Poster abstracts
Poster number 37 submitted by Nehaal Patrick
A Dual-AAV Ribozyme-mediated Approach to Express a Highly Functional Therapeutic Dystrophin in Duchenne Muscular Dystrophy
Nehaal Patrick (NCH), Liubov Gushchina (NCH), Stefan Nicolau (NCH), Kevin Flanigan (NCH)
Abstract:
The X-linked Duchenne muscular dystrophy (DMD) is caused by frame-truncating mutations in the DMD gene resulting in absence of dystrophin protein expression, leading to progressive muscle wasting, wheelchair dependence, and death in the third decade1. Mutations that allow expression of a partially functional dystrophin are associated with the milder Becker muscular dystrophy (BMD).The full-length DMD cDNA is 10.5 kb but adeno-associate virus (AAV) packaging capacity is limited to 4.7 kb. Current DMD therapies, including exon skipping therapies and micro-dystrophin gene therapies, are directed towards restoring partially functional BMD-like dystrophins.
Here we describe the design and development of two dual-AAV vector systems with vectors carrying either an N-terminal or C-terminal portion of the DMD gene under the control of the CMV promoter, and each containing a small self-cleaving Twister ribozyme gene. The encoded mRNA produced by cleavage and ligation lacks either exons 19-44 (del19-44) or exons 20-50 (del20-50); although neither of these are full-length, both have been found in patients with minimal or no muscle symptoms at all.
Preliminary results suggest robust ribozyme-mediated mRNA ligation of the del19-44 therapeutic mRNA following plasmid transfection into HEK cells, with protein expression studies underway. Similar studies will be performed using del20-50 plasmids. Following confirmation of efficient trans-ligation, they will be cloned into AAV vectors for delivery into DMD mouse models with a goal of therapeutic development.
References:
Lindley, S.R. et al. Ribozyme-activated mRNA trans-ligation enables large gene delivery to treat muscular dystrophies. Science 386(2024-11-15).
Keywords: DMD, Gene Therapy, Ribozyme