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.