Long-term therapeutic efficacy and safety profiles of hpCas13d RNA editing in treating early-onset hypertrophic cardiomyopathy

Through evolving a precise RNA nuclease, hpCas13d, we have successfully inhibited hypertrophic cardiomyopathy in a compound heterozygous model. However, further investigation is needed to assess the long-term therapeutic effects and safety profiles of hpCas13d treatment. AAV-hpCas13d[RQ] was subcutaneously injected into neonatal Myh6 RH/RQ mice. Sequential echocardiography analyses were conducted at 4 months and 12 months to evaluate the sustained therapeutic effects of hpCas13d. Electrocardiography was employed to assess cardiac arrhythmias, and mice were euthanized at 12 months. Quantification of Myh6 RQ degradation induced by hpCas13d[RQ] was performed using digital droplet PCR and cDNA sequencing. Histological analysis, RNA sequencing, and proteomic analyses were utilized to examine the inhibitory effects on pathological phenotypes and downstream signaling pathways. Biodistribution, tissue damage, and host immune response to AAV-hpCas13d[RQ] were assessed to evaluate long-term safety profiles. The allele-specific RNA degradation persisted for 12 months in AAV-hpCas13d[RQ]-treated Myh6 RH/RQ mice. Partial degradation of pathogenic Myh6 RQ transcripts proved adequate for the long-term inhibition of cardiac hypertrophy, arrhythmias, fibrosis, and cellular apoptosis in Myh6 RH/RQ mice. RNA sequencing and proteomic analyses revealed that hpCas13d[RQ] treatment impeded hypertrophy and fibrosis, mitochondrial dysfunction, and abnormalities in ion channels downstream of mutant Myh6. Prolonged treatment with AAV-hpCas13d from the neonatal stage did not induce significant tissue damage, liver toxicity, humoral responses, or cellular immune reactions against the AAV9 capsid and bacterial hpCas13d. These results underscore the promising translational potential of AAV-hpCas13d in treating cardiovascular diseases and advancing in vivo gene therapy. • AAV-hpCas13d[RQ] enables long-term RNA degradation with high precision. • AAV-hpCas13d[RQ] inhibits cardiac hypertrophy and arrythmias long-term. • AAV-hpCas13d[RQ] treatment maintained transcriptional and proteomics homeostasis. • Sustained expression of hpCas13d results in no obvious tissue damage.