Zinc-finger Nucleases as a Novel Therapeutic Strategy for Targeting Hepatitis B Virus DNAs

Abstract

Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. We targeted HBV DNA for cleavage using zinc-finger nucleases (ZFNs), which cleave as dimers. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. After 3 days in culture, 26% of the target remained linear, whereas approximately10% was cleaved and misjoined tail-to-tail. Notably, ZFN treatment decreased levels of the hepatitis C virus pregenomic RNA by 29%. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the ZFN sites. Targeted cleavage and misrepair destroys the XbaI site. After 3 days in culture, approximately 6% of plasmids were XbaI-resistant. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using ZFNs.

Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections

“…We believe that these same endonucleases might also be used to directly target latent or persistent viral genomes, conceptually offering the possibility of a cure for otherwise lifelong infections (8,39). We will consider the differing biologies of three such infections (HIV, HBV, and HSV) and the implications that their biology has on the potential for curative therapy using targeted DNA endonucleases.…”

“…ZFNs can cleave both episomal and proviral DNA, followed by repair through highly errorprone nonhomologous end joining. In cell culture experiments, an HBV-specific ZFN showed site-specific cleavage leading to a reduction in pregenomic RNA levels without a loss in cell viability (39).…”

“…Off-target cleavage has been reduced by the development of FokI variants that can function only as heterodimers, thus preventing cleavage by undesired homodimers (176), but questions remain regarding the ultimately achievable specificity of ZFNs. Despite these potential limitations, ZFNs have achieved major successes, having been used extensively in the study of knockout of the HIV coreceptor CCR5 (75,137,204) and more recently to directly target HBV covalently closed circular DNA (cccDNA) in infected hepatocytes (39).…”

Tratamiento actual y nuevas terapias contra la infección crónica por el virus de la Hepatitis B

“…Es por esto que teóricamente unos ZFN diseñados para atacar el ADN del VHB no tendrían efecto sobre el genoma del hospedero. 2 estudios in vitro han demostrado que mediante este mecanismo se disminuye la concentración de ARN pregenómico y ADNccc significativamente (69,70). No obstante, el principal reto es lograr encontrar un mecanismo de entrega eficiente a los hepatocitos (66).…”

Zinc Finger Nucleases Targeting the Human Papillomavirus E7 Oncogene Induce E7 Disruption and a Transformed Phenotype in HPV16/18-Positive Cervical Cancer Cells

“…ZFNs disrupting the promoter function of human T-cell leukemia virus type 1 (HTLV-1) specifically killed HTLV-1-infected cells in a model of adult T-cell leukemia (13). In addition, it was reported that effective cleavage of vital DNA targets by HBVspecific ZFNs decreased HBV pregenomic viral RNA levels by 29% (14).…”