327. Genetic Editing for Huntington's Disease

Abstract

Huntington's disease (HD) is a neurodegenerative disorder caused by a pathological CAG expansion at the 3’ end of the first exon of the huntingtin gene (HTT). Currently, there is no efficient treatment for HD. Editing of the mutant HTT gene with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system represents a new and promising approach. Recognition of the HTT target sequence by a single-guide RNA sequences (sgRNA) and the Cas9 protein is inducing DNA double-strand breaks (DSB), which activate endogenous cellular repair pathways. Non-homologous end joining (NHEJ) will introduce small insertion/deletion (indel) that alter the reading frame of HTT gene while homologous directed repair (HDR) is activated in the presence of a DNA template. To validate the approach and optimize the delivery of the CRISPR system with viral vectors, we first targeted artificial sequences containing fluorescent reporter genes in HEK 293T cells. An efficient gene disruption was measured and associated with a loss of fluorescence in neurons, astrocytes, in vitro and in vivo. Furthermore, we developed multiple strategies to disrupt the mutant HTT gene. Quantification demonstrated a high rate of indels, leading to a strong reduction of HTT protein in HEK 293T cells, mouse cortical neurons and human iPS-derived neurons. Blocking HTT expression in vitro HD models is improving several physiopathological parameters. We are currently evaluating the impact of allele or non-allele specific mutant HTT editing in human neurons from HD patients. Altogether, these data demonstrate the potential of the CRISPR technology as therapeutic strategy for HD.