**4. Genotype-targeted therapies**

By directly targeting at specific pathogenic mutations causing DCM, genotypetargeted genetic approaches have emerged as promising strategies for effective and individualized therapies to ameliorate disease phenotypes. To carry out genotypebased therapy, many customized genetic strategies were explored. For examples: for loss-of-function mutations that cause reduced or insufficient protein levels, the straightforward gene replacement strategy can be employed. In this scenario, full-length or partial functional cDNA for corresponding mutated gene can be transferred to cardiac tissue to supplement the reduced gene dosage using appropriate gene delivery approach. For mutations that cause dominant-negative effects on a particular gene, exon-skipping or trans-splicing approaches can be considered to remove or modify the mutant transcripts. For pathogenic mutations that cause other protein dysfunction, the highly efficient CRISPRA/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease) system can be explored to directly correct the mutant variants. In addition, other genotypebased therapeutic strategies such as manipulation of the downstream pathways evoked by specific DCM mutations were also explored. Below, we summarize several examples of gene-targeted therapeutic strategies that produced encouraging results in the treatment of DCM (**Table 2**), which hold the opportunities to ultimately improve patient outcome in the future.

#### **4.1 Dystrophin mutations-targeted gene therapies**

Mutations in the dystrophin (*DMD*) gene cause muscular dystrophy patients and X-linked familial DCM in early childhood and patients often die of cardiac and respiratory failure [44]. Currently, there are no effective cures for DMD yet and management

**111**

*Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic…*

of DMD mostly focuses on preserving the limited muscle strength and ameliorating disease symptoms [64]. Mechanistically, dystrophin protein deficiency due to *DMD* gene mutations is the primary cause of DMD and subsequent DCM and heart failure. And the disease severity is mostly correlated with the dystrophin protein level. Thus, a plausible treatment strategy would be to restore the expression level of dystrophin protein [44]. To cure this devastating disease, dystrophin gene-targeted therapeutic strategies such as gene replacement, exon skipping and CRISPRA/Cas9 genome editing techniques are mostly employed, which have shown encouraging results in restoration of dystrophin protein expression and recovery of dystrophin protein function in both

Gene replacement strategy involves delivering a functioning gene to replace or supplement the mutant gene to the target organ and cells to ease the disease phenotype caused by genetic mutation. This approach often utilizes the adeno-associated virus (AAV) as a vector to mediate gene transfection into the skeletal and cardiac muscle cells. However, as the dystrophin gene has 79 exons and its transcript is about 14 Kb, its size is too large to fit in currently available gene construction vector for gene transfection. Alternatively, a mini- or micro- dystrophin gene coding for a functionally similar to dystrophin but smaller in size was thus used. The authors Wang and colleagues firstly defined the minimal functional region of the dystrophin protein, later referred as mini-dystrophin. The authors then packed this min-dystrophin gene into an AAV vector to mediate muscle transfection and demonstrated the effectiveness of gene delivery and restoration of dystrophin gene function [65]. After this study, several other independent groups latter confirmed the effectiveness of a shortened albeit functional dystrophin gene replacement strategy mediated by the AAV gene delivery system in preservation of cardiac and skeletal muscle function and extending the lifespan in the dystrophic mice [66, 67]. Notably, further studies in animal models of muscular dystrophy and human patients detected certain immunologic responses to the shortened dystrophin peptide, which need to be carefully considered

Exon skipping is an RNA-based splice-switching approach that causes cells to "skip" the exon containing the pathogenic variant, resultant in-frame transcripts that produce a shorter peptide, albeit still at least partially functional protein to ameliorate disease phenotypes. This approach was initially developed to mask the pathogenic mutations containing exon 51 using an antisense oligonucleotide (AON), resulting in genetic correction of the open reading frame of the *DMD* gene and partial restoration of dystrophin protein expression and improvement of muscle pathology and function [71–73]. These encouraging studies lead to at least two ongoing clinical trials to evaluate the efficacy and safety of this exon skipping approach targeting to the pathogenic exon 51 or 53 (NCT02255552, NCT02310906). With the revolutionary development of the CRISPRA/Cas9 genome editing technologies that enable precise and efficient genetic modifications from single-nucleotide alternation, insertion of gene of interest to deletion of chromosomal regions, numerous studies have demonstrated the feasibility and high efficiency of this technique in restoration dystrophin expression and function in skeletal and cardiac muscle, both in vitro and in vivo [74, 75]. For example, a recent study carried out by El Refaey and colleagues packaged SaCas9 (Cas9 from *Staphylococcus aureus*)/gRNA constructs into an AAV serotype rh74 and delivered it to the mdx/Utr+/− dystrophic mice through a retro-orbital approach [76]. The authors showed this CRIPRA-mediated genome editing strategy could efficiently excise the mutant exon 23 of dystrophin in the mice model, resulting in the restoration of the dystrophin protein expression and cardiac myofiber architecture, and significantly improved the cardiac contractility in vivo. A follow up study using this AAV-mediated CRISPR genome editing approach found that restored dystrophin expression and improved cardiac function were consistently

*DOI: http://dx.doi.org/10.5772/intechopen.94434*

animal models and clinical trials.

in future clinical application [68–70].

#### *Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94434*

of DMD mostly focuses on preserving the limited muscle strength and ameliorating disease symptoms [64]. Mechanistically, dystrophin protein deficiency due to *DMD* gene mutations is the primary cause of DMD and subsequent DCM and heart failure. And the disease severity is mostly correlated with the dystrophin protein level. Thus, a plausible treatment strategy would be to restore the expression level of dystrophin protein [44]. To cure this devastating disease, dystrophin gene-targeted therapeutic strategies such as gene replacement, exon skipping and CRISPRA/Cas9 genome editing techniques are mostly employed, which have shown encouraging results in restoration of dystrophin protein expression and recovery of dystrophin protein function in both animal models and clinical trials.

Gene replacement strategy involves delivering a functioning gene to replace or supplement the mutant gene to the target organ and cells to ease the disease phenotype caused by genetic mutation. This approach often utilizes the adeno-associated virus (AAV) as a vector to mediate gene transfection into the skeletal and cardiac muscle cells. However, as the dystrophin gene has 79 exons and its transcript is about 14 Kb, its size is too large to fit in currently available gene construction vector for gene transfection. Alternatively, a mini- or micro- dystrophin gene coding for a functionally similar to dystrophin but smaller in size was thus used. The authors Wang and colleagues firstly defined the minimal functional region of the dystrophin protein, later referred as mini-dystrophin. The authors then packed this min-dystrophin gene into an AAV vector to mediate muscle transfection and demonstrated the effectiveness of gene delivery and restoration of dystrophin gene function [65]. After this study, several other independent groups latter confirmed the effectiveness of a shortened albeit functional dystrophin gene replacement strategy mediated by the AAV gene delivery system in preservation of cardiac and skeletal muscle function and extending the lifespan in the dystrophic mice [66, 67]. Notably, further studies in animal models of muscular dystrophy and human patients detected certain immunologic responses to the shortened dystrophin peptide, which need to be carefully considered in future clinical application [68–70].

Exon skipping is an RNA-based splice-switching approach that causes cells to "skip" the exon containing the pathogenic variant, resultant in-frame transcripts that produce a shorter peptide, albeit still at least partially functional protein to ameliorate disease phenotypes. This approach was initially developed to mask the pathogenic mutations containing exon 51 using an antisense oligonucleotide (AON), resulting in genetic correction of the open reading frame of the *DMD* gene and partial restoration of dystrophin protein expression and improvement of muscle pathology and function [71–73]. These encouraging studies lead to at least two ongoing clinical trials to evaluate the efficacy and safety of this exon skipping approach targeting to the pathogenic exon 51 or 53 (NCT02255552, NCT02310906).

With the revolutionary development of the CRISPRA/Cas9 genome editing technologies that enable precise and efficient genetic modifications from single-nucleotide alternation, insertion of gene of interest to deletion of chromosomal regions, numerous studies have demonstrated the feasibility and high efficiency of this technique in restoration dystrophin expression and function in skeletal and cardiac muscle, both in vitro and in vivo [74, 75]. For example, a recent study carried out by El Refaey and colleagues packaged SaCas9 (Cas9 from *Staphylococcus aureus*)/gRNA constructs into an AAV serotype rh74 and delivered it to the mdx/Utr+/− dystrophic mice through a retro-orbital approach [76]. The authors showed this CRIPRA-mediated genome editing strategy could efficiently excise the mutant exon 23 of dystrophin in the mice model, resulting in the restoration of the dystrophin protein expression and cardiac myofiber architecture, and significantly improved the cardiac contractility in vivo. A follow up study using this AAV-mediated CRISPR genome editing approach found that restored dystrophin expression and improved cardiac function were consistently

*Cardiac Diseases - Novel Aspects of Cardiac Risk, Cardiorenal Pathology and Cardiac Interventions*

AV blocker as significant risk factors for disease malignancy [61, 62]. Because of this well-determined genotype–phenotype knowledge, an actionable prognostic genotype–phenotype association, implementation of a lower threshold and earlier implantable cardioverter defibrillator (ICD) therapy than current guidelines recommend in patients with LMNA mutations, was demonstrated to significantly

Based on the Genetic Testing Registry (https://www.ncbi.nlm.nih.gov/gtr/), there are from 40 to 80 genes included in the testing panels for most commercially available genetic testing for DCM. In familial DCM, the yield of genetic testing, resulting in identification of pathogenic mutations, can thus far reach up to 40%, in a comparable level to that of other inherited cardiac disorders such as HCM and long QT syndrome. The sensitivity of genetic testing is compromised partially due to that not all genes implicated in DCM are included in the gene panels for testing. This is especially the case for those identified from sporadic DCM cases or single family. Furthermore, limited by human genetics approaches that heavily rely on pedigree availability and candidate gene approaches, variants for more than half of familial DCM cases have not been identified yet. As technique advances in genetics of cardiomyopathy, identification of the remaining genetic causes in inherited DCM cases and elucidation of the underlying pathogenic mechanisms leading to the phenotype are evolving rapidly. Targeted gene panels for genetic testing are increasing in an unprecedented scale. With further characterization and functional validation, the ever expanding gene panels of genetic testing promise to increase the rate of positive identification and provide individuals and families with a more

By directly targeting at specific pathogenic mutations causing DCM, genotypetargeted genetic approaches have emerged as promising strategies for effective and individualized therapies to ameliorate disease phenotypes. To carry out genotypebased therapy, many customized genetic strategies were explored. For examples: for loss-of-function mutations that cause reduced or insufficient protein levels, the straightforward gene replacement strategy can be employed. In this scenario, full-length or partial functional cDNA for corresponding mutated gene can be transferred to cardiac tissue to supplement the reduced gene dosage using appropriate gene delivery approach. For mutations that cause dominant-negative effects on a particular gene, exon-skipping or trans-splicing approaches can be considered to remove or modify the mutant transcripts. For pathogenic mutations that cause other protein dysfunction, the highly efficient CRISPRA/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease) system can be explored to directly correct the mutant variants. In addition, other genotypebased therapeutic strategies such as manipulation of the downstream pathways evoked by specific DCM mutations were also explored. Below, we summarize several examples of gene-targeted therapeutic strategies that produced encouraging results in the treatment of DCM (**Table 2**), which hold the opportunities to ulti-

Mutations in the dystrophin (*DMD*) gene cause muscular dystrophy patients and X-linked familial DCM in early childhood and patients often die of cardiac and respiratory failure [44]. Currently, there are no effective cures for DMD yet and management

improve patient outcome and survival [63].

comprehensive and conclusive genetic testing.

mately improve patient outcome in the future.

**4.1 Dystrophin mutations-targeted gene therapies**

**4. Genotype-targeted therapies**

**110**

detected at up to 19 months [77]. Similarly, Hakim and colleagues modified the dose of AAV.SaCas9 and AAV-9.gRNA vector that directs Cas9 to introns of 22 and 23 and performed the AAV-9 CRISPRA gene therapy in the mdx mice that carry a nonsense mutation in exon 23 of the dystrophin gene [78, 79]. The authors found significantly increased dystrophin restoration and reduced fibrosis in both skeletal and cardiac muscle and improved muscle function and cardiac hemodynamics at up to 18 months.

### **4.2** *LMNA* **mutations-tailored therapeutic strategy**

A genetic approach referred as trans-splicing approach was initially carried out to correct *LMNA* mutations in the *Lmna* DelK32 knock-in mouse harboring a *LMNA*-related congenital muscular dystrophy mutation in the exon 1 [80, 81]. The *Lmna* DelK32/DelK32 mice exhibited severe muscular and cardiac defects and early premature death. The trans-splicing approach allows converting the targeted endogenous mutated pre-mRNA to a therapeutic pre–trans-spliced molecule containing the wildtype coding sequence, resulting in a processed mRNA transcript devoid of the exon harboring the pathogenic variant [82, 83]. To perform the trans-splicing rescue in the *Lmna* DelK32 knock-in mouse model, 5'-RNA pre-trans-splicing molecules containing the first five exons of *Lmna* and targeting intron 5 of *Lmna* pre-mRNA were firstly developed. After confirmed the induced trans-splicing events on endogenous *Lmna* mRNA in vitro, AAV mediated delivery system was then evaluated in vivo in the newborn mice. Similarly, trans-splicing events were successfully detected in both skeletal and heart muscle of mice up to 50 days after AAV delivery. However, the tans-splicing occurring efficiency needs to be further improved to exert significant rescue effects on cardiac function and premature death.

To target at the *LMNA* pathogenic variants resulting in deficiency of the protein level, another strategy referred as stop codon readthrough was explored through inactivating the molecules participating in nonsense-mediated decay at the RNA level [84]. This approach applies to the scenario when pathogenic variants result in frameshifts and premature stops, subsequently leading to protein deficiency because of increased nonsense-mediated decay of mutant RNAs. PTC124, also known as ataluren, is a chemical compound that can selectively induce ribosomal readthrough of premature but not normal termination codons [85]. PTC124 was tested in iPSC-CMs derived from patients carrying different nonsense and frameshift mutations in the *LMNA* gene [85]. In one of the three frameshift mutants tested, administration of PTC124 significantly increased the translation of full-length LMNA protein and partially restored the protein function, as shown by reduced cardiomyocyte apoptosis and improved excitationcontraction coupling [84].

#### **4.3** *TTN* **truncating variants-targeted gene therapy**

While the pathogenicity of *TTN* missense mutations to DCM remains largely undetermined, *TTN* truncating variants (*TTNtvs*) are the most common cause to DCM. To date, experimental evidence generated largely from rodent animal models, human induced pluripotent stem cell-derived cardiomyocyte, and patient tissues mostly support the mechanism of titin protein haploinsufficiency. Correction of the titin protein deficiency by using traditional viral-mediated gene replacement strategy to increase the expression of titin protein, just as that has been successfully employed to restore the dystrophin deficiency, however, is challenging. Because the *TTN* gene spans 294 Kb of genomic region and its spliced transcript is more than 100 Kb, a size that is way bigger than any currently available cargo capacity for AAV

**113**

capability.

*Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic…*

mediated gene transfection. Alternatively, exon skipping, a genetic approach that had been initially employed to treat the DMD, through using an AON to mask the pathogenic mutations containing exon 51 or 53 and restoration of DMD phenotype [72], as aforementioned, was explored to treat *TTNtvs* related DCM. Notably, this strategy had been also successfully utilized for treating Mybpc3 mutations caused

To employ exon skipping strategy for treating *TTNtvs* caused DCM, Gramlich and colleagues targeted to the Ser14450fsX4 variant located in the exon 326 identified in a DCM patient that caused autosomal-dominant truncating frameshift mutation [87]. They used an AON-mediated exon skipping approach to remove the exon 326 in both patient cardiomyocytes in vitro and mouse heart in vivo. In vitro, skipping of the exon 326 containing the Ser14450fsX4 pathogenic variant restored the impaired myofibril assembly and stabilized its structural integrity and normalized expression of sarcomeric protein in the patient-specific cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) model. Furthermore, in the corresponding Ttn knock-in mice model, skipping of the exon 326 significantly improved sarcomere formation and contractile function and prevented subsequent development of DCM phenotype. These results provide strong evidence to support that the RNA-based exon skipping strategy could be a potential treatment option for DCM caused by gene variants that are otherwise technically difficult to be

Mutations in the δ- sarcoglycan coding gene (*SGCD*) can lead to DCM without

An earlier version of genome editing technique referred as transcription activatoraffected nuclease (TALEN) was explored to directly correct the *PLN* R14del mutation that is associated with high risk for malignant ventricular arrhythmias and end-stage heart failure in DCM patient carriers [89]. The authors Karakikes and colleague firstly derived iPSC-CMs from a DCM patient harboring the *PLN* R14del mutation, followed by detailed phenotypic characterization of this iPSC-CMs model. Next, the authors made the effort to correct the *PLN* R14del mutation in the iPSC-CMs model using TALEN gene editing strategy. This approach successfully corrected the R14del mutation and attenuated the R14del-associated disease phenotypes in the iPSC-CMs model. Furthermore, the authors engineered an AAV6 vector that enabled knock-down the endogenous *PLN*, while simultaneously overexpress a codon-optimized *PLN*, to effectively reverse the disease phenotype in the iPSC-CMs model as well. Thus, this study provides another successful example to using genome editing approach to directly target at the pathogenic mutations associated with DCM

obvious involvement of skeletal muscle disorders [57]. The gene replacement strategy was firstly applied in the treatment of DCM caused by a deletion mutation in the *Sgcd* gene that disrupted the dystrophin-associated glycoprotein complex in a hamster model [88]. The authors Kawada and colleague constructed a recombinant AAV vector containing the full-length cDNA of *Sgcd* gene driven under the cardiac specific expression promoter of CMV and intramurally injected it to the *Sgcd* mutation induced DCM model in the hamster*. They detected* robust expression of the *Sgcd* gene and other types of sarcoglycans as well in the transfected myocardium. The restored expression of *Sgcd* and other sarcoglycans normalized the diameter of transduced cardiomyocytes, improved the contractile function and ultimately prolonged the life span of the animals harboring the *Sgcd* gene mutation. This study provides one of the earliest evidence to employ gene replacement strategy to treat DCM caused by a specific gene mutation with efficient and sustained transfection

*DOI: http://dx.doi.org/10.5772/intechopen.94434*

HCM in a knock-in mouse model [86].

delivered due to size limitation for gene replacement therapy.

**4.4 Genotype-targeted therapies for other DCM genes**

*Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94434*

mediated gene transfection. Alternatively, exon skipping, a genetic approach that had been initially employed to treat the DMD, through using an AON to mask the pathogenic mutations containing exon 51 or 53 and restoration of DMD phenotype [72], as aforementioned, was explored to treat *TTNtvs* related DCM. Notably, this strategy had been also successfully utilized for treating Mybpc3 mutations caused HCM in a knock-in mouse model [86].

To employ exon skipping strategy for treating *TTNtvs* caused DCM, Gramlich and colleagues targeted to the Ser14450fsX4 variant located in the exon 326 identified in a DCM patient that caused autosomal-dominant truncating frameshift mutation [87]. They used an AON-mediated exon skipping approach to remove the exon 326 in both patient cardiomyocytes in vitro and mouse heart in vivo. In vitro, skipping of the exon 326 containing the Ser14450fsX4 pathogenic variant restored the impaired myofibril assembly and stabilized its structural integrity and normalized expression of sarcomeric protein in the patient-specific cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) model. Furthermore, in the corresponding Ttn knock-in mice model, skipping of the exon 326 significantly improved sarcomere formation and contractile function and prevented subsequent development of DCM phenotype. These results provide strong evidence to support that the RNA-based exon skipping strategy could be a potential treatment option for DCM caused by gene variants that are otherwise technically difficult to be delivered due to size limitation for gene replacement therapy.

#### **4.4 Genotype-targeted therapies for other DCM genes**

Mutations in the δ- sarcoglycan coding gene (*SGCD*) can lead to DCM without obvious involvement of skeletal muscle disorders [57]. The gene replacement strategy was firstly applied in the treatment of DCM caused by a deletion mutation in the *Sgcd* gene that disrupted the dystrophin-associated glycoprotein complex in a hamster model [88]. The authors Kawada and colleague constructed a recombinant AAV vector containing the full-length cDNA of *Sgcd* gene driven under the cardiac specific expression promoter of CMV and intramurally injected it to the *Sgcd* mutation induced DCM model in the hamster*. They detected* robust expression of the *Sgcd* gene and other types of sarcoglycans as well in the transfected myocardium. The restored expression of *Sgcd* and other sarcoglycans normalized the diameter of transduced cardiomyocytes, improved the contractile function and ultimately prolonged the life span of the animals harboring the *Sgcd* gene mutation. This study provides one of the earliest evidence to employ gene replacement strategy to treat DCM caused by a specific gene mutation with efficient and sustained transfection capability.

An earlier version of genome editing technique referred as transcription activatoraffected nuclease (TALEN) was explored to directly correct the *PLN* R14del mutation that is associated with high risk for malignant ventricular arrhythmias and end-stage heart failure in DCM patient carriers [89]. The authors Karakikes and colleague firstly derived iPSC-CMs from a DCM patient harboring the *PLN* R14del mutation, followed by detailed phenotypic characterization of this iPSC-CMs model. Next, the authors made the effort to correct the *PLN* R14del mutation in the iPSC-CMs model using TALEN gene editing strategy. This approach successfully corrected the R14del mutation and attenuated the R14del-associated disease phenotypes in the iPSC-CMs model. Furthermore, the authors engineered an AAV6 vector that enabled knock-down the endogenous *PLN*, while simultaneously overexpress a codon-optimized *PLN*, to effectively reverse the disease phenotype in the iPSC-CMs model as well. Thus, this study provides another successful example to using genome editing approach to directly target at the pathogenic mutations associated with DCM

*Cardiac Diseases - Novel Aspects of Cardiac Risk, Cardiorenal Pathology and Cardiac Interventions*

detected at up to 19 months [77]. Similarly, Hakim and colleagues modified the dose of AAV.SaCas9 and AAV-9.gRNA vector that directs Cas9 to introns of 22 and 23 and performed the AAV-9 CRISPRA gene therapy in the mdx mice that carry a nonsense mutation in exon 23 of the dystrophin gene [78, 79]. The authors found significantly increased dystrophin restoration and reduced fibrosis in both skeletal and cardiac muscle and improved muscle function and cardiac hemodynamics at up to 18 months.

A genetic approach referred as trans-splicing approach was initially carried out to correct *LMNA* mutations in the *Lmna* DelK32 knock-in mouse harboring a *LMNA*-related congenital muscular dystrophy mutation in the exon 1 [80, 81]. The *Lmna* DelK32/DelK32 mice exhibited severe muscular and cardiac defects and early premature death. The trans-splicing approach allows converting the targeted endogenous mutated pre-mRNA to a therapeutic pre–trans-spliced molecule containing the wildtype coding sequence, resulting in a processed mRNA transcript devoid of the exon harboring the pathogenic variant [82, 83]. To perform the trans-splicing rescue in the *Lmna* DelK32 knock-in mouse model, 5'-RNA pre-trans-splicing molecules containing the first five exons of *Lmna* and targeting intron 5 of *Lmna* pre-mRNA were firstly developed. After confirmed the induced trans-splicing events on endogenous *Lmna* mRNA in vitro, AAV mediated delivery system was then evaluated in vivo in the newborn mice. Similarly, trans-splicing events were successfully detected in both skeletal and heart muscle of mice up to 50 days after AAV delivery. However, the tans-splicing occurring efficiency needs to be further improved to exert significant rescue effects on cardiac function and

To target at the *LMNA* pathogenic variants resulting in deficiency of the protein level, another strategy referred as stop codon readthrough was explored through inactivating the molecules participating in nonsense-mediated decay at the RNA level [84]. This approach applies to the scenario when pathogenic variants result in frameshifts and premature stops, subsequently leading to protein deficiency because of increased nonsense-mediated decay of mutant RNAs. PTC124, also known as ataluren, is a chemical compound that can selectively induce ribosomal readthrough of premature but not normal termination codons [85]. PTC124 was tested in iPSC-CMs derived from patients carrying different nonsense and frameshift mutations in the *LMNA* gene [85]. In one of the three frameshift mutants tested, administration of PTC124 significantly increased the translation of full-length LMNA protein and partially restored the protein function, as shown by reduced cardiomyocyte apoptosis and improved excitation-

While the pathogenicity of *TTN* missense mutations to DCM remains largely undetermined, *TTN* truncating variants (*TTNtvs*) are the most common cause to DCM. To date, experimental evidence generated largely from rodent animal models, human induced pluripotent stem cell-derived cardiomyocyte, and patient tissues mostly support the mechanism of titin protein haploinsufficiency. Correction of the titin protein deficiency by using traditional viral-mediated gene replacement strategy to increase the expression of titin protein, just as that has been successfully employed to restore the dystrophin deficiency, however, is challenging. Because the *TTN* gene spans 294 Kb of genomic region and its spliced transcript is more than 100 Kb, a size that is way bigger than any currently available cargo capacity for AAV

**4.2** *LMNA* **mutations-tailored therapeutic strategy**

**112**

premature death.

contraction coupling [84].

**4.3** *TTN* **truncating variants-targeted gene therapy**

to achieve therapeutic benefit. Notably, while it provides evidence to support the effectiveness of genome editing technique to correct pathogenic variants causing DCM in vitro, germline correction of embryos harboring DCM variants had not been reported yet.
