CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants

Area of Science:

• Cardiovascular Genetics
• Gene Therapy
• Molecular Cardiology

Background:

• Truncating variants in the TTN gene (TTNtvs) are the leading genetic cause of dilated cardiomyopathy, a severe heart condition.
• TTNtvs lead to reduced functional titin protein, impacting sarcomere integrity and cardiac function, with limited therapeutic options.
• Challenges in developing therapies include the large size of the TTN gene, the diversity of TTNtvs, and incomplete understanding of their pathogenicity.

Purpose of the Study:

• To adapt CRISPR activation technology for functional interrogation of TTNtv pathogenicity.
• To develop a potential therapeutic strategy for dilated cardiomyopathy caused by TTNtvs using CRISPR activation.
• To investigate the molecular consequences and therapeutic efficacy of TTN gene activation in patient-derived cardiac models.

Main Methods:

• Utilized CRISPR activation with dCas9-VPR in human cardiomyocytes and 3D cardiac microtissues derived from induced pluripotent stem cells harboring a TTNtv.
• Employed guide RNA screening with custom TTN reporter assays and agarose gel electrophoresis to quantify TTN protein levels and isoforms.
• Conducted RNA sequencing and cardiomyocyte epigenetic assays to identify molecular effects and nominate regulatory elements for cardiomyocyte-specific TTN activation.

Main Results:

• CRISPR activation of TTN, targeting its promoter or regulatory elements, successfully rescued TTN protein deficits caused by TTNtvs.
• Increased TTN protein levels normalized sarcomere content and cardiac contractile function, even in the presence of elevated truncated TTN protein.
• CRISPR activation upregulated TTN transcripts along with transcripts involved in myofibril assembly and sarcomere structure.

Conclusions:

• CRISPR activation of TTN effectively reversed functional deficits associated with TTNtvs, supporting haploinsufficiency as a key mechanism.
• This study demonstrates the potential of TTN CRISPR activation as a therapeutic strategy for a broad spectrum of TTNtv-related dilated cardiomyopathy.
• The findings pave the way for developing gene-based therapies targeting TTN variants in heart failure.