Recent Progress and Future Prospective in HBV Cure by CRISPR/Cas

Abstract

Hepatitis B virus (HBV) infection remains an important issue of global public health. Although current antiviral therapy has dramatically reduced the mortality and morbidity of chronic hepatitis B (CHB), it fails to cure it. Rebound viremia often occurs after stopping antiviral therapy. Persistent HBV covalently closed circular DNA (cccDNA) and integrated DNA under antiviral therapy form the major barrier to eradication of HBV infection. CRISPR-mediated genome editing has emerged as a promising therapeutic approach to specifically destroy persistent HBV genomes, both cccDNA and integrated DNA, for HBV cure. However, the cleavage of integrated HBV DNA by CRISPR-Cas9 will cause double-strand break (DSB) of host genome, raising a serious safety concern about genome instability and carcinogenesis. The newly developed CRISPR-derived base editors (BEs), which fuse a catalytically disabled nuclease with a nucleobase deaminase enzyme, can be used to permanently inactivate HBV genome by introducing irreversible point mutations for generation of premature stop codons without DSBs of host genome. Although promising, CRISPR-mediated base editing still faces daunting challenges before its clinical application, including the base-editing efficacy, the off-target effect, the difficulty in finding conserved target HBV sequences, and in vivo delivery efficiency. Several strategies have been adopted to optimize the efficiency and specificity of CRISPR-BEs and to improve in vivo delivery efficacy through novel viral and non-viral delivery approaches. Particularly, the non-viral delivery of Cas9 mRNA and ribonucleoprotein by lipid nanoparticles exhibits attractive potential for liver-targeted delivery in clinical. Along with all progress above, the CRISPR-mediated gene therapy will ultimately achieve HBV cure.

Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo

“…31 In recent years, gene editing, particularly CRISPR-Cas9 and variants, has been explored as a potentially curative therapeutic approach for HBV but is hampered by CRISPR-Cas9 specificity, the potential for chromosomal translocations after a DSB, and delivery. 32 Here, we have developed an ARCUS nuclease with high on-target activity and specificity against the HBV polymerase gene with no detectable chromosomal translocations in a PHH infection model. Our studies show that ARCUS-POL can target cccDNA and integrated HBV DNA in vitro, resulting in robust HBsAg decline.…”

Advantages of CRISPR-Cas9 combined organoid model in the study of congenital nervous system malformations

“…Compared with the traditional gene-editing technology, CRISPR-Cas9 has a higher gene-editing efficiency, lower offtarget effect, and no DNA integration, so it is an ideal geneediting technology (Yang and Yang, 2021). In addition, Some Frontiers in Bioengineering and Biotechnology frontiersin.org CNSM is caused by multiple genes, so it is necessary to repair multiple genes in order to completely reverse the disease.…”

Gene Editing Technologies to Target HBV cccDNA

“…Altogether, compared with recent reviews of the topic [142,143], significant advances have been made in the last few years regarding the development of cccDNA targeting strategies and the comprehension of cccDNA fate after editing. Major concerns regarding host genome instability issues that might follow DSBs in HBV-integrated sequences prompted the use of newly developed base editing strategies which have shown promising preliminary results.…”