Adenine base editing of the DUX4 polyadenylation signal for targeted genetic therapy in facioscapulohumeral muscular dystrophy

Darina Šikrová,Vlad A Cadar,Yavuz Ariyurek,Jeroen F.J Laros,Judit Balog,Silvère M Van Der Maarel

doi:10.1016/j.omtn.2021.05.020

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation of the D4Z4 repeat resulting in misexpression of the D4Z4-encoded DUX4 gene in skeletal muscle. One of the key genetic requirements for the stable production of full-length DUX4 mRNA in skeletal muscle is a functional polyadenylation signal (ATTAAA) in exon three of DUX4 that is used in somatic cells. Base editors hold great promise to treat DNA lesions underlying genetic diseases through their ability to carry out specific and rapid nucleotide mutagenesis even in postmitotic cells such as skeletal muscle. In this study, we present a simple and straightforward strategy for mutagenesis of the somatic DUX4 polyadenylation signal by adenine base editing in immortalized myoblasts derived from independent FSHD-affected individuals. We show that mutating this critical cis-regulatory element results in downregulation of DUX4 mRNA and its direct transcriptional target genes. Our findings identify the somatic DUX4 polyadenylation signal as a therapeutic target and represent the first step toward clinical application of the CRISPR-Cas9 base editing platform for FSHD gene therapy.

Highlights

Validation of single guide RNA (sgRNA) targeting DUX4 polyadenylation signal in HAP1 cells In myonuclei, the Facioscapulohumeral muscular dystrophy (FSHD) disease gene DUX4 is transcribed from the distal unit of the D4Z4 repeat on the 4qA subtelomere, where its transcripts are stabilized by a PAS in exon 3
The adjacent SpCas[9] protospacer adjacent motif (PAM) site (TGG) downstream of this PAS allows for the design of an single guide RNA that places the last three adenines of the DUX4 PAS (ATTAAA) in the activity window of nSpABE (Figure 1A)
We did not detect editing of adenines at positions 5 to 7 (A5-7) despite these adenines still fitting into the reported activity window of nSpABE7.10.35 In nSpABEmax-transfected cells, we achieved more efficient adenine base editing at A4 (36.5% ± 3.8%) as well as at downstream adenines A5 (22.5% ± 2.25%) and A6 (7.3% ± 3.6%), which is in agreement with a previous report that nSpABEmax is superior to nSpABE7.10 in terms of editing efficiency and processivity.[40]

Summary

Introduction

DUX4 derepression is caused by a malfunction of D4Z4 chromatin modifiers.[16,17,18] Most FSHD2 individuals can be explained by heterozygous mutations in the gene encoding for the Structural Maintenance of Chromosomes flexible Hinge Domain-Containing protein 1 (SMCHD1),[17] a protein involved in, among other pathways, epigenetic inactivation of the X chromosome in mammals.[19,20,21,22,23] A small number of SMCHD1 mutation-negative FSHD2 families have been reported in which mutations in the genes encoding for the chromatin modifiers DNA Methyltransferase 3B (DNMT3B) or Ligand Dependent Nuclear Receptor Interacting Factor 1 (LRIF1) were shown to cause D4Z4 chromatin relaxation and DUX4 expression in skeletal muscle.[16,18]

Objectives

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Results

Conclusion