A New Kid on the Playground of CRISPR DMD Therapy.

Abstract

DUCHENNE MUSCULAR DYSTROPHY (DMD) is the most common lethal muscle disease, affecting approximately 250,000 boys worldwide. The disease is caused by mutations in the dystrophin gene. Genetic approaches that can repair or replace the mutated gene may radically change the disease course and improve quality of life. Several mechanistically distinctive types of genetic manipulation strategies are currently being explored for treating DMD. These include small molecule read-through of the nonsense stop codon, antisense oligonucleotide–mediated exon skipping of the RNA transcript, adeno-associated virus (AAV)–mediated gene replacement with a <4-kb microdystrophin gene and dual-AAV–mediated 6to 8-kb minidystrophin gene therapy, transplantation of heterologous or genetically corrected autologous muscle stem cells, and clustered regularly interspaced short palindromic repeats (CRISPR)–mediated genome editing. Readthrough strategy targets the translation step, and it only works for a sub-population of patients. Exonskipping targets splicing and has to be designed personally for the specific mutation. Both readthrough and exon-skipping treatments require repeated administration in order to achieve therapeutic benefits. One read-through drug has been approved inEurope, and one exon-skipping drughas been approved in the United States. Dual AAV minidystrophin therapy has the potential to deliver a genetically optimized minigene that is derived from a naturally existing therapeutic gene in mildly affected Becker muscular dystrophy patients. Success has been achieved in the mouse model of DMD by local and systemic delivery. AAV microgene therapy delivers a synthetic, highly abbreviated gene that encodes a protein about one-third the size of full-length dystrophin. Systemic microgene therapy has been conducted in the mouse and dog models, and a human trial is slotted for later this year. Preclinical studies suggest that a single intravenous injection of an AAV microgene vector may provide lifelong protection in rodents. CRISPR therapy is a new type of therapy that has emerged in the last few years. It can remove the mutation from the genome. CRISPR therapy has two major components: an endonuclease called CRISPR-associated protein (Cas) and a guide RNA (gRNA) that directs the Cas to the target site for genome cutting. The Cas protein can be divided into two classes and five types. Up to now, CRISPR therapy is mainly based on Cas9, a class 2, type II Cas protein. A flurry of papers published in the last 3 years have established the proof of principle for CRISPR DMD therapy using Cas9. Collectively, these studies show effective editing of patient cells in vitro and mouse cells in vivo. Of high relevance to the development of CRISPR as a therapeutic modality for DMD, several groups delivered the gRNA and Cas9 expression cassette with AAV in mouse models of DMD. Encouragingly, treatment resulted in excellent restoration of dystrophin expression in skeletal muscle and the heart by immunostaining and western blot analysis. Physiological assays also demonstrated improvement of skeletal muscle function. A large collection of Cas endonucleases was discovered in the last couple of years. Many of these newly emerged Cas proteins are capable of genome editing in eukaryotic cells. They represent a rich mine for CRISPR therapy. The unique properties of different Cas proteins offer unlimited opportunities to meet different therapeutic needs.