A New Kid on the Playground of CRISPR DMD Therapy

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.^1,2 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 6- to 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. Read-through strategy targets the translation step, and it only works for a sub-population of patients. Exon-skipping targets splicing and has to be designed personally for the specific mutation. Both read-through and exon-skipping treatments require repeated administration in order to achieve therapeutic benefits. One read-through drug has been approved in Europe, and one exon-skipping drug has been approved in the United States.^3–5 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.^6,7 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.^8–11 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.¹² 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.¹³ 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.^14–24 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.^15–17,21

A large collection of Cas endonucleases was discovered in the last couple of years.^25–28 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. A study published in Science Advances on April 12 explored Cpf1, a class 2 type V Cas protein, for DMD therapy in induced pluripotent stem cells derived from a DMD patient and the mdx mouse model for DMD.²⁹ This is the first report demonstrating Cpf1 editing in a mammalian model of a human disease. Cpf1 creates sticky ends in the genome and favors pyrimidine-rich sequences. The Cpf1 gRNA is simpler than that of Cas9. The use of a different protospacer-adjacent motif sequence by Cpf1 expands versatility to genome editing. The authors showed that Cpf1 editing effectively restored the disrupted reading frame and yielded a near-full-length dystrophin protein. In induced pluripotent stem cells–derived cardiomyocytes, Cpf1 editing normalized the mitochondrial number and rescued the respiration rate. Re-implantation of Cpf1-corrected mdx zygotes in pseudopregnant females yielded mutation-corrected mice with normal muscle histology and improved muscle function. Off-target analysis suggests that Cpf1 is highly specific. It is also worth pointing out that in contrast to the published CRISPR strategy, which uses two gRNAs to direct Cas9 to two separate locations in the genome for mutation removal, Zhang et al. utilized a very creative approach by directing Cpf1 to cut the splicing acceptor site. This allows restoration of dystrophin expression by one cut, instead of two cuts, and hence may greatly improve the efficiency. Taken together, the results reported by Zhang et al. raise a high hope to complement existing Cas9 DMD therapy with Cpf1 (and likely other Cas proteins) for future clinical translation.²⁹

It took about 8 years from the initial description of exon-skipping in mdx myoblasts to the first in-human clinical trial.^30,31 Similar time was taken from the report of the first engineered microdystrophin gene to the AAV-mediated local injection in DMD boys.^32,33 It remains to be seen whether CRISPR DMD therapy can follow the same path or may be on the fast track. However, it should be pointed out that there are significant roadblocks ahead of CRISPR therapy, such as off-target toxicity and the immunogenicity of bacteria-derived Cas protein. In the case of CRISPR DMD therapy, there are additional sets of challenges to overcome; for example: How long will therapy last? Will the immune reaction to the Cas protein eventually eliminate the treated dystrophin-positive muscle cells? Will CRISPR therapy improve heart function? Will gene editing effectively target muscle stem cells? Will AAV CRISPR cause any toxicity following long-term in vivo expression? Will CRISPR editing work in large mammals? For Cpf1, one still has to show that it can be used as a postnatal therapy for muscle diseases using viral and/or nonviral mediated gene transfer.³⁴

Acknowledgments

The author thanks Anna Azvolinsky, Rhonda Bassel-Duby, Shi-jie Chen, Chengzu Long, and Eric Olson for helpful discussion. DMD CRISPR therapy in the Duan lab is supported by the National Institutes of Health (AR-69085), Department of Defense (MD150133) and Hope for Javier.

Author Disclosure

D.D. is a member of the scientific advisory board for Solid Biosciences and an equity holder of Solid Biosciences. The Duan lab has received research support from Solid Biosciences.