Genetic Therapy Solution for Duchenne Muscular Dystrophy
Abstract
Duchenne muscular dystrophy is a genetic disease that arises from a mutation of the dystrophin gene. Dystrophin has important functions in protecting muscle from mechanical damage. A loss of functional of dystrophin causes progressive degeneration of muscle tissue. Affected patients ultimately die due to progressive loss of muscle function, particularly in the heart and diaphragm. Current clinical treatment options are very limited and a cure is not available. Several therapy options are being explored in preclinical and clinical research. Two gene therapy approaches have turned out to be very promising. First, (1) gene editing: here, very targeted changes to the patient’s genetic information are made to repair the mutation in the dystrophin gene. This approach requires a large “gene editing protein complex” based on next-generation CRISPR prime editor technology be introduced into muscle cells. This complex precisely edits the faulty portions of the dystrophin gene in order to replace or bypass the site of mutation. Second (2), gene replacement therapy: here, a full-length normal copy of dystrophin is introduced into muscle cells. Once introduced, the functional copy of dystrophin can substitute for the cell’s mutant genes, restoring normal cytoskeletal function in the muscle tissue. One of the main limitations for both gene editing and gene replacement therapies is the amount of genetic information that needs to be introduced into muscle cells. The current ideal DNA delivery “vehicle” is an engineered class of viruses called adeno-associated virus (AAV), which has a limited cargo capacity of about 5,000 nucleotides. Both the information required to introduce next-generation CRISPR gene-editing technology as well as a fixed copy of full-length dystrophin exceed the cargo capacity limitation of AAV. Although strategies to circumvent AAV’s limited cargo capacity are undergoing testing using truncated versions of dystrophin (i.e., micro-dystrophin), they appear to have a limited efficacy. Our focus has been on addressing the cargo capacity limitation of the AAV-vehicle in order to make better DMD treatments possible. Recently, we have developed a novel technology to overcome these packaging constraints. After introduction of the adeno-associated virus into a cell, the information in the form of DNA is unpacked. This DNA information is then transcribed into RNA (an intermediate messenger molecule) to be finally translated from RNA to protein (for example, a CRISPR protein or the dystrophin protein). One way to overcome the cargo constraints of adeno-associated virus is to break the cargo into smaller pieces that are individually packaged and delivered to the cell. These fragments are subsequently joined back together to result in the full “coding information” being reassembled. We have developed a technology that can achieve very efficient joining of RNA molecules (the intermediate messengers). This will benefit both the gene editing and gene replacement therapy approach by delivering either the CRISPR gene editing complex or the full-length dystrophin itself. The technology uses a novel engineered RNA element and otherwise relies on cellular machinery present in every cell of the body to produce the proteins. In this proposal, we will adapt our technology to either deliver the CRISPR gene editing complex (first goal) or deliver the full-length dystrophin replacement gene (second goal) to mice with DMD. Dosing, safety, and injection routes of viral-mediated gene therapy will be explored in a small group of non-human primates. Our experiments will help a broad range of patients with dystrophin mutations in early stages of the disease (our treatment aims to prevent disease progression but may be inadequate to replace lost tissue). In the case of an early intervention, it has the potential to be curative. From recent clinical trials for Duchenne muscular dystrophy, it has become obvious that ther
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010423
Entities
People
- Samuel L Pfaff
Organizations
- Salk Institute for Biological Studies
- United States Army