Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases.

Magdalena Dabrowska,Wlodzimierz J Krzyzosiak,Wojciech Juzwa,Marta Olejniczak

doi:10.3389/fnins.2018.00075

Abstract

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of CAG repeats in the first exon of the huntingtin gene (HTT). The accumulation of polyglutamine-rich huntingtin proteins affects various cellular functions and causes selective degeneration of neurons in the striatum. Therapeutic strategies used to date to silence the expression of mutant HTT include antisense oligonucleotides, RNA interference-based approaches and, recently, genome editing with the CRISPR/Cas9 system. Here, we demonstrate that the CAG repeat tract can be precisely excised from the HTT gene with the use of the paired Cas9 nickase strategy. As a model, we used HD patient-derived fibroblasts with varied numbers of CAG repeats. The repeat excision inactivated the HTT gene and abrogated huntingtin synthesis in a CAG repeat length-independent manner. Because Cas9 nickases are known to be safe and specific, our approach provides an attractive treatment tool for HD that can be extended to other polyQ disorders.

Highlights

Expansions of short tandem repeat sequences in functionally unrelated genes are causative factors of numerous human hereditary neurological diseases
Multiple therapeutic approaches have been described for the treatment of Huntington’s disease (HD) and other polyQ diseases’ these approaches suffer from specific limitations that hinder their introduction to the clinic
It has been shown using RNAi and antisense oligonucleotides that the knockdown of huntingtin, either the mutant or both mutant and normal is beneficial in mouse models of HD (Harper et al, 2005; Boudreau et al, 2009; Kordasiewicz et al, 2012)

Summary

Introduction

Expansions of short tandem repeat sequences in functionally unrelated genes are causative factors of numerous human hereditary neurological diseases. There are nine known neurodegenerative disorders caused by the expansion of CAG repeats within the coding regions of associated genes. These disorders include Huntington’s disease (HD) (Bates et al, 2015); spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 (SCA) (Paulson et al, 2017); spinal-bulbar muscular atrophy (SBMA) (Spada et al, 1991); and dentatorubral-pallidoluysian atrophy (DRPLA) (Koide et al, 1994). Expanded polyglutamine (polyQ) protein may form intracellular aggregates and affects numerous cellular activities inducing pathogenesis via a gain of toxic function. Despite many years of research on an effective treatment method, HD and other polyQ diseases are incurable, and only their symptoms can be controlled. Several different strategies have already been employed in cellular and animal models of polyQ diseases to achieve the desired therapeutic effects

Methods

Results

Conclusion