immune system of bacteria against viruses and Over 250 million people worldwide are chronically plasmids. When a virus enters a bacterium, it will infected by the hepatitis B virus (HBV). Symptoms insert its own DNA in the host DNA for further of HBV are fatigue, fever, nausea, joint pain, replication. Some bacteria are able to copy a short abdominal pain and yellowing of the skin and eyes. sequence of the virus’s DNA (target sequence), to Prolonged HBV infection can lead to severe liver serve as a detection mechanism for future viral failure, cirrhosis, and hepatocellular liver cancer. It infections. The complex in which the detection can be fatal when not treated. Up to 600.000 mechanism operates is called CRISPR. The CRISPR people a year are likely to die from a hepatitis B complex is attached to the nuclease Cas9. In short, infection (Dienstag et al., 2008). CRISPR is able to recognize the viral DNA through HBV is a double-stranded DNA virus of the its target sequence and Cas9, in turn, is able to Hepadnaviridae family. The virus binds to the destroy the viral DNA by breaking the double helix. hepatocyte membrane in the liver after which the Genome engineering has made it possible to HBV core is being released in the cytoplasm and design target sequences of CRISPR, which is called subsequently transported to the nucleus where it guide RNA (gRNA). So, in principle any desired will form a covalently closed circular DNA (cccDNA) DNA sequence can be recognized and cleaved by (figure 1). This cccDNA is crucial for the CRISPR-Cas9. Moreover, extra nucleotides can be persistence of the infection, since it serves as a inserted to CRISPR-Cas9 and accordingly built in template for transcription of all viral genomes and into the cell’s genome. Therefore CRISPR-Cas9 can sub-genomes. be applied to transform DNA that causes life threatening diseases. In this study we want to investigate the application of CRISPR-Cas9 in the treatment of hepatitis B by direct targeting and cleavage of HBV cccDNA. We hypothesize that HBV can be suppressed by CRISPR-Cas9 through cleavage of the HBV genome. Hence, we first examine the efficiency of several gRNAs in HBV infected cell lines by measuring an important indicator for viral expression and replication in the medium, specifically the secretion of surface antigen HBsAg. Next, we sought to evaluate the antiviral effect of the most effective gRNAs in a mouse model of Figure 1. Schematic overview of the HBV life cycle. HBV, to ensure that it functions properly in the hepatocytes of a living organism. Finally, we Treatment of hepatitis B is currently based on wanted to examine the long-term anti-HBV effect inhibiting viral replication by acting on post- of CRISPR-Cas9 in a special cell line that mimics transcriptionally processes (Schlomai & Rice, the HBV life cycle components and consequently 2014). Consequently, cccDNA is not targetted, so it produces the infectious virus (Sells et al., 1987). remains stable and persists in the hepatocyte’s Therefore, we measured the amount of cccDNA at nucleus. Hence, medication must be taken day 24 and 34 after treatment. In all experiments chronically to prevent further viral outbreaks. we expect CRISPR-Cas9 to cause a decrease in Infected liver cells may contain up to 50 cccDNA HBV expression compared to control conditions. molecules (Balsano et al., 2008), therefore agents acting directly on cccDNA might be more desirable Materials and Methods and possibly even curative. For example, specific nucleases that are able to break the viruses CRISPR-Cas9 design and validation double-stranded DNA, without damaging the host Whole-genome sequences from HBV were inquired genomic DNA, would be ideal for this purpose. from the GenBank sequence database to The recently developed CRISPR-Cas9 determine several target sequences. We made use technology makes it possible to manipulate DNA of a CRISPR online design tool Dharmacon (https://horizondiscovery.com/en/resources/featur Hydrodynamic injection of ed-articles/dharmacon-editr-crispr-cas9-gene- 1.3x WT HBV and Cas9/gRNA engineering-system) to generate and order four different gRNAs (table 1), that were able to target the HBV DNA at different locations. To evaluate the efficiency of the gRNA’s we added the four different designs of CRISPR-Cas9 to HBV infected cell lines, that were maintained in DMEM and 10% calf serum. We used mismatching target sequences as control. To determine the efficiency Draw blood to analyze of the gRNA after 72 hours, we measured an serum virus load indicator of HBV genomic expression in the Figure 2. Schematic overview of in vivo experiment. medium of the cell lines, namely hepatitis B surface antigen (HBsAg). The two most efficient Statistical analyses gRNAs were used for follow-up experiments. All analyses were performed in SPSS 14.0. Table 1. Sequences of the manufactured gRNAs. Comparisons between two groups were analyzed by Students t-test. Comparisons among more groups were analyzed by one-way ANOVA followed by Tukey’s post-hoc test to compare between two groups.
Anti-HBV effect in vivo
We used a mouse model of HBV to evaluate the anti-viral effect of CRISPR-Cas9 in vivo. NRG mice were administered 15 µg 1,3xHBV plasmid and 20 µg CRISPR-Cas9 through tail vain hydrodynamic injection in 7-9 seconds (figure 2). We took measures on HBV titer in a blood sample to determine the anti-viral effects of CRISPR-Cas9 at day 2 and 4 post injection. Mismatching target sequences were used as control. Mice were obtained from Jackson laboratory and were housed in an accredited facility with ad libitum access to food and water. All animal protocols were approved by the University of Massachusetts.
Long term anti-HBV effect
For this experiment we used the HepG2.2.15 hepatoblastoma cell line, which contains HBV cccDNA and is able to produce infectious virions. In contrast to other cell lines, HepG2.2.15 is ideal for taking measures of cccDNA. We measured the effect of CRISPR-Cas9 mediated cleavage on the abundance of cccDNA in cells after 24 and 34 days. Control constructs were made of mismatching target sequences.