Key result
CRISPR activation increases titin levels ~2-fold and rescues contractile function in TTN-truncation DCM models.
Why the study?
Therapeutics targeting TTN truncation variants in dilated cardiomyopathy have been elusive due to TTN size, variant rarity, and incomplete knowledge of pathogenicity.
This preclinical study demonstrates that using CRISPR activation to address haploinsufficiency reverses the structural and functional deficits caused by TTN truncation variants, suggesting a potential future therapeutic strategy for this genetic form of dilated cardiomyopathy.
Answers are grounded in this paper and cite their sources.
No immediate clinical use; leaves open CRISPR activation as a hypothesis-generating strategy for TTN-DCM pending human translation.
BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity. METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy–associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts. CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.
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Ghahremani et al. (2024) studied Dilated cardiomyopathy associated with TTN truncation variants. CRISPR activation (dCas9-VPR) vs. Non-targeting control gRNA was evaluated on TTN protein levels, sarcomere content, and contractile function. CRISPR activation of TTN increased TTN protein levels by 2-3x, which normalized sarcomere content and rescued contractile functional deficits in models of TTN truncation variant dilated cardiomyopathy.
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