advancing muscular dystrophy research

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University of Washington
Department of Neurology

Joel Chamberlain, Ph.D.

Recent Presentations:

Delivery of a lacZ shRNA to Muscles of the ROSA26 Mouse

Erin S. Kirkegaard, Leonard Meuse, Eric Finn, Joel R. Chamberlain. Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA; Department of Neurology, University of Washington, Seattle, WA

RNA interference (RNAi) is a conserved cellular process whereby double-stranded RNAs direct post-transcriptional gene silencing through sequence-specific degradation of the target RNA. Inhibitory RNAs can be expressed in cells as naturally occurring microRNAs or small interfering RNAs (siRNAs). An efficient alternative to transfection of siRNAs synthesized in vitro is the introduction of recombinant viral vectors expressing short hairpin RNAs (shRNAs) that take advantage of the cellular RNAi pathways for subsequent processing to active siRNAs in vivo. AAV vectors have proven useful for systemic delivery of genes to a variety of tissues. We are investigating the potential of systemic delivery of AAV vectors for gene knockdown to study gene function and for gene therapy of dominant genetic disorders. In the present study we have developed a recombinant AAV6 vector expressing an shRNA directed against E. coli lacZ that allows efficient knockdown of lacZ mRNA. We asked whether total b -galactosidase (b-gal) activity could be decreased following systemic delivery of the shRNA targeting lacZ mRNA in a variety of tissues in the ROSA26 mouse. Our initial studies focus on the reduction of constituitively expressed b -gal in cardiac and skeletal muscles. Intravenous injection of the vector led to a time-dependent decrease in b -gal activity compared to uninjected controls with a greater reduction in cardiac versus skeletal muscle. This difference most likely reflects the variability in transduction of the heart and quadriceps muscles. The ability to decrease b -gal activity with RNAi in vivo suggests that AAV6 vectors delivering siRNAs could be a useful approach for dominantly inherited diseases of muscle and other tissues.


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