Duchenne Muscular Dystrophy (DMD) is a relatively common degenerative disease that results from mutation of the gene encoding the dystrophin protein.  The genetic nature of DMD has led to substantial interest in gene therapy-based approaches to this disease, including several clinical trials.  However, these therapies typically require the random integration of exogenous DNA into the genome or the lifelong re-administration of transient gene therapy vectors, 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 exciting alternative to these approaches is the targeted editing of the human genome to repair the endogenous mutant dystrophin gene.  This concept represents a potential cure to DMD without the need for random integration of or repeated exposure to foreign biological material.

The focus of our work is to develop and implement strategies for directed modification of the genome for the treatment of genetic disorders.  Engineered nucleases, including zinc finger nucleases (ZFNs), TALE nucleases (TALENs), and CRISPR/Cas9 constitute powerful tools for coordinating the site-specific manipulation of genomic DNA sequences.  The ZFN and TALEN technologies have been developed by biomolecular engineering of novel enzymes comprised of synthetic DNA-binding domains fused to the catalytic domain of a restriction endonuclease.  Engineering of the DNA-binding domain to target specific sites in the human genome can be used to direct nuclease activity and endogenous DNA repair machinery to any locus of interest.  More recently, the RNA-guided nuclease Cas9, which has natural role in bacterial adaptive immunity, has been used in human cells as a method to direct nuclease activity to new targets without protein engineering. Using any of these systems, site-specific nuclease-mediated DNA cleavage can be used to frameshift or excise gene sequences via DNA re-ligation.  Alternatively, DNA sequences can be added or exchanged at targeted loci via the nuclease-mediated enhancement of homologous recombination.  Our goal is to use these genome editing technologies to repair mutated DNA sequences responsible for genetic diseases such as DMD. 

We have engineered and optimized ZFNs, TALENs, and CRISPR/Cas9 systems that can mediate efficient manipulation of the dystrophin gene sequence in human cells. This includes the direct correction mutations or the introduction of dystrophin cDNA into the endogenous dystrophin locus under control of the natural promoter.  We have used these approaches to restore dystrophin expression in human muscle progenitor cells derived from DMD patients as well as dermal fibroblasts that can be reprogrammed to the myogenic lineage.  Substantial levels of dystrophin are expressed in the DMD cells following genetic correction.  We further show that these nucleases are non-toxic with minimal off-target effects.  Corrected cells have been transplanted into immunodeficient mice. This project represents an exciting new avenue for DMD therapy that can permanently correct the underlying genetic mutations.