Multiplex CRISPR/Cas9-Based Genome Engineering for the Genetic Correction of Duchenne Muscular Dystrophy
Abstract
Gene therapy is a promising approach to treating Duchenne Muscular Dystrophy (DMD). However, current methods typically require the addition of extra dystrophin genes to the genome or the lifelong re-administration of foreign genetic material that works transiently to restore dystrophin expression, both of which have significant safety and practical concerns. Furthermore, these strategies have been limited by an inability to deliver the large and complex dystrophin gene sequence. An appealing alternative to these gene replacement approaches is the targeted repair of the endogenous mutant dystrophin gene. This concept, known as genome editing, represents a potential cure to DMD without the need for permanent integration of or repeated exposure to foreign biological material. Furthermore, it corrects the problem at the source by correcting the mutation to the naturally occurring dystrophin gene. Genome editing has been made a reality for human gene therapy by the recent development of transformative technologies that use engineered enzymes to cut and paste DNA sequences at specific sites in the genome. In fact, genome editing is now in clinical trials for treating cancer and HIV. The most recently developed genome editing technology, known as CRISPR, is much more robust than previous technologies and has rapidly transformed all areas of biomedical research and biotechnology in less than 2 years. Several efforts are underway to use CRISPR to correct genetic diseases, and we have demonstrated that it is possible to restore dystrophin expression in muscle cells from DMD patients. However, for this to be viable for clinical translation, we must demonstrate successful genome editing in skeletal and cardiac muscle tissue in animal models of the disease. In this study, we will use adeno-associated virus to delivery CRISPR to skeletal and cardiac muscles of a mouse model of DMD and a mouse model carrying the human dystrophin gene. The overall objective of this research proposal is to develop methods to restore dystrophin expression via targeted genome editing in vivo. The central hypothesis is that nuclease-mediated gene correction will lead proper dystrophin expression and function in mouse models of DMD. This approach capitalizes on some of the most innovative recent developments in biomedical research and gene therapy for a unique therapeutic strategy for DMD pursued by our group that is uniquely qualified in the relevant technologies and has established collaborations with experts in the necessary delivery methods. In principle, genome editing can be used to change any DNA sequence and therefore is theoretically applicable to any DMD patient with any mutation. However, the CRISPR must be customized for the target gene sequence. Therefore, for our initial studies we will focus on excision of exon 51 from the genome, which will be applicable to 13% of all DMD patients, similar to current exon skipping studies. However, we have also demonstrated effective excision of exons 45-55 in our preliminary results, which would address 62% of all DMD patients. Once proof-of-principle of in vivo correction of the dystrophin gene and restoration of dystrophin expression is demonstrated in the course of this 2-year project, this technology will be ready for lead candidate development and optimization to prepare for filing an Investigational New Drug Application with the Food and Drug Administration. The necessary additional follow-on studies will include evaluation of safety, immunogenicity of the CRISPR system, and potentially large animal models before moving to clinical trials. This research plan is innovative because it capitalizes on the unfulfilled potential of the CRISPR genome editing technology to address the fundamental limitations of conventional gene therapies and the unmet need for a safe and effective permanent cure to DMD. Importantly, this approach is also broadly applicable to numerous genetic diseases in
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 04, 2016
- Source ID
- W81XWH1510469
Entities
People
- Charles A. Gersbach
Organizations
- Duke University
- United States Army