Abstract Details

Title Dual Vector Co-Delivery of nNOS-Recruiting Minidystrophin (?H2-R15/?R18-19) as a Therapeutic Strategy for DMD

Topic Muscle Disease/Neuromuscular Junction

Presentation(s) S05 - (-)

Poster/Presentation
Number 007

Objective

Background Gene therapy by rAAV vectors using truncated dystrophins is promising due to their packagable size. Microdystrophin have shown some efficacy but structural abnormalities have been reported in limb muscles. Minidystrophin (?H2-R19), on the other hand, retains more than 50% of dystrophin sequence and reported to provide enhanced physiological function. However, ?H2-R19 lacks nNOS recruiting region. Sarcolemmal nNOS is crucial for normal muscle function and it's absence plays an important role in muscular dystrophy pathogenesis.

Design/Methods nNOS recruiting Minidystrophin transgene was engineered in two different plasmids. First plasmid contained MHCK7 promoter and first part of the transgene encoding N-terminal domain, H1, repeats R1-R3, R16-17, H3 and part of R20. The second plasmid contained rest of the dystrophin sequence and polyA tail. There was a 372 base pair sequence homology at 3' end and 5' of the two plasmids respectively. These were then packaged into two AAV9 vectors and co-injected in TA muscle of mdx mice. Four weeks later muscles were analyzed.

Results Dual minidys vector co-delivery in TA muscle resulted in more than 50% transduction. Minidystrophin (?H2-R15/?R18-19) reconstitution and localization to the sarcolemma was confirmed by western immunoblotting and immunofluorescence respectively. One of the hallmarks of DMD is vasoconstriction & contraction induced ischemia in the muscles due to the absence of nNOS and NO production. With dual vectors, nNOS was recruited to the sarcolemma and nNOS activity was demonstrated. Further studies with vascular delivery via isolated limb perfusion (ILP) to transduce multiple muscles are underway.

Conclusions The preliminary data demonstrates that co-delivery of nNOS recruiting minidystrophin vectors leads to reconstitution and robust expression of transgene and can be optimized towards a therapeutic goal.

Authors/Disclosures
Vinod Malik (Nationwide Childrens Hospital) PRESENTER	No disclosure on file
	No disclosure on file
	No disclosure on file
L J. Greenfield, Jr., MD, PhD, FAAN (UConn Health Center)	Dr. Greenfield has received publishing royalties from a publication relating to health care.
Louise R. Rodino-Klapac (Sarepta Therapeutics)	Ms. Rodino-Klapac has received personal compensation for serving as an employee of Sarepta Therapeutics, Inc.. Ms. Rodino-Klapac has received stock or an ownership interest from Sarepta Therapeutics. Ms. Rodino-Klapac has received intellectual property interests from a discovery or technology relating to health care.
Jerry R. Mendell, MD, FAAN (The Research Institute at Nationwide Children's Hospital)	Dr. Mendell has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Vertex. Dr. Mendell has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Novartis. Dr. Mendell has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Sarepta Therapeutics . The institution of Dr. Mendell has received research support from Sarepta.

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