeting the wild-type TTN allele gene promoter region or the wild-type TTN allele gene enhancer region is an sgRNA having a sequence identified by any one of SEQ ID NOs: 103-105.

[0012] In certain embodiments, the CRISPR-Cas9 complex delivered to cardiac or skeletal muscle tissue in a subject is delivered by one or more expression constructs encoding a nuclease-inactive Cas9 protein linked to an activating factor protein and a guide RNA specific for a regulatory sequence related to wild-type TTN allele gene expression.

[0013] The present invention also provides a composition, specifically a therapeutic composition, for restoring TTN gene function in DCM patients, which may be generalizable to most DCM individuals. In certain embodiments, the composition is a pharmaceutical composition. In a particular embodiment, the present invention provides a therapeutic composition and a pharmaceutical composition comprising a CRISPR-Cas9 complex. In these embodiments, the CRISPR-Cas9 complex comprises a nuclease-inactive Cas9 protein. Further, in these embodiments, the nuclease-inactive Cas9 protein is linked to a eukaryotic transcriptional activator protein and is associated with a guide RNA specific to a regulatory sequence that controls or affects wild-type TTN allele gene expression. In these embodiments, the CRISPR-Cas9 complex comprises a Cas9 protein with reduced or abolished nuclease activity (referred to herein as "nuclease-inactive Cas9 protein"). Further, in these embodiments, the nuclease-inactive Cas9 protein is linked to a eukaryotic transcriptional activator protein and is associated with a guide RNA specific to a regulatory sequence that controls or affects wild-type TTN allele gene expression. In these embodiments, the pharmaceutical composition further comprises a guide RNA molecule identified by any of SEQ ID NOs: 103 to 105. In a specific embodiment, the activator protein is VPR (referred to herein as dCas9-VPR). In an alternative specific embodiment, the activator protein is VP64R (referred to herein as dCas9-VP64). In a further specific embodiment, the activator protein is SunTag (referred to herein as dCas9-SunTag). In a further specific embodiment, the activator protein is SAM (referred to herein as dCas9-SAM). In a specific embodiment, the regulatory sequence that controls or affects TTN gene expression is located within the wild-type TTN allele gene promoter region or the wild-type TTN allele gene enhancer region.In these embodiments, the pharmaceutical composition further comprises a guide RNA molecule that targets the wild-type TTN allele gene promoter region or the wild-type TTN allele gene enhancer region identified by any of SEQ ID NOs: 103-105.

[0014] In certain embodiments, the CRISPR-Cas9 complex delivered to cardiac or skeletal muscle tissue in a subject is delivered by one or more expression constructs encoding a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific for a regulatory sequence involved in wild-type TTN allele gene expression.

[0015] The therapeutic and pharmaceutical compositions of the present invention comprise a CRISPR-Cas9 complex delivered to cardiac or skeletal muscle tissue in an individual, which is preferably an intact CRISPR-Cas9 complex comprising a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific for a regulatory sequence related to TTN gene expression or wild-type TTN allele gene expression. The therapeutic and pharmaceutical compositions of the present invention can be formulated in lipid nanoparticles, lentiviral constructs, or adenovirus or adeno-associated virus constructs.

[0016] These and other features, objects, and advantages of the present invention will become better understood from the following description. In that description, reference is made to the accompanying drawings, which form a part of the description, and in which embodiments of the invention are shown by way of illustration and not limitation. The description of the preferred embodiments is not intended to limit the invention, which includes all modifications, equivalents, and alternatives. Therefore, reference should be made to the claims set forth in this application to interpret the scope of the present invention.

Brief Description of the Drawings

[0017]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

Figure 4F

Figure 4G

Figure 4H

Figure 5A

Figure 5B

Figure 5C

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 6F

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 7E

Figure 8A

Figure 8B

Figure 8C

Figure 8D

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 9E

Figure 9F

Figure 9G

Figure 9H

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 10E

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 12C

Figure 13A

Figure 13B

Figure 14A

Figure 14B

DETAILED DESCRIPTION OF THE INVENTION

[0018] Provided herein, together with this specification, is a more detailed description of the compositions, methods, and kits that make up the present invention, provided not to replace, nor to be an alternative to, the claims set forth below, but to explain and reinforce them.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide general definitions for many of the terms used in this disclosure to one of ordinary skill in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991). These references are intended to be illustrative and exemplary of sources of information known to one of ordinary skill in the art, not limiting thereof. All citations and references described herein are hereby incorporated by reference in their entirety.

[0020] As used herein, unless otherwise specified, the following terms have the meanings ascribed to them below. Note that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The term "about" or "approximately" means within 25% of a given value or range, e.g., within 20% (or 5% or less). As used herein, the terms "or" and "and / or" are used to describe multiple components that are combined with each other or are exclusive. For example, "x, y, and / or z" can refer to "x" only, "y" only, "z" only, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". Note that terms such as "preferably", "generally", and "typically" are not used herein to limit the scope of the claimed invention, nor to suggest that a particular feature is critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to emphasize alternative or additional features that may or may not be utilized in particular embodiments of the present invention.

[0021] Note that for the purposes of describing and defining the present invention, the term "substantially" is used herein to represent the degree of inherent uncertainty that may be attributable to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also used herein to represent the degree to which a quantitative expression may vary without causing a change in the basic function of the subject matter from the stated reference value. As used herein, the term "transcription activation method" is intended to include CRISPR and CRISPRa, TALEN (transcription activator-like effector nuclease; Cermak et al., 2011, Nucl. Acids Res. 39: e82) and TALE-TF (transcription activator-like effector transcription factor; Sanjana et al., 2012, Nat. Protoc. 7: 171-192, Zhang et al., 2011, Nat. Biotechnol. 29: 149-153), ZFN (zinc finger nuclease; Porteus & Baltimore, 2003, Science 300: 763) and ZFN-TF (zinc finger nuclease transcription factor; Beerli et al., 2000, Proc. Natl. Acad. Sci. USA 97: 1495-1500), and any protein comprising a programmable DNA binding domain combined with a transcription activator (see artificial transcription factors described in Ansari & Mapp, 2002, Curr. Opin. Chem. Biol. 6: 765-772), and any protein comprising a programmable DNA binding domain combined with an enzyme that activates transcription via an epigenetic mechanism such as a histone acetyltransferase (e.g., dCas9-p300 as described in Klann et al., 2017, Nat. Biotechnol. 35: 561-568).

[0022] As used herein, the term "CRISPR" (clustered regularly interspaced short palindromic repeats) is intended to encompass all embodiments of targeted delivery of gene modalities or enzyme modalities using guide RNAs, including proteins, specifically bacterial proteins, and most specifically those proteins associated with CRISPR endonucleases, with and without endonuclease activity, derived from bacterial species including, but not limited to, S. pyogenes and S. aureus, as disclosed in Jinek et al., 2012, Science 337: 816-21, Cong et al., 2013, Science 339: 819-823.

[0023] As used herein, the term "CRISPR endonuclease" is intended to encompass Cas9 (referred to as "type II system"), Cas12a (referred to as "type V system"), Cas12f, CasMINI, and CasΦ, derived from several bacterial species including S. pyogenes and S. aureus.

[0024] In certain embodiments, the term "CRISPRa," which refers to a Cas9 protein designated as "dCas9," is a type of Cas9 protein that has reduced or abolished endonuclease activity but retains the ability to bind to a bimolecular (tracrRNA and crRNA) or a unimolecular (sgRNA in which tracrRNA and crRNA are linked by an oligoribonucleotide linker) and direct the specific target of the CRISPR complex to a DNA sequence complementary to the crRNA sequence (Qi et al., 2013, Cell 152: 1173-1183). Thus, the dCas9-CRISPR complex can be used to deliver molecules, including transcriptional activators, to such sites. See, for example, Bikard et al., 2013, Nucl. Acids Res. 41: 7429-7437, Perez-Pinera et al., 2013, Nat. Method 10: 973-976, Tannenbaum et al., 2014, Cell 159: 635-646, Konerman et al., 2014, Nature 517: 583-588, Chavez et al., 2015, Nat. Methods 12: 326-328, Riedmayr et al., 2022, Nature Protocols 17: 781-818.

[0025] As used herein, the term "transcription activation domain" is intended to encompass proteins that can increase the transcription of genes having transcription regulatory elements that respond to such activators. See Ma, 2011, Prot. & Cell 2: 879-888. Specifically, for use as part of a complex with dCas9 to activate TTN gene expression as described herein, transcription activation domains include VPR (a three-factor complex of VP64, P65, and Rta; see Chavez et al., 2015, Nat Methods 12: 326-328), VP64 (see Casas-Mollano et al., 2020, The CRISPR J., https: / / doi.org / 10.1089 / crispr.2020.0064), SunTag (including multiple copies of VP64; see Tanenbaum et al., 2014, Cell 159: 635-646), CBP (histone acetyltransferase domain; see Sajwan & Mannervik, 2019, Sci Rep. 9: 18104), Synergistic Activation Mediator (SAM); see Zhang et al., Sci. Rep. 5: 16227), and SPH (a hybrid containing the epitope tag of SunTag and the P65-HSF activation domain of SAM (see Zhou et al., 2018, Nat Neurosci 20: 440-446, Clouse, 2020, https: / / blog.addgene.org / crispr-activators-dcas9-vp64-sam-suntag-vpr, Chavez et al., 2016, Nat Methods 13: 563-567).

[0026] As used herein, the term "guide RNA" is intended to include bimolecular embodiments (tracrRNA and crRNA) and single-molecule embodiments (sgRNA in which tracrRNA and crRNA are linked by an oligoribonucleotide linker) that can bind to a bacterial-derived Cas9 endonuclease (or its inactivated form, generally referred to as "dCas9") and specifically bind to a DNA sequence complementary to the crRNA. As used herein, "mutation" is intended to include point mutations, including nonsense, missense, and frameshift mutations, as well as insertions, deletions, rearrangements, and splice site variants. As used herein, "transcription start site" (TSS) means the position at which wild-type DNA nucleotides are transcribed into RNA. As used herein, "promoter" is intended to include a region of DNA upstream of a gene that binds a relevant protein to initiate transcription of that gene. The promoter region exists upstream and downstream of the TSS. In some embodiments, the gRNA used with dCas-9 to increase TTN expression is designed to target promoter regions within 500 bp upstream (+500) and 2000 bp downstream (-2000) of the TSS.

[0027] As used herein, "enhancer" is intended to include DNA regulatory elements that activate the transcription of one or more genes to a higher level than in their absence. These elements, such as cis-acting DNA regulatory elements, function at a distance by forming chromatin loops to bring the enhancer and the target gene into proximity. Examples of such elements have been demonstrated to regulate the expression of target genes (Wei et al., 2006, Cell 124: P207-219; Li et al., 2020, Nature 11: 485). As used herein, the term "target tissue" is intended to include any specific tissue in which delivery of the TTN gene activation construct described herein can be advantageously used for therapeutic purposes. Specifically, muscle tissue, particularly skeletal muscle, and most specifically myocardial tissue or heart muscle tissue are the target tissues defined herein.

[0028] As used herein, targets for affecting gene expression specifically include genes encoding titin proteins encoded by the TTN gene in humans or animals, specifically mammals, and most specifically humans. As used herein, the term "polymorphism" refers to the presence of two or more variant forms of a specific DNA sequence that can occur between different individuals or populations. The most common type of polymorphism involves a single nucleotide diversity called single nucleotide polymorphism (SNP). For example, standard methods of long-read DNA sequencing using Oxford Nanopore or PacBio systems (Feng et al., 2021, Nature Communications 12: 3032) can be used to identify SNPs and other polymorphic variants that are specifically present in the WT / full-length TTN allele and not in the TTN mutant allele.

[0029] As disclosed herein, for the delivery vehicles for the therapeutic embodiments of the present invention that can affect gene expression, specifically increase gene expression, in the target tissues of individuals that require an increase in gene expression, include, but are not limited to, lipid nanoparticles, such as their PEGylated embodiments (conjugated with polyethylene glycol) (see Saupe & Rades, 2006, Nanocarrier Technologies, p. 41, Jenning et al., 2000, Intl. J. Pharmaceut. 199: 167-177, Turnbull et al., Mol. Ther. 24: 66-75, Afzelius et al., 1989, Biochim. Biophys. Acta 979: 231-238), adeno-associated virus (AAV) constructs (Fuentes & Schaffer, 2018, Curr. Opin. Biomed. Engin. 7: 33-41, Xu et al., 2019, Viruses 11: 28, Wong et al., 1986, Clin. Exp. Pharmacol. Physiol. 13: 267-270, Tabebordbar et al., 2021, Cell 184: 4919-4938), lentiviral constructs (Yip, 2020, Biomolecules 10:839, Yudovich et al., 2020, Nat. Sci. Report. 10: 22393, Uchida et al., 202, Cell 21: 121-132, Niwano et al., 2008, Mol. Ther. 16: 1026-1032), adenoviral constructs (Ehrke-Schulz et al., 2017, Nat. Sci. Report. 7: 7113, Boucher et al., 2019, J. Control Release 327: 788-800, Raake et al., 2004, J. Am. Coll. Cardiol 44: 1124-1129), modified RNA species (Huang et al., 2015, Molec. Pharmacol.(Hundy et al., 2016, Gene Ther. 23: 380-392, Gyorgy & Maguire, 2017, WIREs 10: e1488, Orefice, 2020, Pharmaceutics 2020 12: 705, Sancho-Albero et al., 2020, RSC Adv. 10: 23975, Pofali et al., 2020, Curr. Cancer Drug Targets 20:821-830, Liu et al., 2021, Front. Cell Dev. Biol. doi.org / 10.3389 / fcell.2021.707607, Metzner & Zaryuba, 2021, Viruses 13: 1238), and endosomes containing the building. Specifically, the lipid nanoparticles can include mono, di, and triglycerides, fatty acids, steroids, and sterols such as cholesterol phospholipids, sphingosine and sphingomyelin, bile salts such as sodium taurocholate, and emulsifying substances. See Shah et al., 2015, Lipid Nanoparticles: Production, Characterization and Stability..

[0030] In various aspects, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the disclosure and one or more pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifying substances, adjuvants, excipients, or carriers. In certain aspects, the disclosure provides a pharmaceutical composition comprising a compound of the disclosure together with one or more pharmaceutically acceptable excipients or vehicles and optionally other therapeutic and / or prophylactic components. Such excipients include liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and the like.

[0031] The term "pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier administered with the compounds of the present disclosure. The term "effective amount" or "pharmaceutically effective amount" refers to an amount of a drug that is non-toxic but sufficient to produce the desired biological result. The result can be the suppression and / or alleviation of the symptoms, signs, or causes of a disease, or any other desired change in a biological system. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0032] "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990). For example, sterile physiological saline at physiological pH and phosphate buffered saline can be used. Preservatives, stabilizers, dyes, and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid can be added as preservatives. Id., 1449. In addition, antioxidants and suspending agents can be used. Id.

[0033] Excipients suitable for non-liquid formulations are also known to those of ordinary skill in the art. A detailed discussion of pharmaceutically acceptable excipients and salts is available in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990). In addition, auxiliary substances such as wetting or emulsifying agents, biological buffers, surfactants, etc. may be present in such vehicles. A biological buffer can be any solution that is pharmacologically acceptable and provides the desired pH for the formulation, i.e., a pH within the physiologically acceptable range. Examples of buffer solutions include physiological saline, phosphate buffered saline, tris buffered saline, Hanks buffered saline, and the like.

[0034] Depending on the desired mode of administration, the pharmaceutical composition can be provided in a unit dosage form suitable for precise single-dose administration. The composition contains an effective amount of a selected drug in combination with a pharmaceutically acceptable carrier and can additionally contain other pharmaceuticals, adjuvants, diluents, buffers, etc. Generally, the compositions of the present disclosure are administered in therapeutically effective amounts by any of the accepted modes of administration. Suitable dosage ranges depend on a number of factors such as the severity of the disease being treated, the age and associated health status of the subject, the potency of the compound being used, the route and form of administration, the indication for which administration is targeted, and the preference and experience of the medical practitioner involved. Those skilled in the art of treating such diseases will be able to confirm the therapeutically effective amount of the compositions of the present disclosure for a given disease based on their personal knowledge and the disclosure of this application without undue experimentation. Accordingly, the compositions of the present disclosure can be administered as pharmaceutical formulations including those suitable for parenteral (including intramuscular, intracardiac, intraarterial, intrathecal, subcutaneous, and intravenous) administration, or in forms suitable for administration by inhalation or spraying. Preferred modes of administration are intravenous, intraarterial, or intracardiac modes of administration using a dosing schedule that can be adjusted according to the degree of pain.

[0035] In yet another embodiment, permeation-enhancing excipients are used that include polymers such as polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin), polyanions (N-carboxymethyl chitosan, poly-acrylic acid), and thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugate).

[0036] Parenteral formulations can be prepared in conventional forms as either liquid solutions or suspensions, solid forms suitable for solubilization or suspension in a liquid prior to injection, or emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents, and suspending agents. Sterile injectable formulations can also be sterile injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterilized fixed oils, fatty acid esters, or polyols have been conventionally used as solvents or suspending media. In addition, parenteral administration can involve the use of sustained release or controlled release systems so that a certain level of dosage is maintained.

[0037] Parenteral administration includes intra-articular, intravenous, intracardiac, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and can include aqueous and non-aqueous isotonic sterile injectable solutions containing antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives. Administration via certain parenteral routes can involve introducing the formulations of the present disclosure into a patient's body via a needle or catheter, which is facilitated by any other mechanical device such as a sterile syringe or continuous infusion system. The formulations provided by the present disclosure can be administered using a syringe, injector, pump, or any other device recognized in the art related to parenteral administration.

[0038] Preferably, the sterile injectable suspension is formulated according to techniques known in the art using a suitable carrier, dispersing or wetting agent, and suspending agent. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterilized fixed oils, fatty acid esters, or polyols have been conventionally used as solvents or suspending media. In addition, parenteral administration can be accompanied by the use of a sustained release or controlled release system so that a certain level of dosage is maintained. Preparations according to the present disclosure for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. They can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition. They can also be manufactured using sterile water or some other sterile injectable medium immediately before use.

[0039] The sterile injectable solutions are prepared by incorporating the required amount of one or more of the compounds of the present disclosure into a suitable solvent containing various other ingredients enumerated above, as required, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is by vacuum drying and lyophilization techniques that yield powders of the active ingredient and any additional desired ingredients from their pre-filter sterilized solutions. Thus, for example, a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of the active ingredient in 10% by volume of propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

[0040] The pharmaceutical compositions of the present disclosure can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulations and can be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents. The compositions of the present disclosure can be formulated, in particular, for aerosol administration, including intranasal administration, to the respiratory tract. The compounds generally have a small particle size on the order of, for example, 5 μm or less. Such particle sizes can be obtained by means known in the art, for example, by micronization. The active ingredient is provided in a pressurized pack containing a suitable propellant, such as chlorofluorocarbons (CFCs), such as dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. The aerosol can also advantageously contain a surfactant such as lecithin. The dose of the drug can be controlled by a metered valve. Alternatively, the active ingredient can be provided in the form of a dry powder, for example, in the form of a powder mix of the compound in a suitable powder base, such as lactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidone (PVP). The powder carrier can form a gel in the nasal cavity. The powder composition can be provided in unit dose form, for example, in capsules or cartridges of gelatin or blister packs, by means of which the powder can be administered, for example, by an inhaler.

[0041] A pharmaceutically effective or therapeutically effective amount of the composition is delivered to the subject. The exact effective amount can vary between subjects and depends on the species, age, size and health of the subject, the nature and extent of the condition being treated, the advice of the physician performing the treatment, and the therapeutic agent or combination of therapeutic agents selected for administration. Thus, the effective amount for a given situation can be determined by conventional experimentation. In the case of AAV, generally, the therapeutic dose is 1×10 13 vg / kg (viral genomes per kilogram of patient) ~ 5×10 14It can be in the range of vg / kg. In the case of lipid nanoparticles, this can be 1 mg / kg or more (mg of nanoparticles per kilogram of patient) (estimated dosage from Manso et al., 2020, Sci Transl Med 12: eaax1744 regarding targeting LAMP2 in the heart using AAV in mice, and Rothgangl et al., 2021, Nat. Biotechnol. 39: 949-957 as a reference regarding targeting the liver for PCKS9 in non-human primates). The subject can be administered only the dosage necessary to suppress and / or alleviate the signs, symptoms, or causes of the disorder, or to bring about any other desired change in the biological system. If desired, the formulation can be prepared using an enteric coating configured for sustained-release or controlled-release administration of the active ingredient.

[0042] The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form can be a packaged preparation, and the package can contain tablets, capsules, and powders packaged in separate amounts of the preparation, for example, in vials or ampoules. Also, the unit dosage form can itself be a capsule, tablet, cachet, or lozenge, or can be an appropriate number of any of these in a packaged form.

[0043] Definitions The following terms and expressions used herein have the indicated meanings. "Pharmaceutically acceptable salt" refers to both acid addition salts and base addition salts. "Therapeutically effective amount" refers to the amount of a compound that, when administered to a subject, is sufficient to effect treatment of a disease or disorder described herein. The amount of the compound that will be a "therapeutically effective amount" will vary depending on the compound, the disorder and its severity, and the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art. "To modulate" or "modulating" refers to treating a function, condition, or disorder, or preventing, suppressing, enhancing, or inducing them. For example, the compounds of the present disclosure are thought to be able to modulate atherosclerosis in humans by stimulating the removal of atherosclerotic lesions of cholesterol. As used herein, the term "ameliorating" when used with respect to the effects of the methods and pharmaceutical compositions provided herein will be understood by those skilled in the art to mean any beneficial clinical effect on a patient with DCM in which the disease-related symptoms are reduced, alleviated, or treated.

[0044] "To treat" or "treatment" as used herein, when used in a subject, preferably a human, includes the treatment of a disease or disorder described herein, i. inhibiting a disease or disorder, i.e., stopping its occurrence, ii. alleviating a disease or disorder, i.e., causing a reduction of the disorder, iii. slowing the progression of the disorder, and / or iv. inhibiting, alleviating, or slowing the progression of one or more symptoms of a disease or disorder and includes.

[0045] "Subject" refers to a warm-blooded animal, such as a mammal, preferably a human or a human child, that has or is at risk of having one or more of the diseases and disorders described herein.

[0046] Next, various exemplary embodiments of the compositions and methods according to the present invention are described in the following non-limiting examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and the following examples, and these are within the scope of the appended claims.

Examples

[0047] The examples described in this specification incorporate and depend on certain experimental and preparative methods and techniques that are carried out as exemplified herein.

[0048] Induced pluripotent stem cell (iPSc) culture and cardiomyocyte (CM) differentiation All cardiomyocyte (CM) experiments were performed using the parental PGP1 GM23338 iPSC line, a commercially available wild-type control strain previously used to study CM and sarcomere pathobiology, obtained from the Coriell Institute Biorepository (Hinson et al., 2015, Science 349: 982-986; Hinson et al., 2016, Cell Rep. 17: 3292-3304; Chopra et al., 2018, Dev. Cell. 44: 87-96; Ng et al., 2019, JCI Insight 5; Cohn et al., 2019, Stem Cell Reports 12: 71-83). PGP1 iPSCs, and all CRISPR-engineered derivatives, were seeded onto Matrigel-coated tissue culture plates (Corning 354230) in mTeSR1 (STEMCELL Technologies 85875) containing 10 μM ROCK inhibitor Y-27632 (Tocris 1254). mTeSR1 was replenished daily until cells reached a confluency of 80-90%, at which point they were passaged 1:6 using Accutase (BD 561527). Directed differentiation of iPSCs into CMs was achieved via modulation of WNT / β-catenin signaling. Briefly, differentiation of iPSCs (90-100% confluency) was initiated via WNT activation by inhibiting GSK-3 using 9-12 μM CHIR99021 (Tocris 4423) for 24 h in RPMI1640 (Gibco 11875093) containing B27 (insulin-free) supplement (Gibco A1895601), GlutaMAX (Gibco 35050061), and penicillin-streptomycin (Gibco 15140122). On day 3 of differentiation, cells were treated with 5 μM IWP-4 (Tocris 5214) for 48 h to inhibit WNT signaling. On day 9, the medium was changed to RPMI containing B27 (insulin-containing) supplement (Gibco 17504044).On the 13th day, metabolic selection via glucose starvation was performed for 24 hours using glucose-free DMEM (Gibco 11966025) containing 4 mM lactase (Sigma 71718) to obtain more than 95% CM. After selection, the CM was trypsinized (Gibco 25200056) and seeded onto fibronectin-coated tissue culture plates (Gibco 33016015) containing RPMI-B27 supplemented with 2% FBS (GeminiBio 100-106). RPMI1-B27 was replenished every other day until analysis (days 22 to 35 unless otherwise indicated).

[0049] CRISPR experiment To generate the TNNT2-dCas9-VPR iPSc lines described below, the dCas9-VPR open reading frame was PCR amplified from the Lenti EF1a-FLAG-dCas9-VPR vector (Addgene #114195) and cloned into an HR donor plasmid containing TNNT2 homology arms adjacent to the TNNT2 stop codon and the T2A linker sequence. To generate the TTN-tdTomato lines described herein, the tdTomato-FLAG open reading frame was obtained as a gBlock (IDT) and cloned into an HR donor plasmid containing TTN homology arms adjacent to the TTN stop codon. All HR vector propagation steps were performed in DH5α Escherichia coli (E.coli) (NEB C2987). All gene modifications of PGP1 iPSCs were performed using CRISPR / Cas9. The CRISPR manipulation TTNtv used for electroporation described below + / - The iPSC lines have been previously published (Romano et al., 2022, Circulation 145: 194-205). dCas9-VPR and TTN-tdTomato TTNtv + / - For iPSC generation, 8×10 6Individual iPSCs were electroporated with 20 μg of pCas9-GFP (Addgene 44719), 20 μg of hU6-driven gRNA, and 20 μg of an HR donor plasmid carrying either TNNT2-dCas9-VPR or TTN-tdTomato-FLAG (shown in Figures 1B and 1E). The next day, selection was initiated using either G418 or zeocin to isolate single iPSC clones, which were then manually sorted into Matrigel-coated 96-well plates. Once confluent, a portion of each clone was split into 96-well plates, and the remaining cells were harvested for Sanger sequencing and genotyping. Genomic DNA was extracted using prepGem Universal (Zygem 76218), and the region of interest was PCR amplified using Q5 polymerase (NEB M0491S). All PCR products were then screened and confirmed via Sanger sequencing (Eton Bioscience).

[0050] For CRISPRa, guide RNAs (gRNAs) were designed to target TTN (based on the N2BA Ensembl transcript 00000591111) based on the hg38 assembly TTN sequence from the UCSC Genome Browser. All gRNAs were cloned into the lentiGuide-Puro plasmid (Addgene #52963), packaged in 293T cells, titrated, and then used to transduce CM at an MOI = 10 unless otherwise indicated.

[0051] Lentivirus production and CM transduction HEK293T cells (ATCC CRL-3216) were maintained in DMEM (Gibco 11965092) supplemented with 10% FBS (Gemini 100-106), GlutaMAX (Gibco 35050061), 1 mM sodium pyruvate (Gibco 11360070), and penicillin-streptomycin (Gibco 15140122), and passaged using TrypLE (Gibco 12605028). For lentivirus production, 293T cells were grown to approximately 90% confluence, then changed to antibiotic-free medium, and co-transfected using Opti-MEM (Gibco 31985062) and polyethyleneimine (PEI) with the desired lentiviral transfer plasmid, psPAX2 packaging plasmid (Addgene 12260), and pCMV-VSV-G envelope plasmid (Addgene 8454). The next day, the medium was replenished, and the virus-containing medium was harvested on days 2, 3, and 4 after transfection and subsequently concentrated using PEG-6000. The iPSc were transduced with serial-diluted lentivirus, and the iPSc were treated with the appropriate antibiotic (1 μg / mL puromycin or 10 μg / mL blasticidin), and the functional titer was determined by counting the resistant colony-forming units.

[0052] For TTN activation, first, approximately 1×10 6 cells of CM expressing dCas9-VPR were plated into a single well of a 12-well plate pre-coated with fibronectin, and then transduced using RPMI-B27 containing lentivirus at a multiplicity of infection (MOI) of approximately 10 unless otherwise described. The next day, the cells were replenished with RPMI-B27. Analysis of the transduced CM was performed 7 to 14 days after transduction.

[0053] Vertical agarose gel electrophoresis (VAGE), SDS-PAGE, and immunoblot For TTN protein expression analysis, after washing the cells once in PBS, CM was homogenized directly from 12-well culture plates using denaturing urea sample buffer containing 8M urea, 2M thiourea, 3% SDS, 75mM DTT, 0.05M Tris-Cl (pH = 6.8), protease inhibitor cocktail (1 tab / 10 mL) (Roche Diagnostics, Germany 11836170001), and universal nuclease (0.1 μL / 1.0 mL) (Pierce 88700). The CM lysate was heated at 60 °C for 10 minutes and centrifuged at 14,000 rpm, then used or aliquots were frozen to limit freeze-thaw cycles. Samples were normalized against standard curves of sarcomere control, α-actinin (ab9465), or GAPDH (ab8245). Non-TTN blots were performed using precast Bio-Rad Mini-PROTEAN TGX gels and transferred to a PVDF membrane (Bio-Rad 1704272) using the Bio-Rad Trans-Blot Turbo system. TTN blots were performed using a Hoefer Dual Gel Casting system and a 1% agarose gel (Lonza 50152) filled between glass plates with acrylamide gel containing 0.00025% APS and 0.00025% TEMED, 30% glycerol, 0.25M Tris-base, 1.92M glycine, and 0.5% SDS. While containing 50mM Tris-base, 0.384M glycine, and 0.1% SDS in the cold running buffer (the upper buffer also contained 10mM 2-mercaptoethanol), a constant current of 15 mA was supplied for 6 hours using a Hoefer SE600 Chroma Vertical Electrophoresis Unit and a Hoefer PS300B Power Supply Unit.The agarose gel was stained with SYPRO Ruby (Invitrogen S12000) or transferred to a 15×15 cm PVDF membrane (0.45 μm pore size) activated in 100% methanol using a Hoefer TE42 Transfer Electrophoresis Unit and transfer buffer containing 24 mM Tris-base, 192 mM glycine, and 0.1% SDS. The blot was pre-incubated with 5% non-fat milk or 5% bovine serum albumin in Tris-buffered saline with Tween (TBST) (10 mmol / L Tris-HCl, pH 7.6, 75 mmol / L NaCl, 0.1% Tween) at room temperature for 1 hour, followed by incubation with the primary antibody overnight at 4°C. The next day, the blot was washed 3 times in TBST for 5 minutes each, probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling 7076, 7074) at room temperature for 1 hour, and then washed 3 times in TBST for 10 minutes each. Signal detection was performed using an ECL substrate (Thermo 34580) and a Bio-Rad ChemiDoc MP imaging system. The blot images were digitally processed and analyzed using either Bio-Rad Image Lab or ImageJ. The primary antibodies used were as follows: 1:500 anti-TTN-Z1Z2 (TTN-1, Myomedix, Neckargemuend, Germany), 1:500 anti-TTN-M (TTN-9, Myomedix), and 1:1000 anti-GAPDH (5174, Cell Signaling). Additional antibodies used included anti-HaloTag® (G9211, Promega), anti-Cas9 (14697, Cell Signaling), anti-MYH6 / 7 (HPA001239, Sigma), anti-ACTN (ab9465, Abcam), anti-TNNT2 (MA5-12960, Invitrogen), anti-ATF6a (65880, Cell Signaling), anti-phospho (S51) EIFa (9721, Cell Signaling), anti-EIFa (9722, Cell Signaling), and anti-IRE1a (3294, Cell Signaling).

[0054] Cardiac microtissue contraction assay Cardiac microtissues were generated as described by Cohn et al. (2019, Stem Cell Reports 12: 71-83). Briefly, a cantilever device composed of polydimethylsiloxane (PDMS) (Corning Sylgard 184) was molded from an SU-8 silicon master, and fluorescent microbeads (Thermo F8820) were embedded in this device for motion tracking. CM was mixed with normal human cardiac fibroblasts (Lonza) and centrifugally introduced into a PDMS device containing a collagen-based extracellular matrix (ECM). The tissue was maintained in DMEM + 10% FBS replenished daily. For reading frame repair studies, lentiviral SpCas9 and guide RNA (gRNA) were added to CM 5 - 7 days prior to tissue generation. For acquisition of functional data, the tissue was regionally stimulated at 1 Hz using a C-Pace EP stimulation device (IonOptix). Brightfield and fluorescence images were acquired in brightfield mode and 561-RFP laser widefield mode in an Andor Dragonfly microscopy system equipped with a sealed live cell chamber (Okolabs) and a Zyla sCMOS camera. Displacement of fluorescent microbeads was tracked using the ImageJ ParticleTracker plugin, and the maximum single contraction force was calculated using the cantilever spring constant and cantilever displacement values as described by Cohn et al. All tissue experiments included relevant TTN controls using NT (non-target) guide RNA to normalize batch variation in absolute force generation.

[0055] Quantitative PCR, RNA sequencing, and computational analysis RNA was isolated from CM using TRIzol and phenol-chloroform extraction. cDNA was synthesized using Superscript III First-Strand synthesis (Invitrogen 18080-400). Gene-specific PCR primers (primer sequences of SEQ ID NOs: 1-6 listed in Table 1) were designed using the IDT gRNA design tool (provided by IDT at https: / / www.idtdna.com / site / order / designtool / index / CRISPR_CUSTOM), and transcripts were quantified using Fast SYBR Green (Applied Biosystems 4385612) in a ViiA7 real-time PCR system (Applied Biosystems). For RNA sequencing, RNA was isolated using the QIAGEN RNeasy Mini Kit (catalog number 74104). RNA sequencing libraries were prepared using the KAPA mRNA Hyperprep kit (Roche). Illumina NovaSeq S1 flow cell sequencing was performed using a 100-cycle reagent kit. Estimated total single-end reads per sample = 34.5 - 43.1M 100bp SE reads. Reads were aligned to the reference hg38 human genome using STAR, quantified using HTSeq, and analyzed using DESeq2. Gene set enrichment analysis (GSEA) was utilized for gene ontology (GO) terms of the dataset. GO terms of genes related to myocardial contraction are represented in the following and the attached drawings.

[0056]

Table 1

[0057] Fluorescence-activated cell sorting (FACS) To quantify TTN-tdTomato levels in CM, FACS was performed using a BD FACSymphony A5 and FACSDiva software. Prior to FACS, CM was stained with TO-PRO-3 and Hoechst 33342 to gate for viability and single cells, respectively, and then the tdTomato signal was determined (5,000 - 10,000 cells / sample). All TTN-tdTomato experiments were designed and analyzed using a 96-well plate format (∼10 - 30 kCM / well) containing promoter-activating guide RNA or NT control gRNA to analyze TTN-tdTomato levels.

[0058] TTN promoter luciferase assay To develop a reporter assay that can be used to quantitatively detect TTN promoter activity, a lentiviral vector was generated. First, the human TTN promoter was PCR amplified from human CM genomic DNA corresponding to -600 to 0 relative to the TTN N2BA transcription start site (heterozygous for the rs72647838 SNP; see Table 7 for all loci indicated by rs), and cloned into the lentiviral plasmid upstream of the open reading frame of NanoLuc luciferase (Promega) by restriction enzyme digestion followed by sticky-end ligation. After confirming plasmid clones carrying either the rs72647838 allele A (GGGG) or B (GG--) by sequencing, the lentivirus was packaged, precipitated, and titrated (Hunter et al., 209, Nat. Protoc. 4: 495 - 505;). To develop allele-specific TTN activation methods, TTNtv expressing dCas9-VPR + / -CM was transduced with a lentivirus encoding NanoLuc driven by the TTN promoter (multiplicity of infection (MOI += 3)) and a gRNA that recognizes a sequence specific to allele A or B but overlaps with the rs72647838 SNP. After 7 days, the CM was lysed and luciferase activity was measured using the Nano-Glo assay (Promega) and a luminescence plate reader (Biotek). For the dCasMini-VPR TTN activation study, the lentivirus dCasMini-VPR (Addgene #176269) was packaged, precipitated, titrated, and then co-transduced into CM (MOI = 5) along with the TTN promoter-driven NanoLuc (MOI = 3) and the CasMini-compatible gRNA (MOI = 5). After 7 days, the CM was lysed and luciferase activity was measured using the Nano-Glo assay (Promega) and a luminescence plate reader.

[0059] Statistical analysis Data were analyzed and graphed using a combination of the statistical program R and GraphPad Prism. All experiments were performed with at least three biological replicates (n ≧ 3) unless otherwise indicated. Statistical comparisons were made using Student's t-test or ANOVA, and Dunnett's correction was used for multiple comparisons. Statistical significance was defined as P ≧ 0.05 (not significant), P < 0.05 (*), P ≦ 0.01 (**), P ≦ 0.001 (***), and P ≦ 0.0001 (****) as described below and in the associated figures. Experimental results

[0060] (Example 1) Manipulation of DCM-related TTNtv in human cardiomyocytes and mouse models The most common pathogenic DCM-related titin (encoded by TTN) variants are frameshift insertions, deletions, or splice-site mutations, or alternatively, protein-truncating variants (the "tv") that include premature termination codon mutations occurring within a constitutively expressed exon encoding a peptide localized to the A-band structural domain of the protein that titin interacts with sarcomeric myosin (the "TTNtvA") (Schafer et al., 2017, Nat. Genet. 49: 46-53). Using a previously prepared CRISPR-engineered human induced pluripotent stem cell (iPSC) line (frameshift after proline-22582; exon 276 in ENST00000591111; Romano et al., 2022, Circulation 145: 194-205) that harbors a heterozygous single-base deletion variant that results in a premature TTN truncation within the A-band structural domain, a human cell assay was developed to model DCM caused by TTNtv (shown in Fig. 1A). Human cardiomyocytes (CMs) differentiated from these iPSCs using small molecule modulators of WNT signaling in an established method (Lian et al., 2012, Proc. Natl. Acad. Sci. USA 109) were studied as described herein. CMs differentiated from this DCM iPSC line are designated "TTNtvA + / - ".

[0061] TTNtvA + / -And the control CM models were functionally examined in molecular profiling to determine phenotypic abnormalities underlying highly pathogenic TTNtv that can be used as assays for cardiac microtissues (shown in Figure 1B) and for therapeutic drug development. An advanced three-dimensional biomimetic cardiac microtissue platform (VAGE; Cohn et al., 2019, Stem Cell Reports 12: 71-83) has been shown to better predict the in vivo-like cardiac phenotypes of cardiomyopathy mutations compared to other methods for functionally examining TTNtvA (see Cohn et al., Id., Hinson et al., 2015, Science 349: 982-986, Pettinato et al., 2020, Circulation, 142: 2262-2275), and was used in these experiments (shown in Figure 1C). Using cantilever displacement analysis, TTNtvA + / - The microtissues generated a single contraction force (a measure of contractile function) that was reduced by more than 50% compared to controls. This microtissue phenotype was detected using echocardiography and was consistent with the reduced cardiac contractility observed in human hearts with heterozygous TTNtv, which was identified by a decrease in ejection fraction (i.e., the proportion of blood ejected with each heartbeat) (see Herman et al., 2012, N. Engl. J. Med. 366: 619-628). To determine the molecular results of TTNtvA + / - with respect to the levels and sizes of the TTN protein, a specialized vertical agarose gel electrophoresis method (VAGE) configured for studying large proteins such as TTN was used (see Warren et al., 2003, Electrophoresis 24: 1695-197). TTNtvA + / -VAGE analysis of the protein lysates obtained from CM revealed a decrease of more than 40% in the levels of full-length TTN proteins (N2BA and N2B isoforms) (shown in Figures 1D and 1E) compared to the control, and the presence of a large number of truncated TTN proteins not observed in the control lysates (shown in Figures 1D and 1F). TTNtv localized in other structural domains such as the I band also resulted in a decrease in the levels of full-length TTN proteins, but no observable truncated TTN proteins were detected.

[0062] To evaluate the functional relevance of TTNtvA in vivo, CRISPR was used to introduce TTNtvA as P22582fs into an equivalent exon (shown in Figure 1G). To enable a method to track and quantify truncated TTN proteins, a multifunctional HaloTag® (Los et al., 2008, ACS Chem Biol. 3: 373 - 382) was also introduced proximal to TTNtvA (TTNtvA HaloTag / - ). Anti-HaloTag® antibody was used to detect the truncated protein products in vivo. VAGE was used to evaluate the amount of TTN protein and truncated products in wild-type and TTNtvA HaloTag / - mice. A decrease of approximately 15% in the levels of full-length TTN proteins was observed (where N2B is the dominant full-length TTN protein in adult mice; see Cazoria et al., 2000, Circ. Res. 86: 59 - 67), and when immunoprobed with an anti-Z TTN antibody, a small amount of N2B truncated protein was detected. Immunoblotting with an anti-HaloTag® antibody confirmed the presence of TTN truncated protein species (shown in Figure 1H), but TTNtvA + / -It was at a lower level than that observed in human cardiomyocytes (shown in Figure 1D). These results indicated that the physiological results of TTNtv decreased contractile function, which was associated with a decrease in full-length TTN protein levels and the acquisition of truncated TTN proteins. In addition, the decrease in full-length TTN protein levels was a result common to both TTNtv localized in the I band and TTNtv localized in the A band.

[0063] (Example 2) TTN transcriptional activation restores TTN levels and contractile defects due to TTNtv To determine whether the decrease in contractile force associated with TTNtv was mainly due to a decrease in full-length TTN protein levels (since this was a common feature of TTNtv), CRISPR-based transcriptional activation or "CRISPRa" was used. CRISPRa is a recently developed method that increases gene transcript levels through the site-specific recruitment of nuclease-inactive Cas9 ("dCas9") fused to a transcriptional activator domain such as VP64 (see Chavez et al., 2016, Nature Methods 1: 563-567). For CRISPRa, dCas9 fused to a three-activator complex of VP64, p65, and Rta ("dCas9-VPR") was used. VPR has been shown to be a potent transcriptional activator in human cell models (Chavez et al., Id.). CRISPRa TTNtvA + / - To generate CMs from hiPSCs (Figure 3A), standard CRISPR / Cas9 was used with a donor template containing homology arm sequences for cardiac troponin T2 (encoded by TNNT2) to facilitate homologous recombination repair for "knocking in" dCas9-VPR upstream of the stop codon of TNNT2 using a self-cleaving T2A peptide linker (see Kim et al., 2011, PLoS One 6: e18556). TNNT2-T2A-dCas9-VPR + / - iPSc(TTNtvA + / -After array confirmation of the clones, lysates obtained from iPSCs and differentiated CMs were assayed for the expression of Cas9-VPR and cardiomyocyte lineage-specific markers including cardiac myosin heavy chain (MYH6 and MYH7) and cardiac troponin T2 (TNNT2), along with the expression of GAPDH as a loading control (shown in Figure 2D). The expression of dCas9-VPR and TNNT2 was observed only in CMs at the expected scale, demonstrating both cardiomyocyte-specific expression and efficient T2A peptide cleavage.

[0064] Next, the TTN protein reporter was introduced into the CRISPRa TTNtv + / - CM model to achieve a quantitative method for measuring TTN levels in individual CMs. Using standard CRISPR / Cas9, homologous recombination repair was facilitated using a donor template containing TTN homology arm sequences for introducing tdTomato-FLAG upstream of the stop codon of TTN (shown in Figures 2E and 2F). After sequence confirmation, a single TTNtvA | / - iPSC clone was expanded after visualization of TTN-tdTomato expression during CM differentiation, thus confirming that tdTomato had fused with the normal (wild-type, functional) TTN allele rather than the TTNtvA allele.

[0065] Next, to test TTN activation, a single guide RNA (gRNA or sgRNA) that can be introduced into CM together with dCas9-VPR (sequence of SEQ ID NO: 8 shown in Table 3) by lentiviral transduction (lentiGuide-Puro backbone; Addgene #52963; U6 promoter and gRNA scaffold sequence of SEQ ID NO: 7 shown in Table 2) was developed (Figure 3A). The TTN promoter gRNA was designed to be compatible with SpCas9 (NGG PAM) and programmed to recognize a 20-base pair protospacer sequence (described in Table 4) within the N2BA TTN promoter defined by ATAC-seq peak analysis from CM (shown in Figure 3B). TTN promoter gRNA lentivirus and a non-target (NT) gRNA lentivirus that functions as a control were produced and concentrated. CM expressing dCas9-VPR and either the TTN promoter gRNA or NT gRNA was transduced, mRNA was recovered, and TTN transcript levels were quantified using quantitative polymerase chain reaction (qPCR). When normalized to the NT control, 5- to 13-fold activation of TTN transcript levels by a single TTN promoter gRNA was observed using qPCR (shown in Figure 3C). Differences in TTN isoform activation were identified based on qPCR amplification of specific transcript sequences using separate primer pairs that overlap the Z-band TTN sequence that can amplify the N2BA and Novex3 isoforms, and the A-band TTN sequence that can amplify the Novex3 TTN sequence, as well as the N2BA and Cronos isoforms (shown in Figure 3C). All TTN isoforms were activated, with Novex3 being the most strongly activated. Since TTN transcript levels do not always reflect protein levels, the same treatment was performed in TTN-tdTomato expressing dCas9-VPR + / - in CM, and TTN protein levels were quantified using fluorescence-activated cell sorting (FACS) to measure the tdTomato intensity in individual TTNtv + / - in CM. Gating for exclusion of cell debris (FSC high and SSC high) to develop an optimized FACS strategy, which included viable CM (To-pro-3 low and TTN-tdTomato + ). Viable TTNtv that can be activated in a dose-dependent pattern, as demonstrated by the rightward shift of TTN-tdTomato intensity compared to NT controls using histogram analysis, was observed in the viable CM + / - for TTN protein levels (Figure 3E). Collectively, these results showed that CRISPRa using a single gRNA induced by the TTN N2BA promoter was able to increase TTN transcript and protein levels using dCas9-VPR.

[0066]

Table 2

[0067]

Table 3-1

Table 3-2

[0068] Next, by screening a larger panel of single gRNAs using the ends of protospacers targeting +97 to -515 relative to the N2BA transcription start site, a range of TTN activators (with low to high activity) were identified. After transduction into CM, TTN activation was tested by FACS 9 and 12 days after infection (these results are described in Table 4 as SEQ ID NOs: 9 - 32). When normalized to the NT control, several single gRNAs that activated the TTN protein level more than 2-fold (e.g., NPE, NPG, NPP, NPQ, NPS, and NPT), several single gRNAs that activated the TTN protein level 1 - 2-fold (NPA, NPH, NPJ, NPU, and NPX), and a single gRNA that inhibited the TTN protein level (NPB) were observed (shown in Figure 4A). Since the TTN-tdTomato level increased 9 - 12 days after transduction, time-dependent TTN protein level activation was observed (shown in Figure 4A). Using VAGE, after activation of the N2BA TTN promoter with a strong activator, when analyzing the TTN protein isoforms and levels using anti-Z and anti-M TTN antibodies (shown in Figure 4B), an increase in the levels of N2BA, truncated TTN, and Novex3 was observed (Figures 4C - 4E), but no increase in the level of Cronos TTN was observed (Figures 4F - 4H), and dose-dependent activation was observed (shown in Figure 4C).

[0069] Next, in the cardiac microtissue assay, it was evaluated whether TTN activation could rescue TTNtvA-related systolic dysfunction (see Figure 5A). Specifically, due to the treatment resulting in a more than 2-fold increase in the TTN-tdTomato level in TTNtvA compared to the level observed in normal CM (compare with Figure 1E), the NPE gRNA was tested. The cardiac microtissues treated with the NPE gRNA demonstrated more than a 75% increase in single contractile force compared to the NT control (shown in Figure 5A). + / - in which the TTN-tdTomato level was more than 2-fold increased compared to the level observed in normal CM (compare with Figure 1E), the NPE gRNA was tested. The cardiac microtissues treated with the NPE gRNA + / - demonstrated more than a 75% increase in single contractile force compared to the NT control (shown in Figure 5A).

[0070] Shortened TTN has been previously observed to misfold and aggregate in vivo (Fomin et al., 2021, Sci Transl Med. 13:eabd3079), and an increase in TTN levels, specifically shortened TTN levels, can be toxic. To screen for toxicity, the relationship with the onset of cardiomyopathy has been previously shown (Feyen et al., 2021, Circulation 144: 382-392, Wang et al., 2018, Br J Pharmacol. 175: 1293-1304), and the activation status of the unfolded protein response (UPR) (Hetz, 2012, Nat Rev Mol Cell Biol. 13:89-102, Glembotski, 2007, Circ Res. 101: 975-84) was tested. To test UPR activation after TTN CRISPRa in dCas9-VPR-TTNtv+ / -CM, the protein levels of UPR factors including ATF6α, phosphorylated EIF2α, and IRE1α were quantified using immunoblotting of lysates (Borgia et al., 2011, Nature 474: 662-665) (Figure 5B and Figure 5C). The TTN CRISPRa samples did not show a change in the levels of these UPR markers compared to the control. Collectively, these results demonstrate that TTN CRISPRa using dCas9-VPR and a single gRNA induced by the TTN N2BA promoter increases TTN mRNA and protein levels without evidence of UPR activation, despite increasing shortened TTN levels.

[0071]

Table 4

[0072] (Example 3) RNA Sequencing Analysis of TTN Transcriptional Activation Beyond the direct effects on TTN transcripts, RNA sequencing and computational analysis from samples obtained from biological triplicates of NPV and NT control CM were used to evaluate the molecular consequences of TTN CRISPRa at the transcriptome level. Principal component analysis of the samples demonstrated a clear separation from NT biological replicates of NPV (Figure 6A). Differential gene expression (DGE) analysis (cut-off = P adj <0.01 and log2FC ≥ 1 or ≤ -1) demonstrated 1,357 downregulated (Figures 12A–12C) transcripts and 576 upregulated (Figures 13A and 13B) transcripts, although the majority of transcripts were unchanged (Figure 6B). Gene ontology (GO) term enrichment analysis of significantly downregulated (Figures 6C and 6D) and upregulated (Figures 6D and 6E) transcripts revealed changes in biological processes, pathways, and components. GO terms involved in "contractile fiber", "muscle structure development", and "circulatory system development" were enriched in the upregulated gene set (Figure 6E). Plotting of the fold change heatmap of the complete gene set within GO terms including "cardiomyofibril organization", "cardiac muscle contraction", "sarcomere formation", sarcomere "M-line", "I-band / Z-band", and "A-band" (Figure 6F, and Figures 14A–14B) showed a general upregulation of these factors after TTN CRISPRa. GO term enrichment analysis of downregulated transcripts revealed functions in "ion transport" and "intrinsic components of the plasma membrane" (Figure 6C). Summarizing the RNA sequencing results, TTN CRISPRa increased not only the TTN transcript level but also a number of factors involved in sarcomere construction, structure, and formation.

[0073] (Example 4) Activation of TTN transcription utilizing TTN regulatory elements TTNtvA + / -In the CM model, it was also tested whether CRISPRa induced in the DNA regulatory element could activate the TTN level (shown in Figure 7A). Cis-acting DNA regulatory elements such as enhancers have previously been demonstrated to physically contact gene promoters through three-dimensional interactions (see Fullwood et al., 2009, Nature 462: 58-64), and therefore, homing CRISPRa to regulatory elements that physically contact the TTN promoter may also activate the TTN level and function as a treatment for DCM. These experiments were initiated by examining ATAC-seq data regarding peaks upstream of the TTN TSS obtained from CM samples. Five TTN elements (E1-E5; shown in Figure 7B) supported by peak analysis were focused on. To identify TTN elements that contact the TTN promoter, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) using an antibody that recognizes RNA polymerase II (RNAPII), and chromatin immunoprecipitation-sequencing (ChIP-seq) using an antibody that recognizes histone H3 lysine 27 acetylation (H3K27ac) were utilized. ChIA-PET confirmed physical contact between the TTN TSS and E1, E3, and E5 (shown in Figure 7C), while E2 and E4 were supported only by ChIP-seq and ATAC-seq peak analysis (shown in Figure 7B). Single gRNAs (described in Table 5 as SEQ ID NOs: 33-37) that recognize sequences within approximately 250 base pairs from the center of each regulatory element were designed, along with dCas9-VPR and TTNtvA that express TTN-tdTomato + / -It was transduced into CM. CRISPRa embodiments targeting E1 (NEA), E3 (NEC), E4 (NED), and E5 (NEE) were identified, and these increased the TTN-tdTomato signal in a time-dependent manner (shown in Fig. 7D). CRISPRa E2 (NEB) was identified, which resulted in a decrease in the TTN-tdTomato signal suggesting an inhibitory effect. To evaluate the TTN isoforms and levels after TTN element activation, VAGE was used and probed with an antibody against anti-Z TTN. An increase in the expression of N2BA TTN, truncated TTN, and Novex3 isoforms was observed in a pattern similar to TTN promoter activation (compare with Fig. 4C) (shown in Fig. 7E). Collectively, these results supported the conclusion that CRISPRa induced in specific TTN regulatory elements can activate TTN protein, similar to TTN promoter activation.

[0074] (Example 5) TTN Isoform-Specific Transcriptional Activation Utilizing an Internal Promoter In recent years, an internal TTN promoter that regulates a fetal-enriched TTN isoform called Cronos, derived from the hearts of zebrafish (Zou et al., 2015, Elife 4: e09406) and humans (Zaunbrecker et al., 2019, Circulation 140: 1647-1660), has been identified. Cronos TTN has previously been demonstrated to regulate myofibril architecture and contractile function in CMs (see Xu et al., 2021, Mol. Cell. 81: 4333-4345), but a method to increase Cronos TTN levels (e.g., using CRISPRa) has not been developed to date. To test the premise that CRISPRa induced by the Cronos promoter can activate Cronos TTN isoform expression (shown in Figure 8A) (which could be a therapeutic strategy for DCM), we first identified the Cronos TTN promoter by examining ATAC-seq data (shown in Figure 8B) for peaks near the Cronos TSS obtained from CM samples, and designed a single gRNA that recognizes sequences within approximately 500 base pairs from the Cronos TSS (listed in Table 6 as SEQ ID NOs: 38-41). TTNtvA expressing dCas9-VPR and TTN-tdTomato + / - Four candidate gRNAs were transduced into CMs, and an embodiment of CRISPRa targeting the Cronos TSS was identified that included increased TTN-tdTomato signal (shown in Figure 8C) by CPA~D. Since the increase in TTN-tdTomato signal could be subsequent to an increase in N2BA and / or Cronos TTN levels, TTN isoforms were examined using VAGE with CPA gRNA and anti-M TTN antibody compared to NT controls (shown in Figure 8D). In these experiments, it was observed that CPD activated Cronos TTN levels but not N2BA TTN levels. These results demonstrated that the method using CRISPRa and Cronos promoter-induced gRNA can specifically activate the Cronos TTN protein.

[0075]

Table 5

[0076]

Table 6

[0077] (Example 6) TTN allele-specific transcriptional activation utilizing general genetic diversity Activation of the TTN promoter using CRISPRa parallels an increase in the full-length TTN protein level (wild type) with TTNtvA + / -Although it improved contractile dysfunction in the DCM model, it also led to an increase in truncated TTN proteins that are incorporated into the sarcomere and have previously been shown to partially inhibit contractile function (Romano et al., Id.). An improved method for CRISPRa should increase full-length TTN, but not truncated TTN proteins. To achieve this, common genetic variants were identified that localize to the N2BA TTN promoter or regulatory elements (described in Table 7 as SEQ ID NOs: 42-95), which may be configured to achieve allele-specific CRISPRa. Since individuals carrying these common genetic variants are generally heterozygous with respect to the polymorphism (described in Table 8 as an example), allele-specific CRISPRa can be developed by using gRNAs that perfectly align to the major or minor polymorphic alleles (shown in FIG. 9A). This is based on the premise that common genetic variants can provide distinct "landing sites" for dCas9-VPR, which can provide recognition sequences that clearly differ between two alleles (i.e., wild type and TTNtv). See FIGS. 9C and 9F. For example, rs72647838 is located within an ATAC-seq peak and is approximately 300 base pairs upstream of the N2BA TTN TSS (shown in FIG. 9B) and differs in terms of a 2-nucleotide deletion that can be intentionally utilized for allele-specific CRISPRa. Prior studies of CRISPRa have demonstrated that gRNAs further upstream of rs72647838 (e.g., NPX) compared to the TSS can activate TTN protein levels, confirming that the SNP is proximal enough to the TSS to achieve activation using dCas9-VPR. To achieve allele specificity, the gRNAs were designed by using allele-specific PAMs (e.g., gRNA rs72647838A) or spacer sequences (e.g., rs72647838A2 and rs72647838B) (shown in FIG. 9B).Next, two luciferase reporter constructs were generated consisting of the TTN promoter (-600 to 0 relative to the TTN N2BA TSS) harboring either the rs72647838 allele A (major allele or GGGG shown in FIG. 9C) or the rs72647838 allele B (minor allele or GG-- shown in FIG. 9E). By using a luciferase activity assay in CM transfected with either the allele A TTN promoter or the allele B TTN promoter, it was possible to determine whether allele-specific TTN activation was possible using a panel of gRNAs (described in Table 9 as SEQ ID NOs: 103-105). Indeed, the rs72647838A and rs72647838A2 gRNAs (whose recognition sequences perfectly match allele A) were found to activate only the allele A TTN promoter (shown in FIG. 9D), and the rs72647838B gRNA (whose recognition sequence perfectly matches allele B) was found to activate only allele B (shown in FIG. 9F). These results supported the conclusion that CRISPRa can be configured to achieve allele-specific activation by exploiting common genetic diversity within the TTN promoter and other similar regulatory elements.

[0078] Similar beneficial results were obtained when using CRISPR enzymes other than SpCas9 (FIG. 9H). In these experiments, a lentivirus encoding a re-engineered form of Cas12f optimized for eukaryotic function (dCasMini-VPR shown in FIG. 9G, sequence described in Table 10 as SEQ ID NO: 106) was successfully used together with a gRNA recognizing the TTN N2BA promoter, and NT control gRNAs (described in Table 11 as SEQ ID NOs: 107 and 108, and in Table 12 as SEQ ID NO: 109) were co-transfected into CM.

[0079]

Table 7

[0080]

Table 8

[0081]

Table 9

[0082]

Table 10

[0083]

Table 11

[0084]

Table 12

[0085] (Example 7) CRISPRa restored the sarcomere content and systolic dysfunction Since the reduction in sarcomere content and contractility is a functional consequence of TTNtv in CMs (Hinson et al., 2015, Science 349:982-6, Romano et al., 2022, Circulation 145:194-205, Chopra et al., 2018, Dev. Cell. 44:87-96 e5), we determined how TTN CRISPRa affected these functional parameters. To determine sarcomere content, dCas9-VPR-TTNtv+ / - CMs with NPV or NT gRNA were transduced, and the CMs were replated onto 2000-μm fibronectin rectangles (7:1 aspect ratio) as previously described to optimize sarcomere formation and maturation (Clippinger et al., 2019, Proc Natl Acad Sci USA 116:17831-1784037, Ribeiro et al., 2015, Proc Natl Acad Sci USA 112:12705-10). Next, the micropatterned CMs were fixed and immunostained with an anti-sarcomere antibody (anti-TTN). CM sarcomere area was quantified using confocal microscopy and a custom ImageJ script (Figure 10A). TTN CRISPRa using NPV gRNA increased the mean CM sarcomere area compared with the NT gRNA control (Figure 10B). Finally, contractility was quantified after TTN CRISPRa using a custom biomimetic three-dimensional CMT as previously described (Cohn et al., 2019, Stem Cell Reports 12: 71-8311, Romano et al., 2022, Circulation 145: 194-205) (Figure 10C). TTN CRISPRa using NPV gRNA increased the single-CMT contractility compared with the NT gRNA control (Figure 10D). Collectively, it was determined that TTN CRISPRa rescued sarcomere content and CMT contractile deficiency secondary to DCM-related TTNtv (Figure 10E).Furthermore, these functional improvements support the model that haploinsufficiency is the dominant genetic mechanism underlying TTNtv (whereas the dominant-negative hypothesis is involved in gain-of-toxic function through, for example, interference with normal TTN function by toxic truncated TTN protein aggregation or by competitive sarcomere incorporation of truncated TTN toxic peptides, while a single wild-type TTN allele results in insufficient normal TTN protein levels), and that TTN CRISPRa and other similar methods of amplifying TTN protein levels could be a treatment for other DCM-related TTN variants.

[0086] (Example 8) In vivo Ttn activation as a treatment for dilated cardiomyopathy and other types of heart failure To validate the study of TTN activation in a human cardiac microtissue model, a custom knock-in mouse model of dilated cardiomyopathy secondary to a heterozygous Ttn truncation variant (TTNtv+ / -), and a humanized TTN model in which the mouse TTN promoter was replaced with the human TTN promoter were used. The human TTN promoter model enables testing of TTN activation treatments that can be directly applied to humans, which have been validated in human cardiac microtissues. Similar to human hearts (McAfee et al., 2021, Sci Transl Med. 13:eabd7287) and cardiac microtissue models (Romano et al., 2021, Circulation 145:194-205, Hinson et al., 2015, Science 349:982-6), both Ttntv+ / - knock-in mouse models recapitulate the molecular consequences of TTNtv (i.e., expression of truncated Ttn and reduced full-length Ttn protein), functional changes by echocardiography (i.e., reduced ejection fraction and increased ventricular size), and histopathological changes by staining (i.e., increased percentage of myocardial fibrosis) (Gramlich et al., 2009, J Mol Cell Cardiol. 47:352-8). In addition, cardiac stressors (e.g., osmotic pump delivery of chronic isoproterenol, which activates beta-adrenergic signaling, or chronic angiotensin II, which activates the AT1 receptor) worsen the functional consequences of Ttntv in mice after treatment for more than 1 week (Gramlich et al., 2009, J Mol Cell Cardiol. 47:352-8), thus providing an efficient platform for therapeutic screening.

[0087] In this validation study, to accommodate cardiac microtissue research, guide RNAs were tested that were programmed to recognize either dCas9-VPR as an exemplary transcriptional activator, or any of the DNA regulatory elements that control promoter activity, such as the mouse Ttn promoter, the human TTN promoter, or the Ttn enhancer sequence. In addition to dCas9-VPR, other transcriptional activators can be tested, such as other CRISPR / Cas proteins, or other programmable chimeric activator systems (e.g., any programmable DNA-binding protein that conjugates to a transcriptional activator), such as, but not limited to, TALE nucleases or zinc fingers that conjugate to VP64 or other transcriptional activator enzymes. The TTN activator is delivered via transgenic approaches or, in particular, using viral vectors including, but not limited to, adeno-associated vectors such as the strongly cardiac tropic AAV9 and its derivatives, in combination with, for example, a cardiac-specific promoter from troponin T (Prasad et al., 2011, Gene Ther. 18:43-52). Allow sufficient time for Ttn activation, for example, but not limited to, two weeks after injection of AAV delivering dCas9-VPR and the programmed guide RNA, and treat Ttntv+ / - mice with a cardiac stress factor to induce the dilated cardiomyopathy phenotype (Figure 11A). Functional assessments for evaluating the efficacy and safety of TTN activation include echocardiography for cardiac structure and function, histopathology for quantification of myocardial fibrosis, and electrophysiology for arrhythmia risk. Therapeutic efficacy is achieved when TTN activation results in an increase in ejection fraction, and / or suppression of ventricular dilation, and / or suppression of myocardial fibrosis, compared to affected individuals. Based on the DCM c.43628insAT truncation mutation (TTN 2bp insertion mutation) knock-in mouse model, Ttntv+ / - mice are expected to exhibit a stress-induced decrease in ejection fraction of approximately 23% and an increase in ventricular size of approximately 13%. Therapeutic intervention by TTN activation in Ttntv+ / - mice could potentially cause a 15% improvement in ejection fraction and a 10% reduction in ventricular size.TTN activation is also tested in other forms of heart failure in other gene mutations, for example, other gene mutations that cause human DCM, such as RBM20, LMNA, MYH7, TNNT2, TNNI3, TNNC1, TPM1, BAG3, etc. (as described in Hershberger et al., 2021, Circ Res. 128:1514-1532), and other environmental models of heart failure, including but not limited to, aortic banding (TAC) that generates pressure load, or chemical stress factors including but not limited to chronic treatment with isoproterenol or angiotensin II (Figure 11B).

[0088] One of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein using no more than routine experimentation. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition for use in the treatment of dilated cardiomyopathy (DCM), The composition comprises a therapeutically effective amount that can introduce a transcription activator to a site specific to a regulatory sequence that controls or affects TTN gene expression, A pharmaceutical composition that is delivered to cardiac tissue or skeletal muscle tissue, thereby increasing the expression of a functional TTN gene product in the cardiac tissue or skeletal muscle tissue.

2. The pharmaceutical composition according to claim 1, wherein the DCM is associated with a single TTN allele in the target genomic DNA encoding a TTN gene that produces a dysfunctional titin protein gene product.

3. The pharmaceutical composition according to claim 2, wherein the TTN allele encodes a truncated mutation, a nonsense mutation, a frameshift mutation or a splicing variant mutation of the TTN gene, or encodes a variant that reduces the TTN expression level.

4. The pharmaceutical composition according to claim 1, wherein the composition delivered to the cardiac tissue or skeletal muscle 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.

5. The pharmaceutical composition according to claim 4, wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VPR, dCas9-VP64, dCas9-SunTag, or dCas9-SAM.

6. The pharmaceutical composition according to claim 1 or 5, wherein the regulatory sequence for TTN gene expression is located within the TTN gene promoter region.

7. The pharmaceutical composition according to claim 6, wherein the guide RNA targeting 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.

8. The pharmaceutical composition according to claim 6, 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.

9. The pharmaceutical composition according to claim 1 or 5, wherein the regulatory sequence for TTN gene expression is located within the TTN gene enhancer region.

10. The pharmaceutical composition according to claim 9, 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.

11. The pharmaceutical composition according to claim 9, wherein the guide RNA is an sgRNA having a sequence identified by Sequence ID No.

33.

12. The pharmaceutical composition according to claim 4 or 5, wherein the CRISPR-Cas9 complex delivered to the cardiac tissue or skeletal muscle tissue 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.

13. The pharmaceutical composition according to claim 12, wherein the CRISPR-Cas9 complex or one or more expression constructs are constructed for delivery to cardiac tissue or skeletal muscle tissue.

14. An sgRNA molecule identified by any one of SEQ ID NOs: 9, SEQ ID NOs: 13-21, SEQ ID NOs: 23-25, SEQ ID NOs: 27-32, SEQ ID NOs: 33, SEQ ID NOs: 35-37, and SEQ ID NOs: 38-41.

15. 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.

16. The pharmaceutical composition according to claim 15, wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VPR, dCas9-VP64, dCas9-SunTag, or dCas9-SAM.

17. The pharmaceutical composition according to claim 15 or 16, wherein the regulatory sequence for TTN gene expression is located within the TTN gene promoter region.

18. The pharmaceutical composition according to claim 17, wherein the guide RNA targeting 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.

19. The pharmaceutical composition according to claim 17, 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.

20. The pharmaceutical composition according to claim 15 or 16, wherein the regulatory sequence for TTN gene expression is located within the TTN gene enhancer region.

21. The pharmaceutical composition according to claim 20, 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 to 37.

22. The pharmaceutical composition according to claim 20, wherein the guide RNA is an sgRNA having a sequence identified by Sequence ID No.

33.

23. The pharmaceutical composition according to claim 15 or 16, wherein the CRISPR-Cas9 complex delivered to cardiac tissue or skeletal muscle tissue 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.

24. The pharmaceutical composition according to claim 23, wherein the CRISPR-Cas9 complex or one or more expression constructs are constructed for delivery to cardiac tissue or skeletal muscle tissue.

25. The pharmaceutical composition according to claim 15, comprising a guide RNA molecule identified by any one of SEQ ID NOs: 9, SEQ ID NOs: 13-21, SEQ ID NOs: 23-25, SEQ ID NOs: 27-32, SEQ ID NOs: 33, SEQ ID NOs: 35-37, and SEQ ID NOs: 38-41.

26. A pharmaceutical composition for use in the treatment of dilated cardiomyopathy (DCM), comprising a therapeutically effective amount of a composition that can introduce a transcription activator to a site specific to a regulatory sequence that controls or affects a wild-type TTN allele that produces a functional titin protein gene product, A pharmaceutical composition that is delivered to cardiac tissue or skeletal muscle tissue, thereby specifically increasing the expression of functional titin protein in said cardiac tissue or skeletal muscle tissue.

27. ​​The pharmaceutical composition according to claim 26, wherein the DCM is caused by a mutated TTN allele in the target genomic DNA encoding a TTN gene that produces a dysfunctional titin protein gene product.

28. The pharmaceutical composition according to claim 27, wherein the regulatory sequence of the wild-type TTN allele contains a polymorphic variant, and the polymorphic variant is not present in the regulatory sequence of the mutant TTN allele.

29. The pharmaceutical composition according to claim 27, wherein the mutant TTN allele encodes a truncated mutation, a nonsense mutation, a frameshift mutation or a splicing variant mutation of the TTN gene, or a variant that reduces the TTN expression level.

30. The pharmaceutical composition according to claim 26, wherein the composition delivered to the cardiac tissue or skeletal muscle 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 a wild-type TTN allele that produces a functional titin protein gene product.

31. The pharmaceutical composition according to claim 30, wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VPR, dCas9-VP64, dCas9-SunTag, or dCas9-SAM.

32. The pharmaceutical composition according to any one of claims 26 to 31, wherein the regulatory sequence for TTN gene expression is located within the wild-type TTN allele gene promoter region.

33. The pharmaceutical composition according to any one of claims 26 to 31, wherein the regulatory sequence for TTN gene expression is located within the wild-type TTN allele gene enhancer region.

34. The pharmaceutical composition according to claim 32, wherein the guide RNA is an sgRNA that targets a wild-type TTN allele gene promoter region or a wild-type TTN allele gene enhancer region, having a sequence identified by any one of sequence numbers 103 to 105.

35. The pharmaceutical composition according to claim 33, wherein the guide RNA is an sgRNA that targets a wild-type TTN allele gene promoter region or a wild-type TTN allele gene enhancer region, having a sequence identified by any one of sequence numbers 103 to 105.

36. The pharmaceutical composition according to claim 30 or 31, wherein the CRISPR-Cas9 complex delivered to the cardiac tissue or skeletal muscle tissue is delivered by one or more expression constructs encoding a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific to a regulatory sequence relating to a wild-type TTN allele that produces a functional titin protein gene product.

37. The pharmaceutical composition according to claim 36, wherein the CRISPR-Cas9 complex or one or more expression constructs are constructed for delivery to cardiac tissue or skeletal muscle tissue.

38. An sgRNA molecule identified by any one of sequence numbers 103 to 105.

39. A pharmaceutical composition comprising a CRISPR-Cas9 complex, wherein the CRISPR-Cas9 complex comprises a nuclease-inactive Cas9 protein linked to an activator protein, and an sgRNA specific to a regulatory sequence that controls or affects a wild-type TTN allele that produces a functional titin protein gene product.

40. The pharmaceutical composition according to claim 39, wherein the nuclease-inactive Cas9 protein linked to the activator protein is dCas9-VPR, dCas9-VP64, dCas9-SunTag, or dCas9-SAM.

41. The pharmaceutical composition according to claim 39 or 40, wherein the regulatory sequence for TTN gene expression is located within the wild-type TTN allele gene promoter region.

42. The pharmaceutical composition according to claim 39 or 40, wherein the regulatory sequence for TTN gene expression is located within the wild-type TTN allele gene enhancer region.

43. The pharmaceutical composition according to claim 41, wherein the guide RNA targeting the wild-type TTN allele gene promoter region or the wild-type TTN allele gene enhancer region is an sgRNA having a sequence identified by any one of SEQ ID NOs: 103 to 105.

44. The pharmaceutical composition according to claim 42, wherein the guide RNA targeting the wild-type TTN allele gene promoter region or the wild-type TTN allele gene enhancer region is an sgRNA having a sequence identified by any one of SEQ ID NOs: 103 to 105.

45. The pharmaceutical composition according to claim 39 or 40, wherein the CRISPR-Cas9 complex delivered to cardiac tissue or skeletal muscle tissue is delivered by one or more expression constructs encoding a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific to a regulatory sequence relating to a wild-type TTN allele that produces a functional titin protein gene product.

46. The pharmaceutical composition according to claim 45, wherein the CRISPR-Cas9 complex or one or more expression constructs are constructed for delivery to cardiac tissue or skeletal muscle tissue.

47. The pharmaceutical composition according to claim 39, comprising a guide RNA molecule identified by any one of sequence numbers 103 to 105.

48. A CRISPR-Cas9 complex comprising a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific to a regulatory sequence related to TTN gene expression.

49. The CRISPR-Cas9 complex according to claim 48, constructed for delivery to cardiac tissue or skeletal muscle tissue.

50. A CRISPR-Cas9 complex comprising a nuclease-inactive Cas9 protein linked to an activator protein and a guide RNA specific to a regulatory sequence related to a wild-type TTN allele that produces a functional titin protein gene product.

51. The CRISPR-Cas9 complex according to claim 50, which is constructed for delivery to cardiac tissue or skeletal muscle tissue.

Images