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Discussion
The results from this study described for the first time the complete repertoire of cccDNA genomes present in a liver-derived cell line expressing the CRISPR/Cas9 system targeting a locus on HBV DNA. An important finding is that approximately 7% of cleaved cccDNA genomes are repaired in a fashion that yields in frame mutants that might not abrogate function of the targeted gene, in our case HBx (Figure 1d). Hence, multiple sgRNAs targeting different loci on the HBV genome might be required to inactivate cccDNA. Assuming that 10% of hepatocytes in a liver are infected (ca. 5 × 1010 hepatocytes) each carrying four copies of cccDNA, at least 10 loci would have to be targeted to inactivate each cccDNA molecule (2 × 1011 × 0.0710 = 0.56). Therefore, a more practical approach would be to target a sequence motif on the HBV genome known to be required for gene function, such as the active site of the DNA polymerase or RNaseH domains of the pol gene. The terminal protein region of the pol gene where tyrosine 68 is known to be essential for protein priming would be another possibility.21 It is also conceivable that in frame deletions in HBx as observed in our study might hamper the function of this protein to permit efficient transcription from cccDNA,3,13,15 a possibility we have not not yet examined.
While CRISPR/Cas9 is the most efficient method known to date for inactivation of genes, it requires therapeutic administration of at least two components enabling expression of Cas9 and sgRNA in the same cell.16,22 In contrast, editing and inactivation of genes through activation of innate immune pathways could, in theory, be accomplished through oral administration of small molecules. In this regard, activation of APOBEC proteins has been proposed as a potential method to inactivate and even destroy cccDNA.7,23 We have directly compared APOBEC editing of HBV DNA with CRISPR/Cas9 cleavage and found that it is very inefficient compared with CRISPR/Cas9. The results showed that the frequency of cytosine deamination is at least 15,000- to 100,000-fold lower than observed with Cas9. Importantly, they revealed that individual HBV DNA genomes carry only a small number of G:A transitions, which are in most, if not all, cases already present on input virion DNA. Importantly, we found that the 3D-PCR method, often used to measure APOBEC-mediated editing of HBV genomes, yields erroneous results that greatly exaggerate the extent of editing. Hence, our study revealed that 3D-PCR is an unreliable predictor for both, the frequency and in particular the extent of cytosine deamination (Figures 456). The reason lies in the method that selects for amplification of DNA strands with a reduced Tm and hence, high A:T content. The resulting artifact is most likely caused by re-annealing and extension of incomplete PCR products following the denaturation step, leading to a stepwise increase in the number of chimeric products with increasing A:T content. In vitro chimera formation during PCR reactions has been observed previously when input DNA consisted of two or more alleles of the gene targeted for PCR amplification.24,25,26 A curious observation derived from our study is that IFNα treatment did increase the yield of DNA products from the 3D-PCR reaction (Figures 3b and 5a), although NGS did not reveal any significant difference in G:A hypermutations between control and IFNα treated samples (Figure 5b). Perhaps very small differences in the A:T content within the population of input DNA for 3D-PCR accounts for this difference.
A limitation of our study is that it was performed in HepG2 cells, which might not adequately mimic conditions in hepatocytes in the liver. While information about the activity of CRISPR/Cas9 in humans is not yet available, considering that the system depends only on two components, Cas9 and sgRNA and then on the endogenous NHEJ pathway, we believe that our observations demonstrating efficient cleavage of cccDNA are valid also under conditions in patients with CHB. As indicated above, this conclusion is based on the premise that Cas9 and sgRNA can be delivered efficiently to the infected liver. Similarly, it is possible that HepG2 cells, or other hepatocyte cultures do not adequately reproduce conditions for APOBEC-mediated deamination of HBV DNA in infected hepatocytes. However, our results showed that IFNα as well as IFNγ induce STAT1 phosphorylation and translocation into the nucleus, evidence for activation of an innate immune response (Figure 2b,c). In addition, we observed a cytotoxic effect of IFNα when added within 2 days of HBV infections. While the reason for this toxicity is not known, the observation provided further evidence for activation of an innate immune response in HepG2 cells by the cytokine. Finally, we found that APOBEC3G was induced by IFNα as reported previously with HepG2 cells, primary hepatocyte cultures and even in vivo (Supplementary Figure S1).19 In spite of these reservations, it is notable that our results correlate well with reports from in vivo studies. For example, hypermutated genomes could be recovered from some sera of chronically infected HBV patients at a frequency of ca. 10−4, suggesting that deaminated HBV DNA is produced in a very small fraction of HBV-infected hepatocytes.27 A more precise estimate for the fraction of G to A edited HBV genomes was derived from an NGS analysis of virion DNA obtained from CHB patients.28 Median hypermutation frequency in virion DNA from HBeAg-positive patients ranged from 0 to 10−4 depending on the 100 nucleotide long segment analyzed. Notably, editing occurred preferentially in the region of the HBV genome that is single stranded, consistent with the known substrate preference of APOBEC proteins and the prevailing view consistent with our observations that editing occurs on rc, rather than cccDNA.29 Much higher frequencies of editing have so far only been reported in viral DNA from patients with advanced cirrhosis, a condition marked by high cytokine levels.30 Hence deamination of HBV DNA is generally a rare event in infected patients with minimal or no consequences for viral persistence. For this reason it is not surprising that HBV does not encode a protein that inhibits APOBEC activity, like HIV. The latter encodes Vif that causes degradation of APOBEC proteins by the proteasome.31,32 Moreover, all evidence points to a mechanism where deamination of HBV DNA occurs during minus strand DNA synthesis in core particles present in the cytoplasm of infected cells. Our study is in agreement with this consensus because we could not find any evidence for editing of cccDNA following Cas9 cleavage (Figure 7). Lastly, although the Hirt procedure for DNA isolation and the selection of primers flanking the cohesive overlap region were used for PCR amplification of cccDNA, rather than rcDNA (PCR1, Figure 3a), we are aware of the fact that some rcDNA could still have been co-amplified with cccDNA. Hence, given the low frequency of the observed G:A transitions observed with DNA extracted from HBV infected HepG2/NTCP, it is plausible that these mutations were derived from PCR amplified rcDNA, not cccDNA.
Even if editing would occur to some extent on cccDNA, given the low number of deaminated residues present on cccDNA, it is almost certain that removal of the uracil base by uracil-DNA glycosylase is followed by a DNA repair mechanism, not destruction of cccDNA, essentially as observed with chromosomal DNA.8 The present and our recent study demonstrated that cccDNA is efficiently repaired following Cas9 cleavage by the DNA repair machinery, like chromosomal DNA.3 In other words, there is no solid evidence to date for selective recognition of cccDNA by cellular DNA modifying enzymes that would destroy it. Thus, more than 15 years after the proposal that cccDNA could be cleared from infected hepatocytes by a cytokine-mediated non-cytolytic event,4 we still have no insights into a possible mechanism that could explain such an event. For this reason, it appears that exogenous nucleases, such as Cas9, are the sole means known to date to functionally inactivate cccDNA and hopefully cure CHB. |
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发表于 2016-6-23 05:49
