Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA

Kayesh, Mohammad Enamul Hoque; Amako, Yutaka; Hashem, Abul; Murakami, Shuko; Ogawa, S.; Yamamoto, Naoki; Hifumi, Tatsuro; Miyoshi, Noriaki; Sugiyama, Masaya; Tanaka, Yasuhito; Mizokami, Masashi; Kohara, Michinori; Tsukiyama-Kohara, Kyoko

doi:10.1016/j.virusres.2020.198191

Cited by 22 publications

(23 citation statements)

References 52 publications

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“…The adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 were used to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and treated with entecavir, which showed reduction in total liver HBV DNA and cccDNA [141]. Similar studies of anti-HBV effects of the AAV2-/WJ11-Cas9 system in a uPA/scid humanized chimeric mouse model also showed reduced HBV infection [142].…”

Section: Hepatitis Bmentioning

confidence: 91%

Humanized Mice for Infectious and Neurodegenerative disorders

et al. 2021

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Humanized mice model human disease and as such are used commonly for research studies of infectious, degenerative and cancer disorders. Recent models also reflect hematopoiesis, natural immunity, neurobiology, and molecular pathways that influence disease pathobiology. A spectrum of immunodeficient mouse strains permit long-lived human progenitor cell engraftments. The presence of both innate and adaptive immunity enables high levels of human hematolymphoid reconstitution with cell susceptibility to a broad range of microbial infections. These mice also facilitate investigations of human pathobiology, natural disease processes and therapeutic efficacy in a broad spectrum of human disorders. However, a bridge between humans and mice requires a complete understanding of pathogen dose, co-morbidities, disease progression, environment, and genetics which can be mirrored in these mice. These must be considered for understanding of microbial susceptibility, prevention, and disease progression. With known common limitations for access to human tissues, evaluation of metabolic and physiological changes and limitations in large animal numbers, studies in mice prove important in planning human clinical trials. To these ends, this review serves to outline how humanized mice can be used in viral and pharmacologic research emphasizing both current and future studies of viral and neurodegenerative diseases. In all, humanized mouse provides cost-effective, high throughput studies of infection or degeneration in natural pathogen host cells, and the ability to test transmission and eradication of disease.

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Section: Hepatitis Bmentioning

confidence: 91%

Humanized Mice for Infectious and Neurodegenerative disorders

et al. 2021

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“…Simultaneously, the CRISPR/Cas9 system was also applied to target HBsAg or HBV X protein (HBx) in cell culture and in animal experiments, respectively, and demonstrated that the expression levels of HBsAg in cell culture supernatant and mouse serum were both decreased [ 45 ]. Recently, a large number of studies and similar data have been reported on using the CRISPR/Cas9 system to target HBV genes [ 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ], implying that CRISPR/Cas9-based gene editing may be a potential therapeutic method for HBV infection.…”

Section: Crispr/cas System In Virology Researchmentioning

confidence: 99%

Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Teng

Yao

Nair

et al. 2021

Viruses

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In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

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“…Although expensive, highly technically dependent, and immune-deficient, these human hepatocyte chimeric mouse models with HBV infection can generate cccDNA, so they are indeed the gold standard to examine the antiviral effect of the CRISPR-mediated gene editing on HBV infection. Two studies took the AAV delivery system with split SpCas9 or SaCas9 to determine the antiviral activity against HBV [45,46]. Of note, although the results showed the successful gene editing of persistent HBV infection, the efficacy was only modest, suggesting that there remains room for improvement of in vivo delivery efficiency of CRISPR-Cas9.…”

Section: In Vitro and In Vivo Hbv Models For Gene Editing Of Hbv Genomes By Crispr-cas9mentioning

confidence: 99%

“…In addition, the cargo capacity of the AAV vector is limited, so only small Cas9s, such as SaCas9, can fit into the AAVs for treatment of HBV infection as described above [63]. Therefore, to overcome the obstacle of cargo capacity, an alternative strategy utilizes two Cas9-fragment system [46] or splitintein Cas9 system, in which a full functional Cas9 protein is split into two non-functional fragments. Upon delivery to the target cells, the two fragments of Cas9 is reconstituted to a fully functional protein through either recombination [46] or the intein-mediated trans-splicing mechanism [72], but this approach usually requires high viral titers because co-delivery of dual AAVs into the same cells is less efficient.…”

Section: Delivery Of Crispr/casmentioning

confidence: 99%

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

Yang

2021

Viruses

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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.

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Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA

Cited by 22 publications

References 52 publications

Humanized Mice for Infectious and Neurodegenerative disorders

Humanized Mice for Infectious and Neurodegenerative disorders

Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

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

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