Duchenne muscular dystrophy (DMD) affects 1 in every 3,500 boys worldwide. DMD is caused by mutations in the gene encoding dystrophin, a protein key to muscle health. DMD patients are typically weaker than normal by age 3, and with progressive muscle weakness most loose the ability to walk by age 11. DMD progresses to complete paralysis, respiratory insufficiency heart failure, and death, usually before the age of 25. No therapies exist that address the primary defect or dramatically alter the debilitating disease. Exon-skipping is an emerging therapy in which anti-sense oligonucleotide (AO) guided-RNA splicing rescues expression of a partially functional dystrophin; but it is unclear if efficacy will be optimal for clinical gain. We identified a combination therapy that improves the efficacy of exon-skipping in mouse muscle and human DMD patient-stem-cell-derived muscle cells. The DMD mouse model will be used to establish dosing and efficacy. To determine if combination therapy promotes exon skipping in human DMD patient cells with different DMD mutations, DMD patient derived stem cells converted into muscle-like cells in culture and screened for efficacy of combination drug relative to AO alone. The proposed research program will complete studies to identify a single drug/AO combination as a developmental candidate anticipated to treat up to 13% of DMD patients; although the strategy is likely generalizable to enable treatment of 70% of DMD patients.
Statement of Benefit to California:
Duchenne muscular dystrophy (DMD) is a fatal genetic disorder, caused by a defect in the gene that produces dystrophin, a protein critical for normal skeletal muscle function. DMD affects more than 1,000 boys in California. Muscle weakness first appears in boys in the hips and legs and progressively extends to every muscle in the body such that most affected individuals require a wheelchair by age 11, have trouble feeding themselves by their late teens and ultimately loose most muscle function. Patients usually die by age 25 from respiratory or cardiac insufficiency. In addition to the human suffering, DMD places a large economic burden on patients, their families and society. Patients require intensive medical care because they cannot perform the simplest activities of daily living. Eventually, each individual requires ventilation and 24/7 care. The proposed combination therapy is predicted to cause skeletal muscle cells to skip DMD exon 51 and express a partially functional dystrophin protein, lessening the severity of DMD. A therapy that effectively slows or reverses disease will allow patients to lead longer, more productive lives and reduce costly supportive services—progress that will benefit patients, their families and society. Our proposal stands to specifically benefit Californians in another way: Because the University of California owns the intellectual property to the combined therapy, our success could ultimately lead to revenue for a state institution.
Year 1Duchenne muscular dystrophy (DMD) is the most common muscular dystrophies and the most common fatal genetic disorder of childhood. Approximately one in every 5,000 boys worldwide is affected with DMD often caused by spontaneous mutations. Extrapolating from population based studies, there are over 15,000 people currently living with DMD in the US alone. DMD is a devastating and incurable muscle-wasting disease caused by genetic mutations in the gene that codes for dystrophin, a protein that plays a key role in muscle cell health. Children with DMD are typically weaker than normal by age three, and progressive muscle weakness of the legs, pelvis, arms, neck and other areas result in most patients requiring full-time use of a wheelchair by age 11. Eventually, the disease progresses to complete paralysis and increasing difficulty in breathing due to respiratory muscle dysfunction and heart failure, with death usually occuring before the age of 25. While corticosteroids can slow disease progression and supportive care can extend lifespan and improve quality of life, no therapies exist that address the primary defect or dramatically alter the debilitating disease course.
Exon-skipping is a promising therapy that aims to repair the expression of the dystrophin protein by repairing the RNA. We have identified a combination therapy that improves the effectiveness of exon-skipping therapy in mouse muscle and in human DMD patient stem cell derived muscle cells in culture. In exon skipping the genetic defect is directly repaired inside of each muscle cell. Thus, this therapy is predicted to lessen the disease severity.
Early research on this combination therapy for Duchenne used human DMD patient stem cells including: reprogrammed patient fibroblasts converted into muscle-like cells in culture or when transplanted in mice. We have made a panel of these cells with different mutations to assess efficacy in a range of DMD mutations. These cells are necessary because each patient’s mutation in the dystrophin gene is different. In order to know who will or will not benefit from the exon-skipping therapy, individualized cell culture and mouse transplant models from a number of DMD patients must be created to effectively characterize the combination therapy. At 12 months of the CIRM-funded research program, we have established optimal oral dosing of dantrolene that is compatible with 6 month long-term testing in dystrophic mice and optimal dosing of morpholino antisense oligo. The combination therapy is well tolerated by mice, and dystrophin rescue is increased in short term experiments. 6 month treatment experiments are being initiated that will test if the induction of dystrophin can reduce the severity of the disease in the dystrophic mice. Since exon-skipping therapy relies on knowing individual patients exact DNA mutation, this is a form of personalized genetic medicine. While the specific combination therapy being developed here will treat up to 13% of DMD patients, the strategy is likely to be generalized to be able to treat up to 70% of DMD patients.
Year 2DMD remains a devastating and incurable muscle-wasting disease caused by DNA mutations in the gene that codes for dystrophin, a protein that plays a key role in muscle cell health. Most DNA mutations cause a frameshift in the RNA, and result in no expression of functional dystrophin, the protein encoded by the DMD gene. When dystrophin is completely missing from the muscle, the muscle cells are susceptible to repeated cycles of damage, and lead to progressive weakness everywhere in the body including the heart and lungs. Exon-skipping is a promising therapy that aims to repair the expression of the dystrophin protein by repairing the reading frame of the mRNA. This strategy has been demonstrated to successfully restore a small fraction of the normal amount of dystrophin, and is likely to be therapeutically beneficial. However, increasing the amount of dystrophin produced in this strategy is very likely to improve the impact of exon skipping on those affected by DMD. We have identified a combination therapy that improves the effectiveness of exon-skipping therapy in mouse muscle and in human DMD patient stem cell derived muscle cells in culture. In the second year of this project, we have expanded the set of cell lines from DMD patient skin biopsies. These biopsies have been grown in culture and induced into muscle cells using direct reprogramming. We have tested the effectiveness of exon skipping enhancement in these human cell lines to determine if multiple independent mutations are relevant for this potential therapy. We have shown that different cell lines can be repaired by exon skipping strategies, and that the compound can enhance skipping in a dish. At 24 months in the project, we have also completed a 6 month trial of over 180 mice that are in eight different treatment groups including with or without compound (Dantrolene) and either without antisense oligo or with antisense oligo at three different doses administered weekly by IV in mdx mice. Mdx mice are the main mouse model of Duchenne, and we can readily assess dystrophin protein expression and DMD RNA repair in mouse tissues. The experiment is recently completed, but all of the data are blinded. The mouse muscles are being assessed and will be correlated with functional measurements in the 8 different mouse groups. The combination therapy appears to have been well tolerated over the 6 month time frame, and analysis of the muscle specimens to quantitatively assess dystrophin restoration and DMD exon skipping are underway. Additional tissues including blood, kidney, liver and heart are being assessed for potential toxicity related to the combination therapy.