Improved Specificity and Safety of Anti-Hepatitis B Virus TALENs Using Obligate Heterodimeric FokI Nuclease Domains

Abstract

Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral core and surface open reading frames were modified with second- and third-generation FokI nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of FokI homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication.

Hepatitis B Virus Research in South Africa

“…In SA, researchers have been exploring methods of disrupting, degrading, or silencing cccDNA using gene editing tools, such as designer nucleases and epigenetic modifiers. Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with CRISPR associated (Cas) RNA-guided nucleases have been used to cleave both integrated viral DNA and episomal cccDNA (Figure 1) [33][34][35]150]. TALENs are engineered nucleases created by fusing a TALE DNA binding domain to an endonuclease, typically derived from the catalytic region of the FokI enzyme.…”

“…Hence, development of resistance to nucleoside/nucleotide analogs with a low genetic barrier to resistance such as lamivudine poses a challenge to anti-HBV therapy. New strategies to treat HBV infection include gene therapy approaches [33][34][35][36][37]. This review focuses on South African research that is aimed at understanding the biology and epidemiology of HBV, HBV/HIV coinfection, OBI, and new strategies to improve the prevention and treatment of HBV infection.…”

Review article: hepatitis B—current and emerging therapies

“…Zinc finger proteins (ZFP) and transcription activator-like effector nucleases (TALEN) designed for such purpose were able to successfully interfere with the binding of transcription factors in hepatocytes infected with HBV as well as in infected transgenic mice. [60][61][62] Clinical studies with ZFP are already A different non-gene editing approach involves amplification of the apolipoprotein B mRNA editing enzyme catalytic subunit 3A and 3B deaminases (APOBEC3B, A3B/APOBEC3A, A3A). 64,65 A3B was revealed to be upregulated by activation of the inflammatory lymphotoxin β receptor (LTβR) whose ligands include the tumour necrosis factor (TNF) superfamily.…”

“…Another approach involves homing nucleases designed to directly disrupt the cccDNA. Zinc finger proteins (ZFP) and transcription activator‐like effector nucleases (TALEN) designed for such purpose were able to successfully interfere with the binding of transcription factors in hepatocytes infected with HBV as well as in infected transgenic mice 60‐62 . Clinical studies with ZFP are already ongoing for T‐cell gene editing in patients infected with the human immunodeficiency virus (NCT00842634).…”

Cautious optimism in anticipation of hepatitis B curative therapies

“…The authors did a comprehensive review of the available options for gene editing. It is important to note, however, that like with any genetic intervention there is a risk of off-target cleavage[ 5 ], so more studies and large clinical trials are needed to investigate this therapeutic option.…”