Inhibition of replication of hepatitis B virus using transcriptional repressors that target the viral DNA.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is a serious global health problem. Persistence of the virus occurs as a result of stability of the replication intermediate comprising covalently closed circular DNA (cccDNA). Development of drugs that are capable of disabling this cccDNA is vital. METHODS: To investigate an epigenetic approach to inactivating viral DNA, we engineered transcriptional repressors that comprise an HBV DNA-binding domain of transcription activator like effectors (TALEs) and a fused Krüppel Associated Box (KRAB). These repressor TALEs (rTALEs) targeted the viral surface open reading frame and were placed under transcription control of constitutively active or liver-specific promoters. RESULTS: Evaluation in cultured cells and following hydrodynamic injection of mice revealed that the rTALEs significantly inhibited production of markers of HBV replication without evidence of hepatotoxicity. Increased methylation of HBV DNA at CpG island II showed that the rTALEs caused intended epigenetic modification. CONCLUSIONS: Epigenetic modification of HBV DNA is a new and effective means of inactivating the virus in vivo. The approach has therapeutic potential and avoids potentially problematic unintended mutagenesis of gene editing.

Progress With Developing Use of Gene Editing To Cure Chronic Infection With Hepatitis B Virus.

Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population. Carriers of the virus are at risk for life-threatening complications, and developing curative treatment remains a priority. The main shortcoming of licensed therapies is that they do not affect viral covalently closed circular DNA (cccDNA), a stable intermediate of replication. Harnessing gene editing to mutate cccDNA provides the means to inactivate HBV gene expression permanently. Reports have described use of engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) nucleases. Although inhibition of viral replication has been demonstrated, reliably detecting mutations in cccDNA has been difficult. Also, the dearth of murine models that mimic cccDNA formation has hampered analysis in vivo. To reach a stage of clinical use, efficient delivery of the editors to HBV-infected hepatocytes and limiting unintended off-target effects will be important. Investigating therapeutic efficacy in combination with other treatment strategies, such as immunotherapies, may be useful to augment antiviral effects. Advancing gene editing as a mode of treating HBV infection is now at an interesting stage and significant progress is likely to be made in the immediate future.

Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus.

Chronic infection with hepatitis B virus (HBV) remains an important global health problem. Currently licensed therapies have modest curative efficacy, which is as a result of their transient effects and limited action on the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Gene editing with artificial HBV-specific endonucleases and use of artificial activators of the RNA interference pathway have shown anti-HBV therapeutic promise. Although results from these gene therapies are encouraging, maximizing durable antiviral effects is important. To address this goal, a strategy that entails combining gene editing with homology-directed DNA recombination (HDR), to introduce HBV-silencing artificial primary microRNAs (pri-miRs) into HBV DNA targets, is reported here. Previously described transcription activator-like effector nucleases (TALENs) that target the core and surface sequences of HBV were used to introduce double stranded breaks in the viral DNA. Simultaneous administration of donor sequences encoding artificial promoterless anti-HBV pri-miRs, with flanking arms that were homologous to sequences adjoining the TALENs' targets, augmented antiviral efficacy. Analysis showed targeted integration and the length of the flanking homologous arms of donor DNA had a minimal effect on antiviral efficiency. These results support the notion that gene editing and silencing may be combined to effect improved inhibition of HBV gene expression.

Recent advances in use of gene therapy to treat hepatitis B virus infection.

Chronic infection with hepatitis B virus (HBV) occurs in approximately 5 % of the world's human population and persistence of the virus is associated with serious complications of cirrhosis and liver cancer. Currently available treatments are modestly effective and advancing novel therapeutic strategies is a medical priority. Stability of the viral cccDNA replication intermediate is a major factor that has impeded the development of therapies that are capable of eliminating chronic infection. Recent advances that employ gene therapy strategies offer useful advantages over current therapeutics. Silencing of HBV gene expression by harnessing the RNA interference pathway has been shown to be highly effective in cell culture and in vivo. However, a potential limitation of this approach is that the post-transcriptional mechanism of gene silencing does not disable cccDNA. Early results using designer transcription activator-like effector nucleases (TALENs) and repressor TALEs (rTALEs) have shown potential as a mode of inactivating cccDNA. In this article, we review the recent advances that have been made in HBV gene therapy, with a particular emphasis on the potential anti-HBV therapeutic utility of designed sequence-specific DNA binding proteins and their derivatives.

Progress and Prospects of Anti-HBV Gene Therapy Development.

Despite the availability of an effective vaccine against hepatitis B virus (HBV), chronic infection with the virus remains a major global health concern. Current drugs against HBV infection are limited by emergence of resistance and rarely achieve complete viral clearance. This has prompted vigorous research on developing better drugs against chronic HBV infection. Advances in understanding the life cycle of HBV and improvements in gene-disabling technologies have been impressive. This has led to development of better HBV infection models and discovery of new drug candidates. Ideally, a regimen against chronic HBV infection should completely eliminate all viral replicative intermediates, especially covalently closed circular DNA (cccDNA). For the past few decades, nucleic acid-based therapy has emerged as an attractive alternative that may result in complete clearance of HBV in infected patients. Several genetic anti-HBV strategies have been developed. The most studied approaches include the use of antisense oligonucleotides, ribozymes, RNA interference effectors and gene editing tools. This review will summarize recent developments and progress made in the use of gene therapy against HBV.

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 cultured cells and in vivo using 2'-O-guanidinopropyl modified siRNAs.

Silencing hepatitis B virus (HBV) gene expression with exogenous activators of the RNA interference (RNAi) pathway has shown promise as a new mode of treating infection with the virus. However, optimizing efficacy, specificity, pharmacokinetics and stability of RNAi activators remains a priority before clinical application of this promising therapeutic approach is realised. Chemical modification of synthetic short interfering RNAs (siRNAs) provides the means to address these goals. This study aimed to assess the benefits of incorporating nucleotides with 2'-O-guanidinopropyl (GP) modifications into siRNAs that target HBV. Single GP residues were incorporated at nucleotide positions from 2 to 21 of the antisense strand of a previously characterised effective antiHBV siRNA. When tested in cultured cells, siRNAs with GP moieties at selected positions improved silencing efficacy. Stability of chemically modified siRNAs in 80% serum was moderately improved and better silencing effects were observed without evidence for toxicity or induction of an interferon response. Moreover, partially complementary target sequences were less susceptible to silencing by siRNAs with GP residues located in the seed region. Hydrodynamic co-injection of siRNAs with a replication-competent HBV plasmid resulted in highly effective knock down of markers of viral replication in mice. Evidence for improved efficacy, reduced off target effects and good silencing in vivo indicate that GP-modifications of siRNAs may be used to enhance their therapeutic utility.

Production, Characterization, and Assessment of Permanently Cationic and Ionizable Lipid Nanoparticles for Use in the Delivery of Self-Amplifying RNA Vaccines.

Africa bears the highest burden of infectious diseases, yet the continent is heavily reliant on First World countries for the development and supply of life-saving vaccines. The COVID-19 pandemic was a stark reminder of Africa's vaccine dependence and since then great interest has been generated in establishing mRNA vaccine manufacturing capabilities on the African continent. Herein, we explore alphavirus-based self-amplifying RNAs (saRNAs) delivered by lipid nanoparticles (LNPs) as an alternative to the conventional mRNA vaccine platform. The approach is intended to produce dose-sparing vaccines which could assist resource-constrained countries to achieve vaccine independence. Protocols to synthesize high-quality saRNAs were optimized and in vitro expression of reporter proteins encoded by saRNAs was achieved at low doses and observed for an extended period. Permanently cationic or ionizable LNPs (cLNPs and iLNPs, respectively) were successfully produced, incorporating saRNAs either exteriorly (saRNA-Ext-LNPs) or interiorly (saRNA-Int-LNPs). DOTAP and DOTMA saRNA-Ext-cLNPs performed best and were generally below 200 nm with good PDIs (<0.3). DOTAP and DDA saRNA-Int-cLNPs performed optimally, allowing for saRNA amplification. These were slightly larger, with higher PDIs as a result of the method used, which will require further optimization. In both cases, the N:P ratio and lipid molar ratio had a distinct effect on saRNA expression kinetics, and RNA was encapsulated at high percentages of >90%. These LNPs allow the delivery of saRNA with no significant toxicity. The optimization of saRNA production and identification of potential LNP candidates will facilitate saRNA vaccine and therapeutic development. The dose-sparing properties, versatility, and manufacturing simplicity of the saRNA platform will facilitate a rapid response to future pandemics.

Countering hepatitis B virus infection using RNAi: how far are we from the clinic?

Globally, persistent HBV infection is a significant cause of public health problems. Currently available HBV therapies have variable efficacy and there is a need to develop improved treatment to prevent cirrhosis and hepatocellular carcinoma. Although RNA interference (RNAi)-based approaches have shown promise, accomplishing safe and sustained silencing by RNAi activators, as well as their efficient delivery to hepatocytes have hampered clinical translation of this very promising technology. Expressed silencers may be produced in a sustained manner from stable DNA templates, which makes them suited to treatment of chronic HBV infection. DNA expression cassettes can be incorporated into both viral and non-viral vectors, but in vivo delivery of these cassettes with non-viral vectors is currently inefficient. Synthetic short interfering RNAs (siRNAs), which may be chemically modified to improve stability, specificity and efficacy, are more conveniently delivered to their cytoplasmic sites of action with synthetic non-viral vectors. However, the short duration of action of this class of RNAi activator is a drawback for treatment of chronic HBV infection. Despite the impressive progress that has been made in developing highly effective HBV gene silencers, challenges continue to face implementation of RNAi-based HBV therapy. This review will discuss the current status of the topic and consider the developments that are required to advance RNAi-based HBV therapy to clinical application.

Synthesis of 2'-O-guanidinopropyl-modified nucleoside phosphoramidites and their incorporation into siRNAs targeting hepatitis B virus.

Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3' ends. To improve efficacy of siRNAs, chemical modification at the 2'-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immunostimulation have been demonstrated using this approach. Although promising, efficient and controlled delivery of highly negatively charged nucleic acid gene silencers remains problematic. To assess the potential utility of introducing positively charged groups at the 2' position, our investigations aimed at assessing efficacy of novel siRNAs containing 2'-O-guanidinopropyl (GP) moieties. We describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to generate antihepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demonstrated that the GP modifications improved silencing. Moreover, thermodynamic stability was not affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic application of synthetic RNAi activators.

Advancing mRNA technologies for therapies and vaccines: An African context.

Synthetic mRNA technologies represent a versatile platform that can be used to develop advanced drug products. The remarkable speed with which vaccine development programs designed and manufactured safe and effective COVID-19 vaccines has rekindled interest in mRNA technology, particularly for future pandemic preparedness. Although recent R&D has focused largely on advancing mRNA vaccines and large-scale manufacturing capabilities, the technology has been used to develop various immunotherapies, gene editing strategies, and protein replacement therapies. Within the mRNA technologies toolbox lie several platforms, design principles, and components that can be adapted to modulate immunogenicity, stability, in situ expression, and delivery. For example, incorporating modified nucleotides into conventional mRNA transcripts can reduce innate immune responses and improve in situ translation. Alternatively, self-amplifying RNA may enhance vaccine-mediated immunity by increasing antigen expression. This review will highlight recent advances in the field of synthetic mRNA therapies and vaccines, and discuss the ongoing global efforts aimed at reducing vaccine inequity by establishing mRNA manufacturing capacity within Africa and other low- and middle-income countries.

Diversity of Dysregulated Long Non-Coding RNAs in HBV-Related Hepatocellular Carcinoma.

Infection with the hepatitis B virus (HBV) continues to pose a major threat to public health as approximately 292 million people worldwide are currently living with the chronic form of the disease, for which treatment is non-curative. Chronic HBV infections often progress to hepatocellular carcinoma (HCC) which is one of the world's leading causes of cancer-related deaths. Although the process of hepatocarcinogenesis is multifaceted and has yet to be fully elucidated, several studies have implicated numerous long non-coding RNAs (lncRNAs) as contributors to the development of HCC. These host-derived lncRNAs, which are often dysregulated as a consequence of viral infection, have been shown to function as signals, decoys, guides, or scaffolds, to modulate gene expression at epigenetic, transcriptional, post-transcriptional and even post-translational levels. These lncRNAs mainly function to promote HBV replication and oncogene expression or downregulate tumor suppressors. Very few lncRNAs are known to suppress tumorigenesis and these are often downregulated in HCC. In this review, we describe the mechanisms by which lncRNA dysregulation in HBV-related HCC promotes tumorigenesis and cancer progression.

Hepatitis B Virus Research in South Africa.

Despite being vaccine-preventable, hepatitis B virus (HBV) infection remains the seventh leading cause of mortality in the world. In South Africa (SA), over 1.9 million people are chronically infected with HBV, and 70% of all Black chronic carriers are infected with HBV subgenotype A1. The virus remains a significant burden on public health in SA despite the introduction of an infant immunization program implemented in 1995 and the availability of effective treatment for chronic HBV infection. In addition, the high prevalence of HIV infection amplifies HBV replication, predisposes patients to chronicity, and complicates management of the infection. HBV research has made significant progress leading to better understanding of HBV epidemiology and management challenges in the SA context. This has led to recent revision of the national HBV infection management guidelines. Research on developing new vaccines and therapies is underway and progress has been made with designing potentially curative gene therapies against HBV. This review summarizes research carried out in SA on HBV molecular biology, epidemiology, treatment, and vaccination strategies.

Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles.

Expressed polycistronic microRNA (miR) cassettes have useful properties that can be utilized for RNA interference (RNAi)-based gene silencing. To advance their application we generated modular trimeric anti-hepatitis B virus (HBV) Pol II cassettes encoding primary (pri)-miR-31-derived shuttles that target three different viral genome sites. A panel of six expression cassettes, comprising each of the possible ordering combinations of the pri-miR-31 shuttles, was initially tested. Effective silencing of individual target sequences was achieved in transfected cells and transcribed pri-miR trimers generated intended guide strands. There was, however, variation in processing and silencing by each of the shuttles. In some cases the monomers' position within the trimers influenced processing and this correlated with target silencing. Compromised efficacy could be compensated by substituting the pri-miR-31 backbone with a pri-miR-30a scaffold. Inhibition of HBV replication was achieved in vivo, and in cell culture without disruption of endogenous miR function or induction of the interferon response. A mutant HBV target sequence, with changes in one of the guide cognates, was also silenced by the trimeric cassettes. The modular nature of the cassettes together with compatibility with expression from Pol II promoters should be advantageous for gene silencing applications requiring simultaneous targeting of different sites.

Recent Update on the Role of Circular RNAs in Hepatocellular Carcinoma.

After being overlooked for decades, circular RNAs (circRNAs) have recently generated considerable interest. circRNAs play a role in a variety of normal and pathological biological processes, including hepatocarcinogenesis. Many circRNAs contribute to hepatocarcinogenesis through sponging of microRNAs (miRs) and disruption of cellular signaling pathways that play a part in control of cell proliferation, metastasis and apoptosis. In most cases, overexpressed circRNAs sequester miRs to cause de-repressed translation of mRNAs that encode oncogenic proteins. Conversely, low expression of circRNAs has also been described in hepatocellular carcinoma (HCC) and is associated with inhibited production of tumor suppressor proteins. Other functions of circRNAs that contribute to hepatocarcinogenesis include translation of truncated proteins and acting as adapters to regulate influence of transcription factors on target gene expression. circRNAs also affect hepatocyte transformation indirectly. For example, the molecules regulate immune surveillance of cancerous cells and influence the liver fibrosis that commonly precedes HCC. Marked over- or under-expression of circRNA expression in HCC, with correlating plasma concentrations, has diagnostic utility and assays of these RNAs are being developed as biomarkers of HCC. Although knowledge in the field has recently surged, the myriad of described effects suggests that not all may be vital to hepatocarcinogenesis. Nevertheless, investigation of the role of circRNAs is providing valuable insights that are likely to contribute to improved management of a serious and highly aggressive cancer.

In vitro transcribed mRNA for expression of designer nucleases: Advantages as a novel therapeutic for the management of chronic HBV infection.

Chronic infection with the hepatitis B virus (HBV) remains a significant worldwide medical problem. While diseases caused by HIV infection, tuberculosis and malaria are on the decline, new cases of chronic hepatitis B are on the rise. Because often fatal complications of cirrhosis and hepatocellular carcinoma are associated with chronic hepatitis B, the need for a cure is as urgent as ever. Currently licensed therapeutics fail to eradicate the virus and this is attributable to persistence of the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Elimination or inactivation of the viral cccDNA is thus a goal of research aimed at hepatitis B cure. The ability to engineer nucleases that are capable of specific cleavage of a DNA sequence now provides the means to disable cccDNA permanently. The scientific literature is replete with many examples of using designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) to inactivate HBV. However, important concerns about safety, dose control and efficient delivery need to be addressed before the technology is employed in a clinical setting. Use of in vitro transcribed mRNA to express therapeutic gene editors goes some way to overcoming these concerns. The labile nature of RNA limits off-target effects and enables dose control. Compatibility with hepatotropic non-viral vectors is convenient for the large scale preparation that will be required for advancing gene editing as a mode of curing chronic hepatitis B.

Hepatitis B virus: promising drug targets and therapeutic implications.

Introduction: Current therapy for infection with hepatitis B virus (HBV) rarely clears the virus, and viremia commonly resurges following treatment withdrawal. To prevent serious complications of the infection, research has been aimed at identifying new viral and host targets that can be exploited to inactivate HBV replication.Areas covered: This paper reviews the use of these new molecular targets to advance anti-HBV therapy. Emphasis is on appraising data from pre-clinical and early clinical studies described in journal articles published during the past 10 years and available from PubMed.Expert opinion: The wide range of viral and host factors that can be targeted to disable HBV is impressive and improved insight into HBV molecular biology continues to provide the basis for new drug design. In addition to candidate therapies that have direct or indirect actions on HBV covalently closed circular DNA (cccDNA), compounds that inhibit HBsAg secretion, viral entry, destabilize viral RNA and effect enhanced immune responses to HBV show promise. Preclinical and clinical evaluation of drug candidates, as well as investigating use of treatment combinations, are encouraging. The field is poised at an interesting stage and indications are that reliably achieving functional cure from HBV infection is a tangible goal.

Improved Specificity and Safety of Anti-Hepatitis B Virus TALENs Using Obligate Heterodimeric FokI Nuclease Domains.

Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral core and surface open reading frames were modified with second- and third-generation FokI nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of FokI homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication.

In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors.

The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals' potency.

tRNA Lys3 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.