Kristie Bloom

Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases

Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection.

Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the worlds population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realised. Limiting off target effects, improvement of delivery efficiency, dose regulation, and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs, and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

Editing CCR5: a novel approach to HIV gene therapy.

Acquired immunodeficiency syndrome (AIDS) is a life-threatening disorder caused by infection of individuals with the human immunodeficiency virus (HIV). Entry of HIV-1 into target cells depends on the presence of two surface proteins on the cell membrane: CD4, which serves as the main receptor, and either CCR5 or CXCR4 as a co-receptor. A limited number of people harbor a genomic 32-bp deletion in the CCR5 gene (CCR5∆32), leading to expression of a truncated gene product that provides resistance to HIV-1 infection in individuals homozygous for this mutation. Moreover, allogeneic hematopoietic stem cell (HSC) transplantation with CCR5∆32 donor cells seems to confer HIV-1 resistance to the recipient as well. However, since Δ32 donors are scarce and allogeneic HSC transplantation is not exempt from risks, the development of gene editing tools to knockout CCR5 in the genome of autologous cells is highly warranted. Targeted gene editing can be accomplished with designer nucleases, which essentially are engineered restriction enzymes that can be designed to cleave DNA at specific sites. During repair of these breaks, the cellular repair pathway often introduces small mutations at the break site, which makes it possible to disrupt the ability of the targeted locus to express a functional protein, in this case CCR5. Here, we review the current promise and limitations of CCR5 gene editing with engineered nucleases, including factors affecting the efficiency of gene disruption and potential off-target effects.

tRNALys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs

Using exogenous sequences to express RNA interference (RNAi) activators has potential for the treatment of chronic viral infections. However, availability of a variety of suitable of promoter elements is important to optimize transcription control of silencing sequences and facilitate multitargeting. Recent demonstration that tRNA miR genes occur naturally has prompted investigating the incorporation these tRNA Pol III promoters into exogenous RNAi-activating cassettes. We have assessed efficacy of Pol III tRNA(Lys3) short hairpin RNA (shRNA) sequences that target hepatitis B virus (HBV). These cassettes achieved good silencing at low concentrations, and efficacy compared favorably to that of equivalent U6, H1 and CMV expression cassettes. HBV replication in cell culture was inhibited and northern blot hybridization analysis confirmed processing of the tRNA(Lys3) transcripts to form intended antiviral guide sequences. Importantly effects were observed without evidence of disruption of endogenous miR function. Analysis in a murine hydrodynamic injection model of HBV replication confirmed that the tRNA(Lys3) expression cassettes are also effective in vivo. Usefulness of tRNA(Lys3) antiviral expression cassettes expands the repertoire of promoters available for RNAi-mediated HBV silencing and advances the application of expressed sequences for therapeutic gene silencing.

Advances with using CRISPR/Cas-mediated gene editing to treat infections with hepatitis B virus and hepatitis C virus.

Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal. Disabling cccDNA is thus key to curing HBV infections and application of gene editing technology, such as harnessing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, has curative potential. Several studies have reported good efficacy when employing CRISPR/Cas technologies to disable HBV replication in cultured cells and in hydrodynamically injected mice. Recent advances with HCV drug development have revolutionized treatment of the infection. Nevertheless, individuals may be refractory to treatment. Targeting RNA from HCV with CRISPR/Cas isolated from Francisella novicida may have therapeutic utility. Although preclinical work shows that CRISPR/Cas technology has potential to overcome infection with HBV and HCV, significant challenges need to be met. Ensuring specificity for viral targets and efficient delivery of the gene editing sequences to virus-infected cells are particularly important. The field is at an interesting stage and the future of curative antiviral drug regimens, particularly for treatment of chronic HBV infection, may well entail use of combinations that include derivatives of CRISPR/Cas.

A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA

Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.

Transcription Activator-Like Effector (TALE) Nucleases and Repressor TALEs for Antiviral Gene Therapy

Genome modification platforms are fast becoming valuable tools for the development of novel therapies. The use of sequence-specific DNA binding proteins in conjunction with various effector domains enables targeted gene editing to be employed as a mode of therapy. Although this field of research has largely focused on the engineering and repair of mammalian genes, the technology may also be used to disrupt viral DNA or host factors associated with pathogenesis of viral disease. For persistent or latent infections, targeted mutagenesis of the episomal or proviral DNA could render the virus inactive. Alternatively, virus-resistant cells may be generated by disrupting the expression of host factors that are required for viral infection. This review highlights some of the approaches currently used to disable viruses, with a particular focus on TALENs and repressor TALEs for antiviral therapy.

Recent advances in developing nucleic acid-based HBV therapy

Chronic HBV infection remains an important public health problem and currently licensed therapies rarely prevent complications of viral persistence. Silencing HBV gene expression using gene therapy, particularly with exogenous activators of RNAi, holds promise for developing an HBV gene therapy. However, immune stimulation, off-targeting effects and inefficient delivery of RNAi activators remain problematic. Several new approaches have recently been employed to address these issues. Chemical modifications to anti-HBV synthetic siRNAs have been investigated and a variety of vectors are being developed for delivery of RNAi effectors. In this article, we review the potential utility of gene therapy for treating HBV infection.

483. Epigenetic Silencing of Hepatitis B cccDNA In Vitro and In Vivo Using AAV-Delivered Engineered Repressor Transcription Activator-Like Effector

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 Kruppel-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.