CRISPR/Cas9 gene therapy efficiently suppresses hepatitis B virus in mice

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.

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 year

Transiently transfected CRISPR constructs exhibit anti-HBV activity.

Scientific Reports – CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

CRISPR gene therapy has been successfully used in the lab to target HBV (hepatitis B virus) DNA in mammalian cells. they 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 vivo, 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 systems. 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 use.

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, 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.

Ten pages of supplemental information

SOURCE – Nature Scientific Reports

9 thoughts on “CRISPR/Cas9 gene therapy efficiently suppresses hepatitis B virus in mice”

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  8. I’ve had Genital Herpes problem over 1year and. I tried everything of course I went to the hospital in other to see the doctor. about a hundred times probably not that many but it sure seems like it. It never did any good for long and caused all kinds of other issues having to do with taking too many antibiotics. Life wasn’t easy for me until I stumbled upon this natural remedy online which i requested for and it was sent to me here in my country i was told on how to use them and miraculously just after 14 days my life changed completely. I’m feeling so much better now and all of my symptoms are totally gone. I haven’t had an outbreak for over 7 months now All Thanks to Dr Olori the great herbal doctor you can reach him on (droloriherbalcenter@gmail.com)

    Reply
  9. I’ve had Genital Herpes problem over 1year and. I tried everything, of course I went to the hospital in other to see the doctor. about a hundred times, probably not that many, but it sure seems like it. It never did any good for long and caused all kinds of other issues having to do with taking too many antibiotics. Life wasn’t easy for me until I stumbled upon this natural remedy online, which i requested for and it was sent to me here in my country, i was told on how to use them and miraculously just after 14 days my life changed completely. I’m feeling so much better now and all of my symptoms are totally gone. I haven’t had an outbreak for over 7 months now, All Thanks to Dr Olori the great herbal doctor, you can reach him on (droloriherbalcenter@gmail.com)

    Reply

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