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Curing HBV, New Drugs - [HBV at EASL 2016]
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Download the PDF here
Download the PDF here
http://www.natap.org/2014/HBV/012215_01.htm
AASLD: Curing HBV....attacking cccDNA "Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease" - (01/12/15)
A new priority for the ANRS: "HBV Cure" - (01/05/15)
http://natap.org/hbv.htm
http://www.natap.org/2014/AASLD/AASLD_151.htm
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HBV at EASL 2016
EASL: NVR 3-778, a First-in-class HBV Core Inhibitor, Alone and in Combination with Pegylated-interferon (peg-IFN alpha-2a), in Treatment-naÏve HBeAg-positive Patients: Early Reductions in HBV DNA and HBeAg - (04/26/16)
EASL: (NVR 3-778) Potential first-in-class treatment is well-tolerated in patients with chronic hepatitis B - EASL press release - (04/26/16)
EASL: CPI-431-32, A Novel Cyclophilin Inhibitor for Treatment of Chronic Hepatitis B: A Story of Clinical Utility - (05/05/16)
EASL: Combination Therapy for Curing HBV - Arbutus BioPharma - (05/05/16)
EASL: In Vitro and In Vivo Antiviral Activities of AB-423 a Potent Small Molecule Inhibitor of Hepatitis B Virus Capsid Assembly - (05/05/16)
EASL: A Phase 3 Study of Tenofovir Alafenamide Compared With Tenofovir Disoproxil Fumarate in Patients With HBeAg-Positive, Chronic Hepatitis B: Week 48 Efficacy and Safety Results - (04/26/16)
EASL: PRECLINICAL CHARACTERIZATION OF POTENT CORE PROTEIN ASSEMBLY MODULATORS FOR THE TREATMENT OF CHRONIC HEPATITIS B - (04/26/16)
EASL: DIFFERENTIAL REDUCTIONS IN VIRAL ANTIGENS EXPRESSED FROM CCCDNA VS INTEGRATED DNA IN TREATMENT NAÏVE HBEAG POSITIVE AND NEGATIVE PATIENTS WITH CHRONIC HBV AFTER RNA INTERFERENCE THERAPY WITH ARC-520 - (04/26/16)
EASL: TREATMENT OF CHRONICALLY HBV-INFECTED CHIMPANZEES WITH RNA INTERFERENCE THERAPEUTIC ARC-520 LED TO POTENT REDUCTION OF VIRAL MRNA, DNA AND PROTEINS WITHOUT OBSERVED DRUG RESISTANCE - (04/26/16)
EASL: PREDICTING HBSAG CLEARANCE RESPONSES DURING ARC-520 RNA INTERFERENCE (RNAI) THERAPY BASED ON HBSAG EPITOPE PROFILE ANALYSIS - (04/26/16)
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pdf attached
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CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus
Abstract
Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.
"Although largely unexplored in mammalian systems, bacteria and archaea utilize sequence specific DNA nucleases to interfere with viral replication17. Inspired by CRISPR’s evolutionary origins, we aimed to exploit the antiviral activity of Cas9 to target HBV DNA in mammalian cells. We show that targeting multiple conserved regions of HBV with Cas9 results in robust suppression of viral replication and direct mutagenesis and depletion of cccDNA. While integrated forms of HBV DNA were not depleted by Cas9 cleavage, these forms should not contribute to viral rebound in vivo18, and Cas9-driven mutagenesis of these sequences nonetheless would damage the viability of viral proteins generated from integrants. The unique advantages of the CRISPR/Cas9 system (such as multiplexed targeting) are of interest in developing antiviral applications, and indeed, very recently other groups have published examples of Cas9 cleavage of HBV in multiple model systems19,20,21,22. Our work provides an extension beyond these complementary studies, by demonstrating the anti-HBV effects of sgRNAs specifically targeting highly conserved regions of HBV in vitro and in vivo, by directly confirming mutagenesis in cccDNA in a de novo infection model of HBV, and extending this antiviral activity to patient-derived virus. Additionally, our finding that appropriately chosen virus-targeting sgRNAs can avoid inducing off-target cleavage, even upon sustained Cas9/sgRNA expression, strengthens the case for selecting viral targets as good candidates for CRISPR/Cas9 therapeutic use23."
Introduction
Hepatitis B virus (HBV) chronically infects over 250 million people worldwide. Chronically infected individuals are at an increased risk for deadly complications, including cirrhosis, end-stage liver disease and hepatocellular carcinoma, resulting in approximately 600,000 deaths per year1. HBV is a member of the Hepadnaviridae family and its life cycle involves both DNA and RNA intermediates. The HBV genome exists in the nuclei of infected hepatocytes as a 3.2kb double-stranded episomal DNA species called covalently closed circular DNA (cccDNA). cccDNA is a key component in the HBV life cycle, since it is the template for all viral genomic and subgenomic transcripts2. Currently approved HBV therapies act post-transcriptionally to inhibit viral replication and thus fail to target or eliminate the cccDNA pool, which exhibits extraordinary stability and persistence3. Consequently, these drugs must often be taken indefinitely to prevent viral rebound. Agents that act directly on viral DNA to deplete this reservoir may represent more desirable and possibly curative therapeutic alternatives4.
To this end, targeted nucleases may provide an efficient and specific way to damage the HBV genome while sparing host genomic DNA5,6,7. Targeted nucleases catalyze double-stranded DNA break (DSB) formation, which leads to the formation of mutagenic insertions and deletions (indels) through error-prone nonhomologous end-joining (NHEJ) at the target DNA locus. Recently, the type II CRISPR-Cas system of Streptococcus pyogenes SF370 has been adapted as an RNA-guided, sequence-specific DNA nuclease for use in mammalian cells8,9. CRISPR/Cas9 and other genome engineering technologies have been employed to design candidate therapeutics via gene targeting, knockout of beneficial host genes, and mutation of integrated viruses10, and we sought to further study the application of CRISPR/Cas9 to direct targeting and cleavage of HBV cccDNA. We hypothesized that by directly targeting the HBV genome for cleavage using CRISPR/Cas9, we could suppress HBV by mutagenizing critical genomic elements or decreasing the stability of cccDNA and other viral intermediates through repeated linearization of the circular genomes (Fig. 1a).
Results
CRISPR/Cas9 design and validation
Using the CRISPR online design tool ( http://www.genome-engineering.org/crispr/), we generated 24 single guide RNAs (sgRNAs) targeting the HBV genome (Fig. 1b, Table S1). Target sequences were chosen in order to maximize conservation across viral genotypes (Fig S1) and minimize homology to the human genome. Based on these criteria, we only designed guides targeting the core, polymerase and X ORFs, but numerous Cas9 target sites also exist in the S ORF (Fig. 1b). To evaluate the efficacy of selected sgRNAs (Fig. 1b) in targeting HBV, we co-transfected the HepG2 hepatoma cell line with an HBV-expressing plasmid and constructs expressing Cas9 and individual gRNAs, and measured the production of HBV 3.5kb RNA (encoding pre-genomic RNA (pgRNA), the template for reverse transcription) as well as the secretion of HBV surface antigen (HBsAg) into the medium, two reliable indicators for viral gene expression and replication (Fig. 1c). sgRNAs 17 and 21 (sg17 and sg21) consistently led to a decrease in pgRNA levels and HBsAg production (Fig. 1d,e). While other sgRNAs (sg14 and sg19) generated similar decreases in HBV pgRNA, these guides did not have as large an effect on HBsAg secretion as did sg17 and sg21. This source of this discrepancy is not entirely clear, but may be related to targeting different locations along the HBV genome that exert effects on pgRNA transcription but do not suppress HBsAg expression.
Given their strong effect on both viral parameters measured, we proceeded with sg17 and sg21, as well as sg6 - identified from previous pilot experiments. In addition, to investigate the effect of multiplex targeting of HBV DNA in order to impact multiple viral elements, we co-transfected HepG2 cells with control sgRNA, sg17, sg21, or a combination of sg17/sg21. The combination of two guide RNAs targeting HBV led to stronger reductions in HBsAg and HBV 3.5kb RNA as compared to the single guide RNAs (Fig S2).
Confirmation of anti-HBV effect in vivo
We next sought to evaluate the antiviral effect of Cas9 in vivo, to ensure that our anti-HBV constructs functioned appropriately in primary hepatocytes. To do this, we used a mouse model of HBV, where HBV and Cas9/sgRNA plasmids were introduced to the liver of immunodeficient mice (NRG) by hydrodynamic injection (HDI)11 (Fig. 1f). In the case of proof-of-concept studies such as this, we endeavor to minimize the use of animal subjects. Thus, the complete battery of in vivo experiments described below were performed with only sg21 and its mutated control, although similar results were replicated with other guides (data not shown). Animals expressing Cas9 and sg21 in this model showed a progressive suppression of HBV expression as compared to controls expressing Cas9 and a mutated sgRNA (sg21M; 3’ 5 bp mismatch), reflected by a decrease in HBsAg secretion and a 4-fold decrease in viremia at day 4 post injection (Fig. 1g,h).
Sustained Cas9/sgRNA expression dramatically inhibits HBV
Recent genome-wide CRISPR knockout studies have shown that sustained Cas9/sgRNA expression induces progressively greater indel formation over time in mammalian cells12. Based on this information and encouraged by our initial results, we evaluated the efficacy of sustained Cas9/sgRNA expression in inhibiting HBV using a model that more reliably recapitulates HBV life cycle components. For these studies, we used the HepG2.2.15 hepatoblastoma cell line, which harbors both a functional HBV integrated form and cccDNA, and constitutively produces infectious virions13 (Fig S3). Because cccDNA cannot be reliably quantified or detected in plasmid co-transfection or HDI systems, the HepG2.2.15 system is more ideal for investigating CRISPR/Cas9-mediated clearance of this viral species.
We transduced HepG2.2.15 cells with Cas9-2A-Puro lentiviruses encoding Cas9 and individual sgRNAs (sg6, sg17, sg21) chosen based on our initial results, and treated cells with puromycin to select for transduced cells (Fig. 2a). As controls, cells were also transduced with constructs containing sgRNAs and a nuclease deficient Cas9 (D10A/H840A; dCas9) to control for nuclease-independent effects of Cas9 on viral fitness, or WT Cas9 with mutated sgRNAs (gXM) to control for guide sequence-independent effects. Cas9/sgRNAs induced robust suppression of HBV DNA release (77-95% decrease across different sgRNAs), HBeAg secretion, and viral mRNA production (greater than 50%) (Fig S4). We next analyzed the effect of Cas9-mediated cleavage on the abundance of non-integrated viral forms, composed mainly of cccDNA (See Methods). Quantitative PCR showed a robust reduction in total HBV DNA and in cccDNA. Pooling the data from sg6, sg17, and sg21, cccDNA reduction progressed from 71 + /-7% reduction at day 21 to 92 + /-4% at day 36 post transduction (Fig. 2b,c, data for individual sgRNAs in Fig S5). These results were confirmed by directly analyzing low molecular weight DNA from transduced cells by Southern blot (Fig. 2d). cccDNA and its deproteinated relaxed circular form (dpRC DNA) precursor were greatly depleted in Cas9/sgRNA transduced cells. In contrast, when total HBV DNA was analyzed, no substantial reduction in the levels of integrated HBV DNA was detected (Fig S6).
We then performed the Surveyor assay on HBV, to directly determine whether the viral DNA was cleaved and repaired via error-prone NHEJ similar to genomic targets of CRISPR/Cas9. Interestingly, analysis of total HBV DNA forms for indel formation, an indirect measure of Cas9-mediated cleavage, revealed a substantial mutagenesis rate (Fig. 2e top). When we performed the same analysis after depleting integrated genomic HBV, we observed a lower rate of indel formation (0% vs 32%, 62% vs 88% and 21% vs 66% for guides sg21, sg17 and sg6, respectively) (Fig. 2e bottom). Notably, however, this analysis method cannot detect Cas9-mediated cleavage of cccDNA followed by exonuclease-mediated degradation from the newly-formed free DNA ends (instead of re-ligation by NHEJ), and may be limited by the very small amount of episomal HBV remaining at late time points (Fig. 2d, Fig S4). Consistent with high levels of indel formation in the core ORF targeted by sg17, immunostaining for HBV core protein (HBc) revealed a robust reduction in HBc levels in sg17-expressing cells as compared to controls (Fig. 2f). Because long-term expression of Cas9 and guide RNAs can lead to off-target cleavage at sites with homology to the target sequence, we then performed next-generation sequencing at several computationally predicted off-target sites for sg6, sg17, and sg21. Within the sensitivity of our assay (< 0.3% based on read depth), we detected no indel formation at the 8 off-target sites that we surveyed after constitutive expression of Cas9 and sgRNAs for over four weeks (Fig. S7a). This observed specificity may be due to the large sequence differences between viral and human genomic DNA (Fig. S7b-d). These encouraging results still did not exclude the possibility that some of the antiviral effects of Cas9 in the HepG2.2.15 system occur through mutations in integrated HBV DNA, thereby reducing the fitness and/or persistence of virions produced from mutated loci rather than acting directly on episomal DNA. Since integration of HBV DNA into the host human genome is not part of the canonical HBV life-cycle, we next evaluated the effects of Cas9 targeting in the context of de novo HBV infection, where episomal cccDNA serves as the only template for viral gene expression and replication.
Cas9 cleaves cccDNA and inhibits de novo HBV infection
To evaluate our anti-HBV CRISPR/Cas9 strategy in a setting of de novo infection, we used HepG2 cells overexpressing the HBV receptor NTCP (Hep-NTCP)14, which are permissive to infection with HBV. Because sg17 showed the highest levels of cccDNA mutagenesis in our HepG2.2.15 experiments, these cells were transduced with Cas9/sg17, Cas9/sg17M, or dCas9/sg17 lentiviruses, co-cultured with HBV producing HepG2.2.15 cells, and selected with puromycin to get rid of non-transduced Hep-NTCP and co-cultured HepG2.2.15 cells (Fig S8 left). Alternatively, Hep-NTCP cells were selected with puromycin following transduction and subsequently infected with HBV-positive patient serum (Fig S8 right). When the transduced Hep-NTCP were infected with cell culture-produced virus, Cas9/sg17 greatly abrogated productive HBV infection, as reflected by reduction in HBsAg and HBV DNA secretion, as well as 3.5kb RNA and cccDNA levels, compared to controls (Fig. 2g); this was confirmed by infection with patient-derived virus (Fig S9). While nuclease-deficient Cas9 also reduced viral 3.5kb RNA abundance in this system, this finding fits with other reports that dCas9 binding can inhibit transcription in mammalian cells15. Surveyor assay performed using DNA from cells infected de novo with HepG2.2.15-derived virus confirmed direct Cas9-mediated mutagenesis of HBV episomal DNA (Fig. 2h). Although some mutagenesis was also detected when the mutated sg17M was used, this most likely was due to low-level cleavage with DNA bulge-containing guide RNAs16. This finding provides direct evidence that Cas9 is capable of targeting episomal forms of the virus, and exerting anti-HBV effects by directly targeting cccDNA.
Discussion
Although largely unexplored in mammalian systems, bacteria and archaea utilize sequence specific DNA nucleases to interfere with viral replication17. Inspired by CRISPR’s evolutionary origins, we aimed to exploit the antiviral activity of Cas9 to target HBV DNA in mammalian cells. We show that targeting multiple conserved regions of HBV with Cas9 results in robust suppression of viral replication and direct mutagenesis and depletion of cccDNA. While integrated forms of HBV DNA were not depleted by Cas9 cleavage, these forms should not contribute to viral rebound in vivo18, and Cas9-driven mutagenesis of these sequences nonetheless would damage the viability of viral proteins generated from integrants. The unique advantages of the CRISPR/Cas9 system (such as multiplexed targeting) are of interest in developing antiviral applications, and indeed, very recently other groups have published examples of Cas9 cleavage of HBV in multiple model systems19,20,21,22. Our work provides an extension beyond these complementary studies, by demonstrating the anti-HBV effects of sgRNAs specifically targeting highly conserved regions of HBV in vitro and in vivo, by directly confirming mutagenesis in cccDNA in a de novo infection model of HBV, and extending this antiviral activity to patient-derived virus. Additionally, our finding that appropriately chosen virus-targeting sgRNAs can avoid inducing off-target cleavage, even upon sustained Cas9/sgRNA expression, strengthens the case for selecting viral targets as good candidates for CRISPR/Cas9 therapeutic use23.
Interestingly, while Cas9/sg17 was efficient in suppressing infection and in directly cleaving nuclear cccDNA, Cas9/sg21 efficiently cleaved only integrated but not episomal DNA, which resulted in a lack of activity for Cas9/sg21 in de novo infection experiments (data not shown). The reason for this is unclear and warrants further study. Cas9 is a large multi-domain protein, and thus one hypothesis is that particular regions of the HBV genome are differentially accessible to Cas9 because of the tightly packed physical architecture of cccDNA. This underscores the importance of using models of authentic cccDNA to investigate therapeutic applications of targeted nucleases for HBV, and suggests that a careful selection of targets and guides will be required to achieve a substantial mutagenesis and depletion of viral DNA. In addition, our proof of concept experiments show that multiplexing sgRNAs can generate stronger antiviral effects (Fig S2), suggesting that this strategy may further maximize CRISPR-mediated restriction of components of the viral life cycle, possibly including cccDNA stability.
This study provides a proof of concept, but clinical translation of CRISPR/Cas9 systems to cure HBV will require some advances over the work described here. First, an exhaustive profiling of possible Cas9 target sites on cccDNA can uncover optimal target sites based on cccDNA accessibility and sgRNA binding properties. Secondly, delivery of Cas9/sgRNA constructs in vivo will require the use of clinically relevant delivery vectors such as AAV, which may require additional modifications such as switching to smaller Cas9 orthologs to save packaging size30. Finally, although we could not find evidence of off-target cutting in our directed sequencing, possibly due to the low homology between viral and human genomic Cas9 targets, an extensive genome-wide profiling of off-target effects is warranted.
The unusual persistence of cccDNA is currently the major obstacle for curing chronic HBV infection. To eliminate the virus and to prevent possible re-activation, it is probably necessary to eliminate all or at least the vast majority of episomal DNA from hepatocytes through a combination of exogenous treatment (presented here) and immune-mediated endogenous clearance. CRISPR/Cas9-mediated therapy may synergize with currently-used RT inhibitors, which should block the formation of new molecules of cccDNA via re-entry of newly synthesized replicative forms to the nucleus. The developments proposed above represent an active area of investigation for groups looking for ways to use CRISPR in a therapeutic fashion more broadly, which may accelerate progress toward an anti-HBV CRISPR therapeutic.
In summary, these results constitute the first example of CRISPR/Cas9 systems directly targeting an authentic pathogenic virus with episomal DNA, and demonstrate the potential for cccDNA-directed antiviral therapy using Cas9, which may represent a significant step towards the cure of chronic HBV infection. The results demonstrated here may also be used to inform the development of CRISPR/Cas9-based therapeutics for other DNA viruses, such as herpesviruses and papillomaviruses that use an episomal DNA as a template for their gene expression and replication.
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