Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice.

BACKGROUND: There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA-expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA). RESULTS: Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA. CONCLUSIONS: Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.

Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes.