Studies of Efficacy and Liver Toxicity Related to Adeno-Associated Virus–Mediated RNA Interference

Abstract

Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6-promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the overexpressed shRNAs from AAV-U6 vectors. Our results demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side effects.

Caspase-3 and GFAP as early markers for apoptosis and astrogliosis in shRNA-induced hippocampal cytotoxicity

“…We suggest that the design of shRNA-based studies should involve careful consideration of several parameters, such as shRNA dose (Grimm, 2011), promoter choice (Lebbink et al, 2011;Sun et al, 2013), AAV serotype (Ehlert et al, 2010) and construct backbone (Boudreau et al, 2009;Han et al, 2011;McBride et al, 2008). These factors should subsequently be tested with respect to titer, construct and specificity in vivo.…”

Thyroid hormone suppresses hepatocarcinogenesis via DAPK2 and SQSTM1-dependent selective autophagy

“…17,18 To further determine whether the protective effect of T 3 is through autophagy, genetic inhibition of hepatic Atg7 via adeno-associated virus serotype 8 (AAV8) vector-delivered shRNA in livers of eu-and hyper-thyroid mice was performed. As expected, knockdown of ATG7 promoted accumulation of SQSTM1 along with suppressed LC3-II production, indicating the inhibition of autophagy in mouse livers (Fig.…”

“…The construction and production of these pseudotyped AAV8 vectors, which contain the H1 promoter and the shRNA coding sequence, has been reported previously. 18 To determine the T 3 effect on DAPK2 expression in vivo, hyperthyroid mice were generated via intraperitoneal injection of T 3 (10 mg/100 g body weight) for 14 d. To induce hypothyroidism in mice, 0.02% methimazole plus 0.1% sodium perchlorate was added to drinking water for 14 d.…”

Progress and Prospects of Anti-HBV Gene Therapy Development

“…Expressed shRNAs or pri-miR mimics have the advantage of sustained supply of RNAi activators and are hence more relevant for targeting chronic diseases such as HBV infection. However, several studies reported toxicities that were caused by saturation of the RNAi pathway following overexpression of shRNAs from RNA polymerase (Pol) III promoters [ 85 , 86 , 87 ]. Use of cassettes containing artificial pri-miRs may alleviate this problem as these templates may be expressed from Pol II promoters to afford better transcription control.…”