Targeting Hepatitis B Virus with Zinc Finger Nucleases

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy

ZFN-induced target site disruption in HepAD38 cells.

<p>(<b><i>a, b</i></b>) Cells were transduced with scAAV2 vectors expressing ZFN pairs 1, 2, 3 or all three pairs together (1–3) at a total MOI of 10000 genomes/cell. The presence of mutations was analyzed in DNA isolated from transduced cells 72 hours later using the Surveyor nuclease assay. (<b><i>c</i></b>) For ZFN site 3, an analysis of DNA mutagenic events disrupting the internal NcoI cleavage site was also performed. Above the gel images, the sizes of PCR amplicons and the cleavage products produced upon Surveyor nuclease cleavage (indicating mutations at the indicated target site) or NcoI cleavage are shown. bp – base pairs; UC – untreated control; ZFN – zinc finger nuclease. Bands indicating mutations are highlighted with an asterisk and the percentage of ZFN-mediated DNA mutation for each targeted site is indicated. (<b><i>d</i></b>) DNA mutations that were detected at ZFN target sites 1, 2 and 3 within HepAD38 HBV sequences are shown above the wild-type ZFN site. Nucleotides with differences in at least one sequence are shown in color. Spacer regions are underlined. The rates at which DNA mutations were detected are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097579#pone-0097579-t001" target="_blank">Table 1</a>. wt – wild type.</p

scAAV-ZFN induced cytotoxicity.

<p>(<b><i>a</i></b>) HepAD38 cells were transduced with scAAV2 vectors expressing GFP and mCherry reporter genes, ZFN pairs 1, 2 or 3, or mismatched ZFN pairs at a total MOI of 10000 genomes/cell, or all 3 ZFN pairs at a total MOI of 10000 (low) and 30000 (high) genomes/cell. At 48 hours post transduction, cell viability was measured by MTT assay and reported as percent of control. (<b><i>b</i></b>) HepAD38 cells transduced with scAAV2 vectors expressing individual ZFN half sites at a MOI of 5000 genomes/cell were also analyzed. (<b><i>c</i></b>) Untreated cells and cells treated with scAAV2 vectors expressing reporter genes, ZFN pairs 1, 2 or 3 or a mismatch ZFN pair were monitored for cell viability at 3, 5, 7 and 14 days post transduction. eGFP – enhanced green fluorescent protein; ZFN – zinc finger nuclease.</p

HBV-ZFN target sites.

<p>(<b><i>a</i></b>) HBV rcDNA genome map showing HBV ORFs and ZFN target site locations. (<b><i>b</i></b>) HBV target site sequence heterogeneity for HBV-ZFN pairs 1–3 across 3847 complete HBV genotype A–H sequences found in Genbank. For each ZFN pair, the target sequence, consensus sequence logo plot and nucleotide Rate4Site (R4S) scores are shown. ZFN spacer nucleotides are highlighted in red and divergent nucleotides between the ZFN target site and the consensus sequence are bold and underlined. Rate4Site scores are graded from low (white) to high (black) sequence heterogeneity. Single nucleotide polymorphisms present in the HepAD38 genomic HBV sequence are shown above each target site in blue. ORF – open reading frame; R4S – Rate4Site; ZFN – zinc finger nuclease.</p

Levels of HBV DNA present in ZFN-treated HepAD38 cells and levels of secreted infectious HBV.

<p>(<b><i>a</i></b>) For experiments shown in panels <b><i>b</i></b> and <b><i>c</i></b>, HepAD38 cells in the presence of dox were transduced with ZFN- or control-expressing scAAV2 vectors (total MOI 10000 genomes/cell) and 3 days later dox was removed from culture medium to enable HBV replication. Cells were left in culture for a further 7 days before HBV genomic levels were quantified in cells (<b><i>b</i></b>) and infectious HBV levels were quantified in supernatants (<b><i>c</i></b>) by ddPCR. (<b><i>d–e</i></b>) HepAD38 cells were passaged every two to three days over the course of 14 days following treatment with ZFN- or control-expressing scAAV vectors and monitored for cellular (<b><i>d</i></b>) and supernatant (<b><i>e</i></b>) HBV levels by ddPCR. ddPCR – droplet digital polymerase chain reaction; dox – doxycycline; eGFP – enhanced green fluorescent protein; scAAV – self-complementary adeno-associated virus; UC – untreated control; ZFN – zinc finger nuclease. *p≤0.05.</p

Summary of HBV-ZFN-induced mutagenesis in HepAD38 cells.

<p>*% indel  =  (1−(1−(a+b)/(a+b+c))∧0.5)*100; where a and b =  cut bands, c =  uncut band.</p

SMRT sequencing results showing on-target and off-target activity of HBV-ZFNs.

<p>LQ, low quality; TS, target site; *Significance between Z1 and Z3 (p<0.01), Z1 and Z2 (p<0.001), Z2 and Z3 (p<0.001).</p

HBV-ZFN activity in mammalian cells.

<p>(<b><i>a</i></b>) HBV-ZFN target site/GFP reporter plasmid containing a ZFN target site (red) placed immediately after the GFP start codon (blue) and preceding the in-frame GFP ORF (green). The HBV-ZFN spacer is shown in lower case. (<b><i>b</i></b>) ZFN reporter constructs and expression plasmids for ZFN pairs 1–3 were transfected into HEK 293T cells. GFP expression was visualized at 72 hours post transfection. (<b><i>c</i></b>) ZFN-mediated target site disruptions within reporter constructs were amplified by PCR from the HEK 293T cells that had been transfected with ZFN-expressing plasmids as indicated. Indels found at each ZFN site are aligned with the wild type target sequence (wt) and nucleotides with differences in at least one sequence are shown in color. The spacer regions are underlined. GFP – green fluorescent protein; SFFV – spleen focus-forming virus promoter; WPRE – woodchuck hepatitis post-transcriptional regulatory element; polyA – polyadenylation signal; wt – wild type; ZFN – zinc finger nuclease.</p

scAAV vector transduction of HepAD38 cells.

<p>(<b><i>a</i></b>) scAAV vector construct organization. (<b><i>b</i></b>) Cells were transduced with scAAV vectors containing a CMV-hrGFP-pA expression cassette at a MOI of 500 (open bars) or 5000 (closed bars) vector genomes/cell, or (<b><i>c</i></b>) were co-transduced with scAAV2-EFS-GFP and scAAV2-EFS-mCherry vectors at indicated MOI. Cells were analyzed for GFP and mCherry expression by flow cytometry at 72 hours post infection. (<b><i>d</i></b>) HepAD38 co-transduction persistence. Cells were transduced with scAAV2-EFS-eGFP and/or scAAV-EFS-mCherry at MOI 5000 genomes/vector/cell and co-transduction levels were monitored by flow cytometry at 2 and 6 days post transduction. ITR – inverted terminal repeat; CMV – cytomegalovirus immediate early promoter; EFS – elongation factor-1a short promoter; hrGFP – humanized Renilla green fluorescent protein; eGFP – enhanced green fluorescent protein; pA – polyadenylation signal; ZFN – zinc finger nuclease.</p

Gene editing and elimination of latent herpes simplex virus in vivo

Herpes simplex virus establishes lifelong latency in ganglionic neurons, which are the source for recurrent infection. Here Aubert et al. report a promising antiviral therapy based on gene editing with adeno-associated virus-delivered meganucleases, which leads to a significant reduction in ganglionic HSV loads and HSV reactivation