Abstract
RNAi has potential for therapeutically downregulating the expression of dominantly inherited genes in a variety of human genetic disorders. Here we used the ROSA26 mouse, which constitutively expresses the bacterial lacZ gene in tissues body wide, as a model to test the ability to downregulate gene expression in striated muscles. Recombinant adeno-associated viral vectors (rAAVs) were generated that express short hairpin RNAs (shRNAs) able to target the lacZ mRNA. Systemic delivery of these rAAV6 vectors led to a decrease of β-galactosidase expression of 30–50-fold in the striated muscles of ROSA26 mice. However, high doses of vectors expressing 21 nucleotide shRNA sequences were associated with significant toxicity in both liver and cardiac muscle. This toxicity was reduced in cardiac muscle using lower vector doses. Furthermore, improved knockdown in the absence of toxicity was obtained by using a shorter (19 nucleotide) shRNA guide sequence. These results support the possibility of using rAAV vectors to deliver RNAi sequences systemically to treat dominantly inherited disorders of striated muscle.
Highlights
RNAi is a post-transcriptional gene silencing phenomenon that is a form of antiviral immune response mounted by many higher eukaryotes such as plants, nematodes, and insects
We previously showed that systemic delivery of recombinant AAV6 (rAAV6) could be used to downregulate FRG1 transcripts body wide in the FRG1 overexpressing mouse model for FSHD [25]. rAAV6 is a serotype with high tropism for muscle in vivo that has shown great promise through its ability to transduce muscles bodywide in the mouse [26,27,28]. Recombinant adeno-associated viral vectors (rAAVs) vectors persist in vivo almost exclusively as episomes and display longterm expression in non-dividing cells such as muscle [29,30]
A previously tested 21 nucleotide short hairpin RNAs (shRNAs) sequence directed against b-gal and expressed from the mouse U6 promoter was introduced into a plasmid carrying the AAV2 inverted terminal repeats (ITRs) and packaging region
Summary
RNAi is a post-transcriptional gene silencing phenomenon that is a form of antiviral immune response mounted by many higher eukaryotes such as plants, nematodes, and insects. An early study of long-term viral expression of shRNAs (19–25 nt recognition sequences) from recombinant vectors derived from adeno-associated virus (rAAV8) in mouse liver resulted in efficient and persistent suppression of target RNA [17].
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