Developing use for recombinant lentiviral vectors for delivery of anti-hepatitis B virus micro RNA mimics

Abstract

Persistent infection with hepatitis B virus (HBV) is associated with an increased risk for development of serious life-threatening complications such as cirrhosis and hepatocellular carcinoma (HCC). This common malignancy is aggressive and has a very poor prognosis. Currently available HBV therapies have variable efficacy and do not diminish the risk for development of HCC associated with infection with the virus. Consequently the development of novel therapies aimed at providing effective treatment for chronic HBV infection remains a medical priority. The therapeutic potential of harnessing the RNA interference (RNAi) pathway to achieve specific and potent silencing of pathology-causing genes has been explored for the development of novel and improved therapy to counter chronic HBV infection. Highly effective expressed anti-HBV RNAi-based sequences have been demonstrated to be capable of mediating impressive silencing of viral gene expression in vitro and in vivo. Nevertheless, accomplishing stable delivery to hepatocytes and sustained, long-term expression of therapeutic effecters remains the greatest challenge impeding the clinical translation of RNAi-based anti-HBV gene therapy. Stable integration of transgenes that may be achieved with recombinant lentiviruses, derived from the human immunodeficiency virus type 1 (HIV-1), makes these vectors particularly useful for attaining sustained expression of RNAi activators. This property is suited to countering HBV persistence, making these delivery vehicles advantageous for gene transfer of expressed RNAi anti-HBV sequences to infected hepatocytes. To advance potential clinical application of RNAi-based anti-HBV gene therapy, second generation self-inactivating (SIN) lentiviral vectors were engineered to include liver specific, RNA polymerase II (Pol II) cassettes that generate HBV-silencing primary micro RNA (pri-miR) mimics. The antiviral pri-miR sequences, placed under the control of the liver-specific mTTR promoter, are derived from natural pri-miR-31 and engineered to generate anti-HBV RNAi guide sequences targeting single (monocistronic pri-miR sequences) or multiple (polycistronic pri-miR sequences) sites within the highly conserved multifunctional HBV X protein (HBx) open reading frame (ORF) of the viral genome. The HBV silencing lentiviruses (LVs) stably transduced liver-derived Huh7 and HepG2.2.15 cells. Northern blot analysis of RNA extracted from stably transduced Huh7 cells verified that the integrated anti-HBV pri-miR sequences were processed to form RNAi-activating guide strands according to the intended design. When stably transduced Huh7 cells were transfected with a HBV replication-competent plasmid, potent inhibition of markers of viral replication was achieved. Sustained, hepatotropic HBV silencing was effected with limited disruption of endogenous miR function. Silencing of a mutated HBV sequence in stably transduced cells was caused by HBV-silencing LV expressing a polycistronic pri-miR effecter. Rigorous evaluation of the anti-HBV efficacy of the polycistronic silencing LV in the HepG2.2.15 HBV replication cell line demonstrated potent antiviral activity. Preclinical analysis of the efficacy of the RNAi-activating lentiviral vector system was assessed using the HBV transgenic mouse model. HBV silencing LVs encoding a liver-specific polycistronic pri-miR 31/5-8-9 sequence was delivered to young adult animals via a tail-vein injection as well as to neonatal mice via a superficial temporal vein injection. The HBV-silencing LV was capable of mediating stable hepatic delivery and sustained expression of anti-HBV pri-miR sequences in vivo. Furthermore, the therapeutic sequences were processed in vivo according to the intended design and mediated powerful knockdown of HBsAg, circulating viral particle equivalents and intrahepatic viral RNA without inducing hepatotoxicity. To our knowledge this is the first study to evaluate the efficacy of RNAi-activating anti-HBV lentiviral vectors in the HBV transgenic mouse model. Although the investigation constitutes a preliminary step towards clinical application of HBV silencing RNAi-based lentiviral vectors, this study has provided proof of principle for a therapeutic strategy that can be potentially useful for the treatment of chronic HBV infection.