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本帖最后由 MP4 于 2015-6-21 21:16 编辑
Inhibition of Hepatitis B Virus Replication In Vivo Following Delivery of Antiviral TALENs With Recombinant Adeno-Associated Viral Vectors
Samantha Nicholson, Buhle Moyo, Kristie Bloom, Claudio Mussolino, Toni Cathomen, Koichi Watashi, Abdullah Ely, Patrick Arbuthnot. Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; Institute for Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
Hepatitis B virus (HBV) is hyperendemic to sub-Saharan Africa where there are approximately 600 000 deaths per year associated with chronic HBV infection. HBV may have long periods of dormancy in carriers of the virus, which is dependent on persistence of the nuclear replication intermediate covalently closed DNA (cccDNA). Current HBV therapies, including nucleot(s)ide analogs, target the actively transcribed virus. The cccDNA is not cleared and may act as a reservoir for re-initiation of viral replication following treatment withdrawal. This poses a significant challenge for the successful treatment of chronic HBV sufferers, and makes the cccDNA intermediate an important target for novel HBV treatments. Employing gene editing technology to disable cccDNA has emerged as a promising novel therapeutic strategy. TALENs targeting the surface and the core viral open reading frames, previously described by our group, demonstrated efficacy against HBV targets. To advance this technology to therapeutic application, efficient delivery and evaluation of the TALENs' safety and target specificity in vivo and in vitro need to be established. This study aimed to evaluate the safety and efficacy of TALENs delivered by adeno-associated viral (AAV) vectors in vivo and in vitro. The HBV-infectable HepG2-hNTCP-C4 cell line was used to establish that the TALENs could disable cccDNA. Capacity for capsid modification, low immunogenicity and good hepatotropic transduction efficiency are favorable features that were considered in selecting these vectors. However, the delivery of TALENs by AAVs is technically complicated, as sequences encoding the two subunits of the complete TALENs exceed the capacity of single stranded AAVs. Consequently, we generated AAVs that encode each of the TALEN subunits. To constitute the complete TALENs, pairs of vectors were thus administered to mice or cultured cells. In vitro testing of AAV-TALENs was undertaken using the HepG2-hNTCP-C4 cell line. These cells present a novel tool for testing anti-HBV therapeutics as they overexpress the human sodium taurocholate co-transporting polypeptide (hNTCP) gene allowing them to be infected by HBV. The viral life-cycle is accurately recapitulated and enables assessment of effects of therapeutic agents on cccDNA. Since viral gene expression is dependent on transcription from the cccDNA intermediate in these cells, measurement of markers of HBV replication may be used as an indicator of cccDNA function. This study evaluated the potential of AAV delivered TALENs in vivo and in vitro by testing their activity and evaluating off-target activity, immunogenicity and liver toxicity. The results provide evidence for the utility of applying AAVs to the delivery of anti-HBV TALENs and offers further support for the feasibility of employing TALENs to treat chronic HBV infection.
Keywords: TALENs; Gene Correction/Modification/Targeting; AAV Vectors
Session: Poster Session: Gene Targeting and Gene Correction I (5:15 PM-6:45 PM)
Date/Time: Wednesday, May 13, 2015 - 5:15 pm
Room: Elite Hall A
Epigenetic Silencing of Hepatitis B cccDNA In Vitro and In Vivo Using AAV-Delivered Engineered Repressor Transcription Activator-Like Effector
Buhle Moyo, Samantha Nicholson, Ilke Roelofse, Carol Crowther, Kristie Bloom, Claudio Mussolino, Toni Cathomen, Koichi Watashi, Abdullah Ely, Patrick Arbuthnot. Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg, Gauteng, South Africa; Institute for Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
Hepatitis B virus (HBV) infection is hyper-endemic (>8 % chronic carriers) to parts of Asia and sub-Saharan Africa. Persistent HBV infection predisposes to liver diseases such as cirrhosis and hepatocellular carcinoma. Current anti-HBV therapies face several limitations. RNA interference-based therapies lack the robustness and specificity required for therapeutic effect while interferon-α and nucleotide/side analogs function post-transcriptionally and thus allow for the persistence of the covalently closed circular (cccDNA). cccDNA may persist indefinitely and enables re-initiation of HBV replication after withdrawal of treatment. Disabling the cccDNA is essential for the successful treatment of chronic HBV. Our group previously described effective anti-HBV transcription activator-like effector (TALE) nucleases (TALENs). Although potentially useful to counter HBV replication, one drawback is that viral sequences integrated into the host genome may be susceptible to digestion by the TALENs. Transcriptional silencing, rather than cleavage, of cccDNA may therefore be preferable to avoid causing undesirable mutations in the host. To this end, TALE binding domains designed to target the viral preS2 promoter and the basic core promoter/enhancer II regions were fused to a Krüppel-associated box repressor domain to generate repressor-TALEs (rTALEs). These rTALEs were shown to inhibit viral replication in vitro and in vivo without inducing significant toxicity. In an in vivo murine model using hydrodynamic transfection with an HBV replication-competent plasmid, a reduction in secreted HBV surface antigen (HBsAg) of 97% and 98% was seen at day 3 and 93% and 96% at day 5 for P1L and P1R respectively. To develop this approach as a feasible therapy, rTALE-encoding sequences were incorporated into recombinant adeno-associated viruses (rAAVs) and assessed in the HepG2.hNTCP-C4 cell line. These cells overexpress the HBV receptor, human sodium taurocholate co-transporting peptide (hNTCP). The HepG2.NTCP-C4 cell line is infectable with HBV and viral gene expression is dependent on formation of cccDNA. Measurement of viral markers of replication may thus be used as an indicator of inhibitory effects on cccDNA. The anti-HBV efficacy of the rTALEs and epigenetic modification of the targeted HBV DNA was characterized. Chromatin immunoprecipitation assays determined the binding of the rTALEs to the cccDNA and induction of epigenetic markers of viral gene suppression. Mobility shift assays confirmed specificity of rTALE binding. In vivo efficacy of rAAV-delivered rTALEs was evaluated in transgenic HBV mice. Our study provides valuable information on the potential therapeutic utility of rTALEs and demonstrates the feasibility of the approach for treatment of HBV.
Keywords: AAV Vectors; Gene Correction/Modification/Targeting; Infectious Diseases
Session: Simultaneous Oral Abstract Sessions: Gene Editing and Gene Regulation II (3:30 PM-5:30 PM)
Date/Time: Friday, May 15, 2015 - 4:30 pm
Room: Empire C
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发表于 2015-6-21 17:20
