Creation of a Six-fingered Artificial Transcription Factor That Represses the Hepatitis B Virus HBx Gene Integrated into a Human Hepatocellular Carcinoma Cell Line

Abstract

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection-related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection-related HCC.

Towards HBV curative therapies

“…Zinc finger nucleases have successfully been used in tissue culture to reduce HBV levels . For example Weber et al developed a zinc finger nuclease that targeted the core and pol regions of cccDNA.…”

“…43 Zinc finger nucleases have successfully been used in tissue culture to reduce HBV levels. [44][45][46][47] For example Weber et al developed a zinc finger nuclease that targeted the core and pol regions of cccDNA. Using an adeno-associated virus (AAV) delivery system, they showed that HBV production from HepAD38 cells could be significantly controlled upon zinc finger nuclease delivery.…”

Virus–host interplay in hepatitis B virus infection and epigenetic treatment strategies

“…In this regard, Hepadnaviruses, which do not carry any integration machinery so their integration is dependent on double‐strand breaks, act along with the cell repair machinery to integrate viral genomes in fragile sites of the host cell genome . In order to overcome these possible off‐targeting effect and the likely increase of potentially disturbing integration events, new approaches using obligate heterodimeric nucleases coupled to two different targeting domains and the targeting of non‐nuclease effector domains, like epigenetic modulators (DNMTs, histone modifiers) or transcription downregulators (KRAB) are of considerable interest.…”

Progress With Developing Use of Gene Editing To Cure Chronic Infection With Hepatitis B Virus

“…Zhao et al, 26 investigated the use of a ZFP-based artificial transcription factor (ATF) to inhibit function of the enhancer I region and diminish X expression. Two three-fingered ZFP motifs were linked to form a six-fingered DNA binding domain that recognized an 18 base pair sequence.…”