Expression of dystrophins with enhanced function

University Of Washington, Seattle WA

Investigators

Abstract

Project Summary/Abstract Duchenne muscular dystrophy (DMD) is an X-linked, lethal recessive genetic disorder resulting from mutations in the DMD gene, which encodes the protein dystrophin (Dys). The 2.2 MB gene displays the highest new mutation rate of any human gene, reflecting the high DMD prevalence (1:5,000). This large size coupled with thousands of unique mutations creates significant complexity for gene therapy. Previous studies by us and others led to the design of ‘micro-dystrophins’ (μDys) that can be delivered systemically to striated muscles using AAV vectors. This approach, along with anti-sense oligonucleotide (ASO) ‘exon-skipping’ methods, have emerged as promising therapies that are being tested in multiple clinical trials, with one µDys approach and several ASO drugs having now been approved by the FDA. Gene editing has garnered significant attention, primarily as an alternate and potentially permanent way to correct Dys expression. While promising, the ASO & editing methods remain comparatively inefficient, and many AAV-mediated editing methods result in genomic rearrangements & vector insertion due to Cas9-induced double-stranded DNA breaks. AAV-μDys has proven to be far more efficient & is not associated with insertional mutagenesis; however, this approach is limited to delivery of dystrophins carrying only one-third of the full-length sequence. Clinical trials, while encouraging, have also revealed dose- related toxicities that limit the AAV dose, which is suboptimal for gene replacement & editing. Our ongoing structure/function studies of Dys have led to the design of numerous micro-, mini- and full-length Dys vectors, & have extended into gene editing methods. Together these approaches offer the potential to express significantly larger and more functional dystrophins. Furthermore, myotropic AAV capsid variants now enable delivery of more potent gene therapies at much lower vector doses. In this proposal we harness previous discoveries and preliminary data to address limitations of current gene therapy approaches for DMD by developing improved gene replacement & editing vectors. In Aim 1, novel technologies for expressing mini- & full-length Dys and/or it’s paralogue utrophin (Utrn) using multiple AAVs will be refined with the goal of efficiently producing highly functional therapies. This approach utilizes known & novel split inteins to enable production of Dys/Utrn proteins larger & more functional than can be delivered with a single AAV vector (i.e. AAV-μDys). In Aim 2, enhanced Dys gene editing methods will be developed to maximize duration and expression levels of large endogenously regulated dystrophins, through delivery of augmented & inducible editing vectors. A major focus is on Satellite cells. Aim 3 critically evaluates the therapeutic impact & longevity of both Aim 1 & 2 approaches in response to aging & injury, and examines the possibility for synergistic application. All aims take advantage of the recently described ‘MyoAAV’ capsids that enable systemic muscle gene delivery at doses 10-20-fold lower than the AAV9 or rh74 serotypes currently in clinical trials. Together these approaches could significantly increase the safety and efficacy of gene therapy for DMD, and should have important applications to other neuromuscular disorders.