Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses AbstractChronic infection with hepatitis B virus (HBV) puts individuals at high risk for complicating cirrhosis and liver cancer, but available treatment to counter the virus rarely eliminates infection. Although harnessing RNA interference (RNAi) to silence HBV genes has shown the potential, achieving efficient and durable silencing of viral genes remains an important goal. Here we report on the propagation of lentiviral vectors (LVs) that successfully deliver HBV-targeting RNAi activators to liver cells. Mono- and tricistronic artificial primary microRNAs (pri-miRs) derived from pri-miR-31, placed under transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, caused efficient inhibition of HBV replication markers. The tricistronic cassette was capable of silencing a mutant viral target and the effects were observed without disrupting the function of an endogenous miR (miR-16). The mTTR promoter stably expressed a reporter transgene in mouse livers over a study period of 1 year. Good silencing of HBV genes, without evidence of toxicity, was demonstrated following intravenous injection of LVs into neonatal HBV transgenic mice. Collectively, these data indicate that LVs may achieve sustained inhibition of HBV replication that is appealing for their therapeutic use. IntroductionThe hepatitis B virus (HBV) is a non-cytopathic human virus that causes both acute and chronic infection of the liver. It is estimated that over 2 billion people have been infected with the virus and more than 350 million individuals have progressed to chronicity.1 Persistent infection with HBV is associated with an increased risk for serious life-threatening complications such as cirrhosis and hepatocellular carcinoma.2 This common malignancy is aggressive and has a very poor prognosis. Currently available HBV therapies, such as nucleoside and nucleotide analogs, have variable efficacy.3,4 Following treatment withdrawal, HBV replication typically rebounds. This is a result of persistence of the viral covalently closed circular DNA, and resistance of this viral replication intermediate to available therapies.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 improved therapy to counter chronic HBV infection. Expressed anti-HBV RNAi-based sequences are capable of mediating impressive silencing of viral gene expression in vitro and in vivo.5, 6, 7, 8 Renewable formation of gene silencers from stable DNA cassettes enables more durable silencing of target genes by expressed RNAi activators, which is a distinct advantage over synthetic small interfering RNAs. Accomplishing stable delivery of expressed HBV gene silencers to hepatocytes with sustained, long-term expression of therapeutic effecters remains the greatest challenge impeding the clinical translation of RNAi-based anti-HBV gene therapy. Adenoviral7, 8, 9 and adeno-associated viral (AAV)10,11 vectors have been used to achieve efficient hepatic delivery and expression of RNAi effecters following systemic administration. However, complications of immunostimulation and expression that may not be sustained compromise the use of these vectors for HBV therapy. Recombinant lentiviral vectors (LVs) derived from HIV-112 have the ability to mediate stable transduction of dividing and non-dividing cells,13, 14, 15 which allows for long-term and potentially indefinite gene expression in hepatocytes in vivo.16, 17, 18, 19 These properties indicate that recombinant LVs may be advantageous for gene transfer of expressed RNAi anti-HBV sequences and for accomplishing long-term sustained silencing of HBV gene expression.To advance RNAi-based anti-HBV gene therapy, second-generation self-inactivating (SIN) LVs were engineered to include liver-specific, RNA polymerase (Pol) II cassettes that generate artificial HBV-silencing primary microRNAs (pri-miRs). The anti-viral pri-miR sequences, placed under the control of the liver-specific murine transthyretin (mTTR) promoter,20 are derived from a natural pri-miR-31 scaffold.5,6 These artificial pri-miR cassettes have the advantage of being safer, amenable to multimerization and compatible with tissue-specific Pol II promoters.6,21 They were engineered to generate anti-HBV RNAi guide sequences targeting one (monocistronic cassette) or three (tricistronic cassette) sites within the conserved multifunctional HBV X (HBx) open reading frame (ORF) of the viral genome. Several lines of evidence implicate HBx in hepatocarcinogenesis.2,22,23 Targeting this ORF should thus counter the hepatocarcinogenic process as well as replication of the virus. The potential utility of the novel HBV silencing LVs to mediate stable and long-term knockdown of HBV gene expression in vitro and in vivo was explored and extensive evaluation of the anti-viral efficacy of the anti-HBV LVs was carried out in cultured liver-derived cells and in HBV transgenic mice. Sustained and good efficacy of inhibition of HBV replication was demonstrated, which indicates that the approach has therapeutic potential.ResultsEngineering second-generation SIN HBV-silencing LVsSecond-generation SIN LVs expressing liver-specific HBV silencing pri-miR mimics were propagated from lentivectors based on the pLVTH19 plasmid backbone (Figure 1a). pLVTH harbors a central polypurine tract (cPPT) sequence, which has been reported to facilitate efficient transduction of quiescent hepatocytes. In addition, the presence of LoxP sites in the 3鈥?long terminal repeat (3鈥?LTR), which are copied to the 5鈥?LTR during reverse transcription, allows for conditional ablation of LV sequences integrated into the hepatocyte genome following Cre-recombinase treatment of transduced cells.24 Engineering the HBV-silencing LVs involved consideration of various factors that could potentially influence the titers of the LV preparations as well as expression of the HBV-targeting RNAi activators. When used to express RNAi activators, the promiscuous cytomegalovirus (CMV) Pol II promoter has been shown to cause severe LV titer reduction.25 In addition, shutdown of CMV promoter activity in the murine liver has been reported,26 which makes this promoter unsuitable for applications where long-term in vivo expression is desirable. To control transcription of previously characterized, effective mono- and tricistronic anti-HBV pri-miR-31 expression cassettes,5,6 the CMV element was replaced with the liver-specific modified mTTR transcriptional regulatory element.20 Achieving liver-specific expression with the mTTR promoter should also eliminate off-target effects that could arise from constitutive expression of the pri-miR mimics in non-targeted tissues. The expressed pri-miR mimics are derived from a natural pri-miR-31 scaffold and have been engineered to generate anti-HBV RNAi guide sequences targeting single (pri-miR-31/5) or three (pri-miR-31/5-8-9) sites within the HBx ORF of the HBV genome (Figure 1b and Supplementary Figure 1).5,6 Targeting of the conserved multifunctional HBx sequence27,28 that is contained within all viral transcripts enables simultaneous knockdown of all HBV mRNAs.Figure 1Schematic illustration of pLVTH-derived HBV silencing LVs, processing and integration of LV-delivered anti-HBV pri-miR mimics in liver-derived cells. (a) Schematic of the pLVTH mTTR miR-31/5 and pLVTH mTTR miR-31/5-8-9 LVs. Monocistronic and tricistronic anti-HBV pri-miR sequences were placed under the control of the mTTR promoter. pLVTH vectors also contain a reporter cassette, under the control of the EF1-伪 promoter, encoding eGFP. The HIV-1 packaging signal (蠄), Rev-response element (RRE), cPPT and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) are indicated. The 3鈥?LTR contains a self-inactivating sequence and LoxP site that is duplicated in the 5鈥?LTR following reverse transcription. (b) Schematic illustration of the artificial anti-HBV mono- and tricistronic pri-miR transcripts that generate guide 5 alone or a combination of guides 5, 8 and 9. (c) Northern blot analysis using RNA extracted from Huh7 cells, which had been stably transduced with LVTH, LVTH mTTR miR-31/5 and LVTH mTTR miR-31/5-8-9 LV preparations. Bands corresponding to the guide (G) or precursor (P) sequences of the putative guides are indicated. Blots were stripped and rehybridized to a probe complementary to endogenous small nuclear U6 RNA to confirm equal loading of cellular RNA. (d) Genomic DNA extracted from LVTH mTTR miR-31/5-8-9-transduced Huh7 cells was analyzed for pri-miR sequence recombination using PCR. Primers, with positions indicated in panel a, generate a band of 493鈥塨p. pLVTH mTTR miR-31/5-8-9 plasmid DNA (lane 1) and genomic DNA extracted from untransduced Huh7 cells (lane 3) were included as positive and negative controls, respectively. SIN 3鈥?LTR, self-inactivating long terminal repeat with a deletion in the U3 region.Full size imageExpression cassettes were incorporated into pLVTH in the sense orientation with respect to the viral genome without a polyadenylation signal (Figure 1a). Exclusion of the polyadenylation signal from the transgene cassette facilitated transcription of the full-length LV genomic transcripts and improvement of the viral titer.29 This vector design was intended to improve stability of the pri-miR transcript and enable robust expression of mature miR sequences.25 Drosha-mediated recognition and processing of miR precursors of the viral genome may result in the production of truncated, non-functional genomic vector transcripts and reduced viral titers.25 Nevertheless, efficient production of anti-HIV LVs expressing miR precursors has been reported.30 In accordance with this, we did not observe significant differences when comparing the titers of control LVTH LV and pri-miR-encoding HBV-silencing LVs. The lack of an effect may be attributed to Drosha saturation in the producer cells and Rev response element-mediated export of unspliced and singly spliced genomic lentiviral RNA from the nucleus.31Processing of LV-delivered anti-HBV pri-miR mimics in transduced liver-derived cellsLiver-derived Huh7 cells were transduced with vesicular stomatitis virus glycoprotein-pseudotyped LVTH, LVTH mTTR miR-31/5 and LVTH mTTR miR-31/5-8-9 lentiviral preparations. Genomic quantitative PCR (qPCR) analysis revealed that the LVTH and LVTH mTTR miR 31/5 LVs were integrated at a copy number of 1.8 per cell, whereas LVTH mTTR miR-31/5-8-9-transduced cells had 1.2 copies of integrated LV per genome of Huh7 cells. To determine whether the integrated artificial anti-HBV pri-miR sequences were being processed according to the intended design, low-molecular-weight Northern blot analysis was carried out. Putative guide sequences of 20鈥?2 nucleotides were detected following hybridization with probes complementary to anticipated mature miR-31/5, miR-31/8 and miR-31/9 sequences (Figure 1c). Quantification of relative band intensities showed variation in the amounts of individual guides derived from the tricistronic pri-miR-31/5-8-9 scaffold. The guide 5 sequence was present in high quantity in stably transduced cells, followed by guide 8, whereas the guide 9 sequence was least abundant. These observations are in agreement with our previous analysis following transient transfection of Huh7 cells with equivalent cassettes.6 The concentration of individual mature guide sequences correlated with silencing efficacy and signal intensity was not dependent on melting temperatures of the probe-guide hybrids. Variability in processing of the components of the tricistronic pri-miRs is likely to be the cause of variation in the concentrations of guide RNAs. Recombination events between repeated sequences in the lentiviral genome may occur during propagation of the recombinant virus,32 and deletion of pri-miR sequence in the context of a tricistronic scaffold has been reported previously.30,31 To assess whether recombination had occurred between equivalent sequences in the tricistronic pri-miR-31/5-8-9 cassette during LV transduction, genomic DNA extracted from LVTH mTTR miR-31/5-8-9-transduced Huh7 cells was analyzed by PCR (Figure 1d). A single band of 493鈥塨p, corresponding to the size of the mTTR pri-miR-31/5-8-9 cassette, was amplified from LVTH mTTR miR-31/5-8-9 genomic DNA. Additional smaller size bands that could be indicative of sequence rearrangements containing deletions of the integrated pri-miR 31/5-8-9 scaffold were not observed. Background smearing is noticeable, which could be interpreted as evidence for some recombination occurring during LV transduction, but this DNA was not abundant. Presence of a dominant band of expected size indicates that an intact tricistronic pri-miR-31/5-8-9 cassette is present in the genome of most transduced cells.Assessment of knockdown of wild-type and a mutant target sequences of HBx in LV-transduced Huh7 cellsSimultaneous expression of several anti-HBV miR sequences from tricistronic cassettes could be potentially valuable in limiting the emergence of viral escape mutants.6 The rationale underlying this approach is that concurrent silencing of multiple targets provides a greater barrier to the virus. Evasion of the inhibitory effects of the trimeric silencer by HBV would require coinciding mutations of the virus, which seems unlikely. To assess the ability of the LV-delivered monocistronic and tricistronic pri-miR mimics to knock down a mutated HBV sequence, a dual-luciferase reporter assay was undertaken (Figure 2). Mutant and wild-type HBx sequences were inserted downstream of the Renilla luciferase ORF in a psiCHECK-derived plasmid. Silencing efficacy was then determined by calculating the ratio of Renilla to Firefly luciferase activity. Although the monocistronic anti-HBV pri-miR-31/5 sequence was capable of mediating potent inhibition of the wild-type HBx target, only modest suppression of the reporter containing the mutant sequence was observed. In contrast, LV-delivered tricistronic pri-miR-31/5-8-9 sequences effectively silenced the expression of both wild-type and mutated HBx, indicating that the tricistronic anti-HBV pri-miR configuration could be potentially useful in improving effectiveness against viral escape mutants.Figure 2Dual-luciferase reporter assay to detect silencing of wild-type and mutant HBx target sequences by mono- and tricistronic sequences. (a) The psiCHECK-HBx and psiCHECK-mHBx dual-luciferase reporter vectors include the entire wild-type or mutant HBx target sequence, respectively, downstream of the Renilla luciferase ORF (RLuc). The guide 5 sequence is indicated, and mutations within the mutant HBx cognate are italicized and underlined. RLuc- and Firefly luciferase (FLuc)-encoding sequences are under the control of constitutively active Simian virus 40 early (SV40) and herpes simplex virus thymidine kinase (HSV TK) promoters, respectively. (b) Assessment of knockdown efficacy of LVs containing monomeric or trimeric HBV-targeting pri-miRs. Data are represented as the means and the error bars indicate the standard errors of the mean. Statistically significant differences are indicated by asterisks (*P 0.05).Full size imageAssessment of disruption of function of an endogenous miR by HBV-targeting artificial pri-miRs in stably transduced Huh7 cellsExpression of artificial pri-miRs from Pol II promoters has been reported to achieve safer silencing than highly active and constitutive Pol III short hairpin RNA expression cassettes.6,21 Evidence of saturation of the natural miR biogenesis pathway following AAV8-mediated anti-HBV short hairpin RNA delivery has highlighted the need for careful analysis of unintended toxic effects following the delivery of exogenous RNAi effecters.10 To address this concern, a dual-luciferase saturation assay (Figure 3), which involved the assessment of translational repression by miR-16, was performed to determine whether the LV-delivered anti-HBV pri-miR mimics cause perturbations in cellular function of miR-16.6,33 A psiCHECK-derived dual-luciferase reporter vector that encodes seven copies of an imperfect miR-16 target downstream of the Renilla luciferase ORF (psiCHECK-miR-16T 脳 7) was used to assess perturbations in miR-16 function (Figure 3a).6 A sponge plasmid encoding seven partly complementary miR-16 target sites (pTZ-U6-miR-16S 脳 7) was used as a control for loss of miR-16 silencing activity (Figure 3b). miR-16 is widely expressed in a range of tissues and is useful to assess disruption of the endogenous miR-processing pathway.33 As expected, co-transfection of the miR-16 target vector (psi-miR-16T 脳 7) and control miR-16 sponge plasmid (pTZ-U6-miR-16S 脳 7) in LVTH-transduced Huh7 cells caused derepression of miR-16 silencing function (Figure 3c). In contrast, no disruption of miR-16 function was observed in LVTH-, LVTH mTTR miR-31/5- and LVTH mTTR miR-31/5-8-9-transduced cells transfected with the psi-miR-16T 脳 7 vector. These data indicate that LV-delivered anti-HBV pri-miR mimics do not have a repressive effect on endogenous miR-16 silencing activity and are not likely to cause saturation of cellular miR function.Figure 3Assessment of effects of lentiviral vector-delivered artificial pri-miRs on endogenous miR-16 function. (a) Schematic illustration to show dual-luciferase psiCHECK-derived vector with seven copies of the miR-16 target inserted downstream of the Renilla luciferase ORF. Firefly luciferase constitutively expressed from the same plasmid was used to normalize data. (b) Schematic illustration of the expression cassette that generates a transcript containing seven copies of an imperfectly matched miR-16 target. The transcript contains 5鈥?27 and 3鈥?stem sequences for improved stability of U6 Pol III transcripts. (c) Analysis of effects of LV-delivered expression cassettes on endogenous miR-16 repression of target reporter sequence using a dual-luciferase assay. The reporter plasmid, containing seven copies of miR-16 target inserted downstream of the Renilla luciferase ORF, was transfected into cells after infection with the control LVTH vector or indicated LVs expressing anti-HBV artificial pri-miRs. Ratio of Renilla to Firefly luciferase activity was measured to assess derepression of endogenous miR-16 by coexpressed miR shuttles. Data are represented as the means and the error bars indicate the standard errors of the mean. Statistically significant differences are indicated by asterisks (*P 0.05 and **P 0.01).Full size imageIntegrated anti-HBV pri-miR mimics mediate inhibition of markers of HBV replication in stably transduced HepG2.2.15 cellsThe HepG2.2.15 cell line was generated by transforming the HepG2 human hepatoblastoma cell line with dimeric HBV DNA34 and is useful for stringent evaluation of anti-HBV agents. HepG2.2.15 cells were transduced with vesicular stomatitis virus glycoprotein-pseudotyped LVTH and LVTH mTTR miR-31/5-8-9 lentiviral preparations. Genomic qPCR analysis revealed that the LVTH LV was integrated at a copy number of 1.5 per cell, whereas LVTH mTTR miR-31/5-8-9-transduced cells had 1.8 copies of integrated LV per genome of Huh7 cells. To assess the anti-viral effects, HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion into the supernatant of transduced cells were measured using a quantitative enzyme-linked immunosorbent assay at 48, 72 and 96鈥塰 after seeding of cells (Figures 4a and b). Significant inhibition of both HBsAg and HBeAg secretion was observed in LVTH mTTR miR-31/5-8-9-transduced HepG2.2.15 cells. In addition, a significant reduction in the number of HBV viral particle equivalents (VPEs), measured using qPCR, was observed in the culture supernatants from HepG2.2.15 cells that had been transduced with the pri-miR-31/5-8-9-encoding LV (Figure 4c). This effect was maintained over the 96-h time period. Importantly, the cell culture medium was not replenished for the duration of the experiment and the LV-delivered pri-miR-31/5-8-9 therapeutic sequence mediated stable inhibition of HBsAg, HBeAg and VPEs despite the rapid rate of virion secretion by HepG2.2.15 cells.Figure 4Anti-viral efficacy of integrated anti-HBV pri-miR mimics in stably transduced liver-derived cells. Concentrations of viral particle equivalents (VPEs) (a), HBeAg (b) and HBsAg (c) in cell culture supernatants of HepG2.2.15 cells measured over a time period of 96鈥塰 after transduction with indicated LVs. HBsAg and HBeAg measurements from quantitative enzyme-linked immunosorbent assay (ELISA) are given as a normalized mean relative to LVTH-transduced cells. HBV VPEs were measured in LVTH- and LVTH mTTR miR-31/5-8-9-transduced HepG2.2.15 cells at 48, 72 and 96鈥塰. HBV DNA secreted from stably transduced cells was determined by real-time quantitative PCR using HBV-specific primers. Data are represented as the means and the error bars indicate the standard errors of the mean. Statistically significant differences are indicated by asterisks (*P 0.05 and **P 0.01).Full size imageThe mTTR promoter is capable of driving long-term stable transgene expression in vivoSeveral groups have demonstrated stable transfer of therapeutic sequences to the liver following systemic delivery of recombinant LVs.19,35, 36, 37, 38 However, the transduction efficiency of recombinant LVs is influenced by proliferation and cell cycle activation of targeted hepatocytes.13 Transduction following systemic delivery of recombinant LVs has been reported to be significantly higher in young and newborn rodents as a result of more rapid hepatocyte proliferation than occurs in adult animals.37,39, 40, 41 To assess long-term transgene expression using bioluminescence imaging in individual animals following LV administration, a Firefly luciferase transgene was included in the recombinant LVs. The reporter gene was placed under the control of the liver-specific mTTR promoter in the pLNT-mTTR-JDG, which was used to generate LVs (Figure 5a). LVs expressing Firefly luciferase under the control of the mTTR promoter were administered intravenously via the superficial temporal vein. Stable expression was recorded in the livers of animals over a period of 12 months (Figures 5b and c). These data corroborate previous reports demonstrating long-term liver-specific activity of the mTTR promoter in vivo.37,42 Moreover, the results indicate that the mTTR promoter is suitable for long-term hepatospecific expression of HBV-targeting artificial pri-miRs.Figure 5Long-term follow-up of Firefly luciferase reporter expression following systemic delivery of LNT-mTTR-JDG to HBV transgenic mice. (a) Schematic of the pLNT-mTTR-JDG lentiviral vector. The vector comprises a 2A-linked Firefly luciferase-eGFP bicistronic expression cassette (FLuc-2A-eGFP) downstream of an internal mTTR promoter. Other lentiviral vector components are similar to those described in Figure 1a. (b) Outline of experiments carried out to measure Firefly luciferase activity. Neonatal HBV transgenic mice received intravenous injections of LNT-mTTR-JDG LV (6 脳 107 transducing units (TU) per mouse) or PBS at days 0 and 1 after birth. (c) Firefly luciferase expression was analyzed using bioluminescence imaging. Images of two LNT-mTTR-JDG LV-injected mice (right on each panel) and control animals injected with saline (left on each panel) are shown.Full size imageAn HBV transgenic mouse model43 was then used to assess the efficacy of the anti-HBV LVs in vivo. These animals contain an integrated unmutated replication-competent viral sequence and constitutive HBV replication in this murine model mimics chronic HBV infection of humans.43,44 pLVTH- and pLVTH mTTR miR-31/5-8-9-derived LVs were further modified to include a 2A-linked Firefly luciferase-eGFP bicistronic expression cassette, thereby generating the LVTH-Luc and LVTH-Luc miR-31/5-8-9 LVs (Supplementary Figure 2a). The reporter gene cassette of these vectors was under the control of the EF1-伪 promoter which, unlike the mTTR transcriptional regulatory element, is not hepatospecific. Control LVTH-Luc or HBV-silencing LVTH-Luc miR-31/5-8-9 LVs were administered to neonatal HBV transgenic mice via the superficial temporal vein. Bioluminescence imaging was performed to analyze Firefly luciferase expression in animals that had received recombinant LV. Stable LV-mediated liver transduction was achieved as evidenced by the variable but sustained Firefly luciferase expression observed in the livers of LV-injected animals (Supplementary Figure 2b). In several animals, transgene expression was also evident at the site of LV injection and is likely to result from transduction of tissue surrounding the injection site. Importantly, reporter gene expression in animals receiving the LVTH-Luc miR-31/5-8-9 vector is constitutive, unlike the liver-specific reporter transcription of the LNT-mTTR-JDG vector (Figure 5a). In animals receiving the LNT-mTTR-JDG LVs, no extrahepatic reporter gene expression was observed (Figure 5c). The content of vector DNA in the livers of LV-injected mice that received recombinant LV was quantified using qPCR. Average vector DNA levels in the livers of mice that received LVs via superficial temporal vein as neonates were 0.45 copies per hepatocyte genome. This indicates that the hepatocytes of the animals were transduced efficiently following administration in vivo.The LVTH-Luc miR-31/5-8-9 LV silences HBV replication in vivo without evidence for toxicityTo determine the anti-viral efficacy of the LVTH-Luc miR-31/5-8-9 LV, the concentration of HBsAg was measured in the sera of LV-injected animals. Significant reduction of serum HBsAg was observed in animals that received the vectors expressing the HBV-targeting LV (Figure 6). Long-term stable inhibition of HBsAg secretion was maintained for the 10-week duration of the follow-up (Figure 6a). Real-time qPCR analysis of circulating VPEs revealed similar trends in this marker of viral replication (Figure 6b). Data generated from the neonatal temporal injection model indicate that immune tolerance of young animals allows for vector persistence and shows that the HBV-silencing LVTH-Luc miR-31/5-8-9 LVs are capable of mediating impressive anti-viral effects.Figure 6Evaluation of long-term effects in vivo of HBV-targeting LVs on HBsAg production, circulating VPEs and intrahepatic viral RNA. (a) One-day-old HBV transgenic mice received LVTH-Luc (open symbols in gray) or LVTH-Luc miR-31/5-8-9 LV (solid black symbols) intravenously via the temporal vein. Each neonate received 2.7 脳 107 transducing units (TU) vector particles. HBsAg in serum samples was measured thereafter for a 10-week period. (b) Circulating VPEs were measured using real-time quantitative PCR. (c) Total RNA was isolated at necropsy from the livers of HBV transgenic mice that had been injected with LVTH-Luc or LVTH-Luc mTTR miR-31/5-8-9 LV. The concentration of surface HBV mRNA was determined by a quantitative reverse transcriptase PCR. Average surface mRNA levels relative to GAPDH mRNA from individual mice are shown (mean卤s.e.m., n猢?/span>3, *P猢?/span>0.05).Full size imageEffects of the HBV-silencing LVs on hepatic viral RNA concentrations were assessed by using quantitative reverse transcriptase PCR. A considerable reduction in intrahepatic HBV surface mRNA concentration relative to GAPDH mRNA was observed in transgenic mice that received therapeutic LVs (Figure 6c). This effect was not as marked as that on circulating VPEs, and is likely to be caused by the surface-containing mRNA being present in great abundance within HBV-replicating hepatocytes. Quantitative reverse transcriptase-PCR was also carried out to measure intrahepatic presence of the sequence corresponding to the mature guide 5 strand (Supplementary Table 1). The guide strand was not detectable in animals receiving the control vectors, but was readily detectable in animals that had received the LVs expressing the anti-HBV pri-miR sequences.To exclude toxicity of LVs, alanine transaminase activity was measured in serum samples from mice that had received the anti-HBV RNAi-activating cassettes (Supplementary Table 2). Values were not raised above the normal range ( 100鈥塙鈥塴鈭?) and indicate that the mTTR Pol II pri-miR expression cassettes are not toxic. This finding is in accordance with the notion that Pol II expression cassettes are safer than their Pol III short hairpin RNA counterparts. Collectively, our findings indicate that the HBV-silencing LV encoding the tricistronic pri-miR-31/5-8-9 sequence mediates highly effective knockdown of markers of HBV replication without evidence for toxicity.DiscussionPersistent infection with HBV remains an important public health problem as the risk for serious life-threatening complications such as cirrhosis and hepatocellular carcinoma is significantly increased in chronic HBV carriers.22 Hepatocellular carcinoma is the sixth most common cancer globally45 and this aggressive malignancy has a very poor prognosis.22 The efficacy of currently available HBV treatments is variable. Although potent suppression of viral replication and an improved barrier to viral resistance has been reported with some of the newer nucleoside/nucleotide analogs, such as entecavir and tenofovir,46 cessation of treatment typically results in rebound of viral replication. This is a result of the inability of this class of therapeutic agents to eradicate nuclear covalently closed circular DNA from infected hepatocytes.4 Persistence of the episomal covalently closed circular DNA minichromosome47 allows for continual transcription of all HBV mRNA transcripts and perpetuation of viral replication.48 Consequently, the risk of developing hepatocellular carcinoma remains unchanged in the majority of chronic carriers receiving treatment and this is perhaps the most significant limitation of currently licensed therapies for HBV. Use of customized HBV-targeting nucleases, such as transcription activator-like effector nucleases49 and the clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated system,35 has emerged as an efficient method of inhibiting viral replication. However, mutagenic effects of cleaving HBV DNA that is integrated into host genomic sequences may cause unintended mutations. Harnessing RNAi to silence HBV replication therefore remains an appealing method of using gene therapy to treat persistent HBV infection.The success observed using anti-HBV RNAi-based gene therapy in preclinical studies has generated enthusiasm for using this gene silencing technology for treating HBV infection.5, 6, 7 Successful treatment of persistent HBV infection with RNAi-based therapy will require long-term, sustained expression of active therapeutic effecters to suppress viral replication successfully. Although effective anti-HBV sequences have been described by several groups, including our own, accomplishing safe and efficient hepatic delivery of anti-viral RNAi effecters remains the greatest hurdle impeding clinical translation of RNAi-based anti-HBV gene therapy. To accomplish durable silencing of persistent HBV infection, long-term and perhaps indefinite expression of therapeutic RNAi effecters, which is not attainable with the existing adenoviral50, 51, 52 and AAV vectors,53,54 is likely to be required. Recombinant LVs derived from HIV-112 have emerged as versatile and efficient transfer tools as a consequence of their unique ability to mediate stable transduction of dividing and non-dividing cells, which allows for long-term and potentially indefinite gene expression. Adenoviral7, 8, 9,55 and adeno-associated viral10,11 vectors have been used to achieve efficient hepatic delivery and sustained expression of RNAi effecters following systemic administration, but the durability of transgene expression may not be as long-lasting as that of LVs. Biosafety of LVs is an important consideration.56 The vectors used here have several modifications that are known to improve their biosafety features: (1) deletion of promoter and enhancer elements to generate SIN vectors;57 (2) splitting genomic sequences necessary for virus propagation to reduce the likelihood of recombination and formation of replication-competent viruses; (3) pseudotyping the vectors with the heterologous viral vesicular stomatitis virus glycoprotein protein58 and (4) incorporation of LoxP sites to enable removal of proviral sequences with Cre recombinase.To advance potential clinical application of RNAi-based anti-HBV gene therapy, we designed and evaluated RNAi-activating recombinant LVs in cultured liver derived cells and in an animal model of HBV replication. This approach enabled sustained knockdown of HBV gene expression, which will be useful to eradicate persistent viral infection. Moreover, we showed that the liver-specific mTTR promoter was capable of mediating stable and long-term (猢?/span>12 months) expression of the Firefly luciferase transgene in HBV transgenic mice, which had received Firefly luciferase-expressing LVs. Artificial anti-HBV pri-miRs were processed according to the intended design and mediated powerful knockdown of circulating markers of viral replication and intrahepatic viral RNA without inducing hepatotoxicity. To our knowledge this is the first study to evaluate the efficacy of RNAi-activating anti-HBV LVs in the HBV transgenic mouse model.The LVs described here may be beneficial for advancing clinical application of RNAi-based gene therapy for other persistent hepatic viral infections, such as hepatitis C virus. Another interesting potential application is through ex vivo modification of hepatocytes or induced hepatocyte progenitors. Feasibility of ex vivo LV transduction of hepatocytes and transplantation has been demonstrated in non-human primates.59 Modifying induced pluripotent stem cells and multipotent progenitor cells60 with LVs ex vivo, to confer RNAi-based resistance to HBV infection, followed by reinfusion into patients, would present a powerful and novel means of treating chronic HBV infection.Materials and methodsConstruction of plasmids encoding LVsThe liver-specific mTTR promoter enhancer sequence was amplified from the pRRLsin.cPPT.mTTR.WPRE lentiviral plasmid61 (Expand High Fidelity PCRPLUS System; Roche Diagnostics GmbH, Mannheim, Germany) using oligonucleotides with the following sequences: mTTR forward, 5鈥?IndexTermGATCAGATCTGTCGACAAAATTTTATCGATCACGAGACTAGC-3鈥?and mTTR reverse, 5鈥?IndexTermGATCGCTCACCATGGTGGTGGCAGGAGCTT-3鈥? The PCR product was inserted into the PCR cloning vector pTZ57R/T (InsTAclone PCR Cloning Kit; Thermo Scientific, Waltham, MA, USA) to form the pTZ-mTTR plasmid. To generate the monocistronic (pmTTR pri-miR-31/5) and tricistronic (pmTTR pri-miR-31/5-8-9) pri-miR expression vectors, the CMV immediate-early enhancer promoter sequence was removed from the previously described pCMV pri-miR-31/55 and pCMV pri-miR-31/5-8-96 vectors after restriction with BglII and SacI. The mTTR promoter was also excised from pTZ-mTTR using BglII and SacI and then ligated to the pCMV pri-miR-31/5 and pCMV pri-miR-31/5-8-9 backbone DNA fragments. To generate the pLVTH-derived plasmids required to propagate HBV-silencing LVs, the monocistronic and tricistronic pri-miR expression cassettes were excised from pmTTR pri-miR-31/5 and pmTTR pri-miR-31/5-8-9 using SalI. Purified fragments were inserted into the SalI site of pLVTH62 to form pLVTH mTTR miR-31/5 and pLVTH mTTR miR-31/5-8-9. Gateway Cloning Technology (Life Technologies Inc., Carlsbad, CA, USA) was used to introduce the modified mTTR promoter into pLNT-GW-JDG to form the pLNT-mTTR-JDG LV plasmid. Briefly, the mTTR promoter sequence was ligated to a pENTR1A entry vector and then transferred into the pLNT-GW-JDG destination vector, kindly provided by the group of Dr Simon N Waddington (University College London, London, UK). To generate pLVTH-Luc and pLVTH-Luc miR 31/5-8-9, a blunted 2A-linked Firefly luciferase-eGFP expression cassette, excised from the pLNT-GW-JDG plasmid, was ligated to blunted pLVTH and pLVTH mTTR miR 31/5-8-9 vector backbones, from which GFP sequences had been removed.Cell cultureThe human hepatoma-derived (Huh7) cells63 and HEK293T cells were maintained in complete Dulbecco鈥檚 modified Eagle鈥檚 medium comprising Gibco Dulbecco鈥檚 modified Eagle鈥檚 medium (Life Technologies Inc.) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies Inc.), penicillin (10鈥?00鈥塙鈥塵l鈭?) and streptomycin (100鈥壩糶鈥塵l鈭?) (Sigma-Aldrich, St Louis, MO, USA). HepG2.2.15 cells34 were maintained in complete William鈥檚 E culture medium comprising Gibco William鈥檚 E Medium GlutaMAX (Life Technologies Inc.) supplemented with 10% heat-inactivated fetal calf serum, penicillin (10鈥?00鈥塙鈥塵l鈭?), streptomycin (100鈥壩糶鈥塵l鈭?) and GibcoGeneticin Selective Antibiotic (400鈥壩糶鈥塵l鈭?) (Life Technologies Inc.). Cells were cultured in a humidified atmosphere containing 5% CO2.LV productionTransient transfection of HEK293T cells, using calcium phosphate as transfection agent, was used to prepare the LVs derived from pLVTH, pLVTH miR-31/5 or pLVTH miR-31/5-8-9 according to previously described methods.64 High-titer LV stocks of LVTH, LVTH miR-31/5 or LVTH miR-31/5-8-9 were prepared by concentrating virus-containing supernatant after ultracentrifugation of virus-containing supernatants at 50鈥?00鈥?i>g for 90鈥塵in at 4鈥壜癈. Pellets were resuspended in Dulbecco鈥檚 phosphate-buffered saline without Ca2+ and Mg2+ (Life Technologies Inc.), frozen and stored at 鈭?0鈥壜癈. To determine virus titers, HeLa cells were transduced with serial dilutions of the viral preparations and real-time qPCR was used to measure the average number of proviral copies in the cells鈥?genomes according to previously described methods.65 Titers of LV preparations were thus calculated as the number of transducing units per ml. The same qPCR assay was used to determine copy numbers of integrated LV in stably transduced Huh7 and HepG2.2.15 cells. LNT-mTTR-JDG, LVTH-Luc and LVTH-Luc miR-31/5-8-9 LV stocks were generated by transient transfection of the transfer lentiviral plasmid and packaging plasmids, gag-pol-expressing pCMV螖R8.74 (LNT-mTTR-JDG) or psPAX2 (LVTH-Luc and LVTH-Luc miR-31/5-8-9) and the pMD.G2 packaging plasmids into HEK293T cells using linear polyethylenimine (MW 25鈥?00) (Polysciences, Warrington, PA, USA) (1鈥塵g鈥塵l鈭?) as a transfection reagent.64 Clarified virus-containing medium was concentrated using Centricon Plus-70 Centrifugal Filter Devices (100鈥?00 MW limit; Merck-Millipore, Billerica, MA, USA) according to the manufacturer鈥檚 instructions. Stocks of concentrated virus were titrated in HeLa cells using the Global UltraRapid Lentiviral Titer Kit (System Biosciences, Mountain View, CA, USA) according to the manufacturer鈥檚 instructions. High-titer virus aliquots were stored at 鈭?0鈥壜癈.Target plasmidsThe pCH-9/3091 replication-competent viral plasmid contains a greater than genome length HBV sequence.66 The psiCHECK-HBx67 and psiCHECK-mHBx6 plasmids have been described previously and, respectively, contain normal or mutant HBx sequences downstream of the Renilla luciferase ORF within psiCHECK-2 (Promega, Madison, WI, USA). The mutated HBx target sequence contains several point mutations in the region targeted by the putative guide 5 of pri-miR-31/5 and pri-miR-31/5-8-9 mimics. The U6-driven sponge plasmid, pTZ-U6-miR-16S 脳 7,6 contains seven copies of an imperfectly complementary target of miR-16. The miR-16 dual-luciferase target plasmid, psiCHECK-miR-16T 脳 7,6 encodes seven miR-16 sites downstream of the Renilla luciferase ORF within psiCHECK-2.HBV antigen measurements, dual-luciferase reporter assay and Northern blot analysisDetermination of the activities of Renilla and Firefly luciferase and quantification of HBsAg was performed as described earlier.67 HBeAg secretion was quantitatively measured using a sandwich-type enzyme-linked immunosorbent assay method (Gentaur, Paris, France), according to the manufacturer鈥檚 instructions. Total RNA for Northern blot analysis was extracted from stably transduced Huh7 cells using Tri reagent (Sigma-Aldrich) and analyzed by Northern blot as described previously.6Animal experimentsAll experiments using HBV transgenic mice were carried out in accordance with protocols approved by the University of the Witwatersrand Animal Ethics Screening committee. LVs was injected into the circulation of anesthetized newborn mice (1 day old) via the superficial temporal vein, as described previously.68 A maximum injectate volume of 60鈥壩糽 was used. Newborn mice each received 6 脳 107 transducing units of LNT-mTTR-JDG LV and 8.7 脳 108 transducing units of LVTH-Luc miR-31/5-8-9 or LVTH-Luc LV. Bioluminescence imaging of animals was carried out using the IVIS Kinetic Imaging System (Caliper Life Sciences Inc., Waltham, MA, USA). Briefly, animals received D-luciferin (Gold Biotechnology, Olivette, MO, USA) at a concentration of 150鈥塵g鈥塳g鈭? by intraperitoneal injection 15鈥塵in before imaging using the Living Image Software (Caliper Life Sciences). Mice were killed at the completion of each study and the harvested livers were cut into three equal pieces. Hepatic genomic DNA was extracted from 25鈥塵g of homogenized tissue using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer鈥檚 instructions. The Global UltraRapid Lentiviral Titer Kit was used to determine the number of integrated vector copies. Total RNA was extracted from mouse livers using Tri reagent. Alanine transaminase activities in serum from blood that had been collected during the course of the experiments were measured using an automated photometric analyzer (Roche Diagnostics, Basel, Switzerland).Quantitation of markers of viral replication and HBV-targeting miRsHBsAg was quantified in mouse serum using the Monolisa HBs Ag ULTRA Assay Kit (Bio-Rad, Marnes-la-Coquette, France) as described previously.5 Viral DNA quantification in culture supernatants of HepG2.2.15 cells and mouse serum was performed using real-time qPCR carried out using the Roche Lightcycler v.2. or the Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and was measured as has been described.8 The Total Nucleic Acid Isolation Kit and MagNA Pure LC instrument (Roche Diagnostics, Switzerland) were used to extract DNA and RNA from serum samples. PCR analysis was performed using the SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) and the following cycling parameters: a 30鈥塻 hot start at 95鈥壜癈 and 40 cycles of denaturation at 95鈥壜癈 for 10鈥塻, annealing at 58鈥壜癈 for 10鈥塻 and extension at 72鈥壜癈 for 10鈥塻. The primers used were as follows: HBV forward, 5鈥?IndexTermTGCACCTGTATTCCATC-3鈥?and HBV reverse, 5鈥?IndexTermCTGAAAGCCAAACAGTGG-3鈥? EuroHep standards were used to generate a standard curve for the quantification of VPEs in the individual samples.8 Total RNA was reverse transcribed with the QuantiTect Reverse Transcription Kit (Qiagen GmbH) according to the manufacturer鈥檚 instructions. To amplify murine GAPDH, HBV surface and core mRNA, respectively, the following primer sets were used: mGAPDH F, 5鈥?IndexTermTTCACCACCATGGAGAAGGC-3鈥?and mGAPDH R, 5鈥?IndexTermGGCATGGACTGTGGTCATGA-3; HBV Surface F, 5鈥?IndexTermTGCACCTGTATTCCATC-3鈥? and HBV Surface R, 5鈥?IndexTermCTGAAAGCCAAACAGTGG-3鈥? and HBV Core F, 5鈥?IndexTermACCACCAAAATGCCCCTAT-3鈥?and HBV Core R, 5鈥?IndexTermTTCTGCGAGGCGGCGA-3鈥? PCR analysis was performed using the SsoFast EvaGreen Supermix and the Bio-Rad CFX96 Touch Real-Time PCR Detection System. A custom TaqMan Small RNA Assay (Life Technologies Inc.) was used to detect and quantify the mature miR-31/5 guide sequences of pri-miR-31/5-8-9 in liver tissue samples. Total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies Inc.) and a specific stem-loop reverse transcription primer according to the manufacturer鈥檚 instructions. 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Financial assistance was received from the South African National Research Foundation (NRF, GUNs 81768, 81692, 68339, 85981 and 77954), Medical Research Council, Poliomyelitis Research Foundation, Stella and Paul Loewenstein Charitable and Educational Trust, German Academic Exchange Service (DAAD). Advice and practical assistance from Drs Tuan Nguyen and Dina Kremsdorf is gratefully acknowledged.Author informationAffiliationsAntiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, Gauteng, South AfricaD Ivacik,聽A Ely聽 聽P ArbuthnotAgence Nationale de S茅curit茅 du M茅dicament et des produits de Sant茅, Saint Denis, FranceN FerryAuthorsD IvacikView author publicationsYou can also search for this author in PubMed聽Google ScholarA ElyView author publicationsYou can also search for this author in PubMed聽Google ScholarN FerryView author publicationsYou can also search for this author in PubMed聽Google ScholarP ArbuthnotView author publicationsYou can also search for this author in PubMed聽Google ScholarCorresponding authorCorrespondence to P Arbuthnot.Ethics declarations Competing interests The authors declare no conflict of interest. Additional informationSupplementary Information accompanies this paper on Gene Therapy websiteSupplementary information Supplementary Information (PDF 1390 kb)Rights and permissionsReprints and PermissionsAbout this articleCite this articleIvacik, D., Ely, A., Ferry, N. et al. Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses. Gene Ther 22, 163鈥?71 (2015). https://doi.org/10.1038/gt.2014.94Download citationReceived: 17 July 2014Revised: 26 August 2014Accepted: 17 September 2014Published: 23 October 2014Issue Date: February 2015DOI: https://doi.org/10.1038/gt.2014.94 Rajvinder Karda, Ahad A. Rahim, Andrew M. S. Wong, Natalie Suff, Juan Antinao Diaz, Dany P. Perocheau, Maha Tijani, Joanne Ng, Julien Baruteau, Nuria Palomar Martin, Michael Hughes, Juliette M. K. M. Delhove, John R. Counsell, Jonathan D. Cooper, Els Henckaerts, Tristan R. Mckay, Suzanne M. K. Buckley Simon N. Waddington Scientific Reports (2020)