A Novel Technology for Full-Length Gene Replacement Therapy of Duchenne Muscular Dystrophy

Project Summary and Abstract Duchenne muscular dystrophy (DMD) is a genetic disease which arises from a nonsense mutation of the dystrophin gene, dmd. Dystrophin has important functions in protecting muscle from mechanical damage and mutations in the gene results in muscle degeneration. The natural progression of the disease involves muscle

degeneration in the limbs leading to loss of mobility and eventual degeneration of cardiac and diaphragm muscle, leading to death. Duchenne muscular dystrophy affects 1 in 5,000 male births, renders most patients paralyzed by age 12, and on average results in death by age 26. There is no known cure and current treatments only delay

disease progression or are aimed at quality-of-life improvements. Gene therapy is an emerging technology that aims to cure genetic diseases. Currently, two main approaches are being explored. Although CRISPR/Cas9 developments have advanced genetic engineering significantly in recent years, many barriers still remain to clinical treatment of diseases such as DMD, including off-target editing,

insertion/deletion mutations, delivery of the large protein complex, and immunogenicity of expressing the bacterial enzyme. Gene-replacement therapy is a promising alternate and potentially curative strategy. Gene- replacement therapy delivered by Adeno-Associated Virus (AAV) offers an elegant solution by replacing, rather

than editing, the defective gene. Using AAVs, exogenous DNA is delivered to target cells and stably resides extra-chromosomally. The dystrophin gene can then be subsequently expressed, functionally replacing the defective endogenous copy. Due to cargo-capacity limitations of AAVs however, current clinical testing have

been restricted to highly truncated versions of dystrophin with resulting limited efficacy. Recently developed technology overcomes the cargo capacity limitation of AAVs, providing an avenue for delivery of full-length gene replacements. First, split-gene constructs are designed by dividing the full gene into

fragments, flanked by regions containing both intronic and base-pairing binding sequences, where each fragment is delivered by a sub-population of an AAV cocktail. Within the cell, the expressed RNA fragments are locally stabilized by the base-pairing of the binding domains, then undergo a spliceosome-mediated joining reaction,

thus concatenating the fragments into full-length mRNA for translation and restoration of functional protein. Preliminary data demonstrates the ability of this approach to deliver three-fragment fluorescent reporters in mouse muscle. The proposed research aims to develop this technology as a gene-replacement therapy through

the engineering of robust constructs for dystrophin gene delivery, optimization of on- and off-target delivery both in vitro and in vivo, and characterization of both therapeutic and unintended host responses in a Duchenne Muscular Dystrophy mouse model. This project will demonstrate the therapeutic potential of a novel class of

gene-replacement therapies. Further, this project lays the groundwork for future studies in higher mammals, and for a potential future treatment and cure of Duchenne Muscular Dystrophy in patients.