Efficient and durable gene activation by Cas9-mediated epigenome editing in vivo

Abstract

Epigenome editing technology holds great promise for treating diverse genetic disorders. While a series of advances has been made on epigenetic silencing using programmable editors, little progress has been made in leveraging epigenetic activation for therapeutic application. Here we demonstrate epigenetic activation of the LAMA1 gene for the treatment of LAMA2-CMD, a severe congenital muscle dystrophy (CMD) caused by biallelic mutations in the LAMA2 gene. LAMA1 is a sister homologue that is known to compensate for the function of LAMA2. However, supplementing LAMA1 or LAMA2 gene via viral platform is not feasible due to the large size of their coding sequences. Through a single administration of our (Adeno-associated virus) AAV vector encoding all the necessary elements for epigenetic activation, a platform termed CRISPR guide-nucleotide directed modulation (GNDM), we observed significant LAMA1 gene upregulation and phenotype improvements in DyW mice, a severe disease model of LAMA2-CMD. Notably, sustained expression of the GNDM gene and subsequent activation of the LAMA1 gene persisted beyond analyzed period of one year despite immune recognition of the GNDM protein by the host immune system. Regulatory T (Treg) cells appeared to facilitate tolerance to GNDM in the transduced muscle tissue. The muscle-tropic AAV capsid exhibited desired vector biodistribution and promising pharmacodynamics with good safety profiles in adult non-human primates (NHPs). Moreover, administration to juvenile NHPs demonstrated superior pharmacodynamics compared to adults, even at half the adult dose, suggesting safer and more effective therapeutic outcomes in mostly pediatric LAMA2-CMD patients. Our approach holds broad applicability for a range of loss-of-function genetic disorders and could offer a therapeutic breakthrough where active epigenome brings clinical benefit.