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1
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Jacobs R, Singh P, Smith T, Arbuthnot P, Maepa MB. Prospects of viral vector-mediated delivery of sequences encoding anti-HBV designer endonucleases. Gene Ther 2025; 32:8-15. [PMID: 35606493 DOI: 10.1038/s41434-022-00342-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 05/05/2022] [Accepted: 05/06/2022] [Indexed: 11/09/2022]
Abstract
Available treatment for chronic hepatitis B virus (HBV) infection offers modest functional curative efficacy. The viral replicative intermediate comprising covalently closed circular DNA (cccDNA) is responsible for persistent chronic HBV infection. Hence, current efforts have focused on developing therapies that disable cccDNA. Employing gene editing tools has emerged as an attractive strategy, with the end goal of establishing permanently inactivated cccDNA. Although anti-HBV designer nucleases are effective in vivo, none has yet progressed to clinical trial. Lack of safe and efficient delivery systems remains the limiting factor. Several vectors may be used to deliver anti-HBV gene editor-encoding sequences, with viral vectors being at the forefront. Despite the challenges associated with packaging large gene editor-encoding sequences into viral vectors, advancement in the field is overcoming such limitations. Translation of viral vector-mediated gene editing against HBV to clinical application is within reach. This review discusses the prospects of delivering HBV targeted designer nucleases using viral vectors.
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Affiliation(s)
- Ridhwaanah Jacobs
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Prashika Singh
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Tiffany Smith
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Mohube Betty Maepa
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
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2
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Yao ZQ, Schank MB, Zhao J, El Gazzar M, Wang L, Zhang Y, Hill AC, Banik P, Pyburn JS, Moorman JP. The potential of HBV cure: an overview of CRISPR-mediated HBV gene disruption. Front Genome Ed 2024; 6:1467449. [PMID: 39444780 PMCID: PMC11496132 DOI: 10.3389/fgeed.2024.1467449] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Accepted: 09/24/2024] [Indexed: 10/25/2024] Open
Abstract
Hepatitis B virus (HBV) infection is a common cause of liver disease worldwide. The current antiviral treatment using nucleotide analogues (NAs) can only suppress de novo HBV replication but cannot eliminate chronic HBV infection due to the persistence of covalently closed circular (ccc) DNA that sustains viral replication. The CRISPR/Cas9 system is a novel genome-editing tool that enables precise gene disruption and inactivation. With high efficiency and simplicity, the CRISPR/Cas9 system has been utilized in multiple studies to disrupt the HBV genome specifically, eliciting varying anti-HBV effects both in vitro and in vivo. Additionally, multi-locus gene targeting has shown enhanced antiviral activity, paving the way for combination therapy to disrupt and inactivate HBV cccDNA as well as integrated HBV DNA. Despite its promising antiviral effects, this technology faces several challenges that need to be overcome before its clinical application, i.e., off-target effects and in vivo drug delivery. As such, there is a need for improvement in CRISPR/Cas9 efficiency, specificity, versatility, and delivery. Here, we critically review the recent literature describing the tools employed in designing guide RNAs (gRNAs) targeting HBV genomes, the vehicles used for expressing and delivering CRISPR/Cas9 components, the models used for evaluating CRISPR-mediated HBV gene disruption, the methods used for assessing antiviral and off-target effects induced by CRISPR/Cas9-mediated HBV gene disruption, and the prospects of future directions and challenges in leveraging this HBV gene-editing approach, to advance the HBV treatment toward a clinical cure.
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Affiliation(s)
- Zhi Q. Yao
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
- Hepatitis (HBV/HCV/HIV) Program, James H. Quillen VA Medical Center, Department of Veterans Affairs, Johnson City, TN, United States
| | - Madison B. Schank
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Juan Zhao
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Mohamed El Gazzar
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Ling Wang
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Yi Zhang
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Addison C. Hill
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Puja Banik
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Jaeden S. Pyburn
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Jonathan P. Moorman
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
- Hepatitis (HBV/HCV/HIV) Program, James H. Quillen VA Medical Center, Department of Veterans Affairs, Johnson City, TN, United States
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3
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Zhang M, Chen H, Liu H, Tang H. The impact of integrated hepatitis B virus DNA on oncogenesis and antiviral therapy. Biomark Res 2024; 12:84. [PMID: 39148134 PMCID: PMC11328401 DOI: 10.1186/s40364-024-00611-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Accepted: 06/29/2024] [Indexed: 08/17/2024] Open
Abstract
The global burden of hepatitis B virus (HBV) infection remains high, with chronic hepatitis B (CHB) patients facing a significantly increased risk of developing cirrhosis and hepatocellular carcinoma (HCC). The ultimate objective of antiviral therapy is to achieve a sterilizing cure for HBV. This necessitates the elimination of intrahepatic covalently closed circular DNA (cccDNA) and the complete eradication of integrated HBV DNA. This review aims to summarize the oncogenetic role of HBV integration and the significance of clearing HBV integration in sterilizing cure. It specifically focuses on the molecular mechanisms through which HBV integration leads to HCC, including modulation of the expression of proto-oncogenes and tumor suppressor genes, induction of chromosomal instability, and expression of truncated mutant HBV proteins. The review also highlights the impact of antiviral therapy in reducing HBV integration and preventing HBV-related HCC. Additionally, the review offers insights into future objectives for the treatment of CHB. Current strategies for HBV DNA integration inhibition and elimination include mainly antiviral therapies, RNA interference and gene editing technologies. Overall, HBV integration deserves further investigation and can potentially serve as a biomarker for CHB and HBV-related HCC.
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Affiliation(s)
- Mingming Zhang
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
- Laboratory of Infectious and Liver Diseases, Institute of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
| | - Han Chen
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
- Laboratory of Infectious and Liver Diseases, Institute of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
| | - Huan Liu
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
- Laboratory of Infectious and Liver Diseases, Institute of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China
| | - Hong Tang
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China.
- Laboratory of Infectious and Liver Diseases, Institute of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China.
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Assefa A, Getie M, Getie B, Yazie T, Enkobahry A. Molecular epidemiology of hepatitis B virus (HBV) in Ethiopia: A review article. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2024; 122:105618. [PMID: 38857639 DOI: 10.1016/j.meegid.2024.105618] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/15/2024] [Revised: 06/06/2024] [Accepted: 06/07/2024] [Indexed: 06/12/2024]
Abstract
Hepatitis B virus (HBV) belongs to the family Hepadnaviridae and is the smallest human DNA virus, with a genome that is only 3200 nucleotides long. The absence of proofreading function in HBV reverse transcriptase provides a wide range of genetic variants for targeted outgrowth at different stages of infection. A number of sub genotypes and ten HBV genotypes (A through J) have been identified through analyses of the divergence of HBV genomic sequences. Numerous clinical outcomes, including the emergence of chronicity, the course of the disease, the effectiveness of treatment, and the response to vaccination, have been related to differences in genotype between HBV isolates. There are just seven studies that have been done in Ethiopia that examine the molecular epidemiology of HBV. Moreover, these studies haven't been compiled and reviewed yet. In this review, we looked at the genetic diversity and molecular epidemiology of HBV, the relationship between HBV genotypes and clinical outcomes, the immunopathogenesis of HBV, and finally the molecular epidemiology of HBV in Ethiopia. PubMed, Embase, and Google Scholar search engines were used to find relevant articles for the review. By using HBV genotyping, clinicians can better tailor vaccination decisions and antiviral therapy for patients with chronic hepatitis B who are more likely to experience the disease's progression.
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Affiliation(s)
- Ayenew Assefa
- Unit of Immunology, Department of Medical Laboratory Science, Debre Tabor University, Debre Tabor, Ethiopia.
| | - Molla Getie
- College of Medicine and Health Science, Medical Laboratory Science Department, Injibara University, Injibara, Ethiopia
| | - Birhanu Getie
- Unit of Medical Microbiology, Department of Medical Laboratory Science, Debre Tabor University, Debre Tabor, Ethiopia
| | - Takilosimeneh Yazie
- College of Health Science, Department of Pharmacy, Debre Tabor University, Debre Tabor, Ethiopia
| | - Aklesya Enkobahry
- College of Medicine and Health Science, Department of Biomedical Science, Injibara University, Injibara, Ethiopia
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Su C, Lin D, Huang X, Feng J, Jin A, Wang F, Lv Q, Lei L, Pan W. Developing hydrogels for gene therapy and tissue engineering. J Nanobiotechnology 2024; 22:182. [PMID: 38622684 PMCID: PMC11017488 DOI: 10.1186/s12951-024-02462-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Accepted: 04/04/2024] [Indexed: 04/17/2024] Open
Abstract
Hydrogels are a class of highly absorbent and easily modified polymer materials suitable for use as slow-release carriers for drugs. Gene therapy is highly specific and can overcome the limitations of traditional tissue engineering techniques and has significant advantages in tissue repair. However, therapeutic genes are often affected by cellular barriers and enzyme sensitivity, and carrier loading of therapeutic genes is essential. Therapeutic gene hydrogels can well overcome these difficulties. Moreover, gene-therapeutic hydrogels have made considerable progress. This review summarizes the recent research on carrier gene hydrogels for the treatment of tissue damage through a summary of the most current research frontiers. We initially introduce the classification of hydrogels and their cross-linking methods, followed by a detailed overview of the types and modifications of therapeutic genes, a detailed discussion on the loading of therapeutic genes in hydrogels and their characterization features, a summary of the design of hydrogels for therapeutic gene release, and an overview of their applications in tissue engineering. Finally, we provide comments and look forward to the shortcomings and future directions of hydrogels for gene therapy. We hope that this article will provide researchers in related fields with more comprehensive and systematic strategies for tissue engineering repair and further promote the development of the field of hydrogels for gene therapy.
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Affiliation(s)
- Chunyu Su
- Key Laboratory of Artificial Organs and Computational Medicine in Zhejiang Province, Institute of Translational Medicine, Zhejiang Shuren University, Hangzhou, 310015, China
- The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325200, China
- College of Biology & Pharmacy, Yulin Normal University, Yulin, 537000, China
| | - Dini Lin
- The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325200, China
| | - Xinyu Huang
- College of Biology & Pharmacy, Yulin Normal University, Yulin, 537000, China
| | - Jiayin Feng
- Key Laboratory of Artificial Organs and Computational Medicine in Zhejiang Province, Institute of Translational Medicine, Zhejiang Shuren University, Hangzhou, 310015, China
| | - Anqi Jin
- Key Laboratory of Artificial Organs and Computational Medicine in Zhejiang Province, Institute of Translational Medicine, Zhejiang Shuren University, Hangzhou, 310015, China
| | - Fangyan Wang
- Key Laboratory of Artificial Organs and Computational Medicine in Zhejiang Province, Institute of Translational Medicine, Zhejiang Shuren University, Hangzhou, 310015, China
| | - Qizhuang Lv
- College of Biology & Pharmacy, Yulin Normal University, Yulin, 537000, China.
| | - Lanjie Lei
- Key Laboratory of Artificial Organs and Computational Medicine in Zhejiang Province, Institute of Translational Medicine, Zhejiang Shuren University, Hangzhou, 310015, China.
| | - Wenjie Pan
- The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325200, China.
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Zhang X, Li Y, Huan C, Hou Y, Liu R, Shi H, Zhang P, Zheng B, Wang Y, Wang H, Zhang W. LncRNA NKILA inhibits HBV replication by repressing NF-κB signalling activation. Virol Sin 2024; 39:44-55. [PMID: 37832719 PMCID: PMC10877346 DOI: 10.1016/j.virs.2023.10.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Accepted: 10/08/2023] [Indexed: 10/15/2023] Open
Abstract
Hepatitis B virus (HBV) infection results in liver cirrhosis and hepatocellular carcinoma (HCC). HBx/nuclear factor (NF)-κB pathway plays a role in HBV replication. However, whether NF-κB-interacting long noncoding RNA (NKILA), a suppressor of NF-κB activation, regulates HBV replication remains largely unknown. In this study, gain-and-loss experiments showed that NKILA inhibited HBV replication by inhibiting NF-κB activity. In turn, HBV infection down-regulated NKILA expression. In addition, expression levels of NKILA were lower in the peripheral blood-derived monocytes (PBMCs) of HBV-positive patients than in healthy individuals, which were correlated with HBV viral loads. And a negative correlation between NKILA expression level and HBV viral loads was observed in blood serum from HBV-positive patients. Lower levels of endogenous NKILA were also observed in HepG2 cells expressing a 1.3-fold HBV genome, HBV-infected HepG2-NTCP cells, stable HBV-producing HepG2.2.15 and HepAD38 cells, compared to those HBV-negative cells. Furthermore, HBx was required for NKILA-mediated inhibition on HBV replication. NKILA decreased HBx-induced NF-κB activation by interrupting the interaction between HBx and p65, whereas NKILA mutants lack of essential domains for NF-ĸB inhibition, lost the ability to inhibit HBV replication. Together, our data demonstrate that NKILA may serve as a suppressor of HBV replication via NF-ĸB signalling.
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Affiliation(s)
- Xi Zhang
- Department of Infectious Diseases, Center of Infectious Diseases and Pathogen Biology, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, 130012, China; Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China; Department of Ophthalmology, The First Hospital of Jilin University, Changchun, 130012, China
| | - Yuanyuan Li
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China
| | - Chen Huan
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China
| | - Yubao Hou
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China
| | - Rujia Liu
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China
| | - Hongyun Shi
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China
| | - Peng Zhang
- Department of Infectious Diseases, Center of Infectious Diseases and Pathogen Biology, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, 130012, China
| | - Baisong Zheng
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China
| | - Yingchao Wang
- Hepatobiliary Pancreatic Surgery, The First Hospital of Jilin University, Changchun, 130012, China.
| | - Hong Wang
- Department of Infectious Diseases, Center of Infectious Diseases and Pathogen Biology, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, 130012, China; Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China.
| | - Wenyan Zhang
- Department of Infectious Diseases, Center of Infectious Diseases and Pathogen Biology, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, 130012, China; Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, 130012, China.
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Abdelwahed AH, Heineman BD, Wu GY. Novel Approaches to Inhibition of HBsAg Expression from cccDNA and Chromosomal Integrants: A Review. J Clin Transl Hepatol 2023; 11:1485-1497. [PMID: 38161502 PMCID: PMC10752814 DOI: 10.14218/jcth.2023.00067] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Revised: 07/19/2023] [Accepted: 08/16/2023] [Indexed: 01/03/2024] Open
Abstract
Hepatitis B virus (HBV) is a widely prevalent liver infection that can cause acute or chronic hepatitis. Although current treatment modalities are highly effective in the suppression of viral levels, they cannot eliminate the virus or achieve definitive cure. This is a consequence of the complex nature of HBV-host interactions. Major challenges to achieving sustained viral suppression include the presence of a high viral burden from the HBV DNA and hepatitis B surface antigen (HBsAg), the presence of reservoirs for HBV replication and antigen production, and the HBV-impaired innate and adaptive immune response of the host. Those therapeutic methods include cell entry inhibitors, HBsAg inhibitors, gene editing approaches, immune-targeting therapies and direct inhibitors of covalently closed circular DNA (cccDNA). Novel approaches that target these key mechanisms are now being studied in preclinical and clinical phases. In this review article, we provide a comprehensive review on mechanisms by which HBV escapes elimination from current treatments, and highlight new agents to achieve a definitive HBV cure.
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Affiliation(s)
- Ahmed H. Abdelwahed
- Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, CT, USA
| | - Brent D. Heineman
- Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, CT, USA
| | - George Y. Wu
- Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington, CT, USA
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Jacobs R, Dogbey MD, Mnyandu N, Neves K, Barth S, Arbuthnot P, Maepa MB. AAV Immunotoxicity: Implications in Anti-HBV Gene Therapy. Microorganisms 2023; 11:2985. [PMID: 38138129 PMCID: PMC10745739 DOI: 10.3390/microorganisms11122985] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2023] [Revised: 11/30/2023] [Accepted: 12/11/2023] [Indexed: 12/24/2023] Open
Abstract
Hepatitis B virus (HBV) has afflicted humankind for decades and there is still no treatment that can clear the infection. The development of recombinant adeno-associated virus (rAAV)-based gene therapy for HBV infection has become important in recent years and research has made exciting leaps. Initial studies, mainly using mouse models, showed that rAAVs are non-toxic and induce minimal immune responses. However, several later studies demonstrated rAAV toxicity, which is inextricably associated with immunogenicity. This is a major setback for the progression of rAAV-based therapies toward clinical application. Research aimed at understanding the mechanisms behind rAAV immunity and toxicity has contributed significantly to the inception of approaches to overcoming these challenges. The target tissue, the features of the vector, and the vector dose are some of the determinants of AAV toxicity, with the latter being associated with the most severe adverse events. This review discusses our current understanding of rAAV immunogenicity, toxicity, and approaches to overcoming these hurdles. How this information and current knowledge about HBV biology and immunity can be harnessed in the efforts to design safe and effective anti-HBV rAAVs is discussed.
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Affiliation(s)
- Ridhwaanah Jacobs
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa
| | - Makafui Dennis Dogbey
- Medical Biotechnology and Immunotherapy Research Unit, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa; (M.D.D.)
| | - Njabulo Mnyandu
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa
| | - Keila Neves
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa
| | - Stefan Barth
- Medical Biotechnology and Immunotherapy Research Unit, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa; (M.D.D.)
- South African Research Chair in Cancer Biotechnology, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa
| | - Mohube Betty Maepa
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa
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9
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Kasianchuk N, Dobrowolska K, Harkava S, Bretcan A, Zarębska-Michaluk D, Jaroszewicz J, Flisiak R, Rzymski P. Gene-Editing and RNA Interference in Treating Hepatitis B: A Review. Viruses 2023; 15:2395. [PMID: 38140636 PMCID: PMC10747710 DOI: 10.3390/v15122395] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2023] [Revised: 12/04/2023] [Accepted: 12/06/2023] [Indexed: 12/24/2023] Open
Abstract
The hepatitis B virus (HBV) continues to cause substantial health and economic burdens, and its target of elimination may not be reached in 2030 without further efforts in diagnostics, non-pharmaceutical prevention measures, vaccination, and treatment. Current therapeutic options in chronic HBV, based on interferons and/or nucleos(t)ide analogs, suppress the virus replication but do not eliminate the pathogen and suffer from several constraints. This paper reviews the progress on biotechnological approaches in functional and definitive HBV treatments, including gene-editing tools, i.e., zinc-finger proteins, transcription activator-like effector nucleases, and CRISPR/Cas9, as well as therapeutics based on RNA interference. The advantages and challenges of these approaches are also discussed. Although the safety and efficacy of gene-editing tools in HBV therapies are yet to be demonstrated, they show promise for the revitalization of a much-needed advance in the field and offer viral eradication. Particular hopes are related to CRISPR/Cas9; however, therapeutics employing this system are yet to enter the clinical testing phases. In contrast, a number of candidates based on RNA interference, intending to confer a functional cure, have already been introduced to human studies. However, larger and longer trials are required to assess their efficacy and safety. Considering that prevention is always superior to treatment, it is essential to pursue global efforts in HBV vaccination.
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Affiliation(s)
- Nadiia Kasianchuk
- Faculty of Biology, Adam Mickiewicz University in Poznań, 61-614 Poznań, Poland
| | | | - Sofiia Harkava
- Junior Academy of Sciences of Ukraine, Regional Branch in Dnipro, 49000 Dnipro, Ukraine;
| | - Andreea Bretcan
- National College “Ienăchiță Văcărescu”, 130016 Târgoviște, Romania;
| | - Dorota Zarębska-Michaluk
- Department of Infectious Diseases and Allergology, Jan Kochanowski University, 25-317 Kielce, Poland;
| | - Jerzy Jaroszewicz
- Department of Infectious Diseases and Hepatology, Medical University of Silesia in Katowice, 41-902 Bytom, Poland;
| | - Robert Flisiak
- Department of Infectious Diseases and Hepatology, Medical University of Białystok, 15-540 Białystok, Poland;
| | - Piotr Rzymski
- Department of Environmental Medicine, Poznan University of Medical Sciences, 60-806 Poznań, Poland
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Korkmaz P, Asan A, Karakeçili F, Tekin S, Demirtürk N. New Treatment Options in Chronic Hepatitis B: How Close Are We to Cure? INFECTIOUS DISEASES & CLINICAL MICROBIOLOGY 2023; 5:267-280. [PMID: 38633851 PMCID: PMC10986727 DOI: 10.36519/idcm.2023.265] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Accepted: 11/18/2023] [Indexed: 04/19/2024]
Abstract
Hepatitis B virus (HBV) infection is the leading cause of chronic liver disease worldwide. HBV-infected patients are at a lifetime risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Today, pegylated interferon (Peg-IFN) and nucleos(t)ide analogs (NAs) are used in the treatment of patients with chronic hepatitis B (CHB). Both treatment options have limitations. Despite effective viral suppression, NAs have little effect on covalently closed circular DNA (cccDNA), the stable episomal form of the HBV genome in hepatocytes. Therefore, the cure rate with NAs is low, and long-term treatment is required. Although the cure rate is better with Peg-IFN, it is difficult to tolerate due to drug side effects. Therefore, new treatment options are needed in the treatment of HBV infection. We can group new treatments under two headings: those that interfere with the viral life cycle and spread and those that modulate the immune response. Clinical studies show that combinations of treatments that directly target the viral life cycle and treatments that regulate the host immune system will be among the important treatment strategies in the future. As new direct-acting antiviral (DAA) and immunomodulatory therapies continue to emerge and evolve, functional cures in HBV treatment may be an achievable goal.
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Affiliation(s)
- Pınar Korkmaz
- Department of Infectious Diseases and Clinical Microbiology, Kütahya Health Sciences University School of Medicine, Kütahya, Türkiye
| | - Ali Asan
- Department of Infectious Diseases and Clinical Microbiology, Bursa Health Sciences University School of Medicine, Bursa, Türkiye
| | - Faruk Karakeçili
- Department of Infectious Diseases and Clinical Microbiology, Erzincan Binali Yıldırım University School of Medicine, Erzincan, Türkiye
| | - Süda Tekin
- Department of Infectious Diseases and Clinical Microbiology, Koç University School of Medicine, İstanbul, Türkiye
| | - Neşe Demirtürk
- Department of Infectious Diseases and Clinical Microbiology, Afyonkarahisar Health Sciences University, School of Medicine, Afyonkarahisar, Türkiye
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Zhang H, Tu T. Targeting Hepatitis B Virus DNA Using Designer Gene Editors. Clin Liver Dis 2023; 27:895-916. [PMID: 37778776 DOI: 10.1016/j.cld.2023.05.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/03/2023]
Abstract
Chronic hepatitis B virus (HBV) infection is a serious disease that currently has no cure. Key forms of HBV include covalently closed circular DNA, which mediates chronic persistence, and integrated DNA, which contributes to immune evasion and carcinogenesis. These forms are not targeted by current therapies; however, gene editing technologies have emerged as promising tools for disrupting HBV DNA. Gene editor-induced double-stranded breaks at precise locations within the HBV genome can induce effects ranging from inactivation of target genes to complete degradation of the target genome. Although promising, several challenges remain in efficacy and safety that require solutions.
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Affiliation(s)
- Henrik Zhang
- Westmead Institute for Medical Research, University of Sydney School of Medicine and Health, 176 Hawkesbury Road, Westmead, NSW 2145, Australia
| | - Thomas Tu
- Westmead Institute for Medical Research, University of Sydney School of Medicine and Health, 176 Hawkesbury Road, Westmead, NSW 2145, Australia.
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12
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Ogunnaike M, Das S, Raut SS, Sultana A, Nayan MU, Ganesan M, Edagwa BJ, Osna NA, Poluektova LY. Chronic Hepatitis B Infection: New Approaches towards Cure. Biomolecules 2023; 13:1208. [PMID: 37627273 PMCID: PMC10452112 DOI: 10.3390/biom13081208] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2023] [Revised: 07/24/2023] [Accepted: 07/28/2023] [Indexed: 08/27/2023] Open
Abstract
Chronic hepatitis B virus (HBV) infection leads to the development of cirrhosis and hepatocellular carcinoma. Lifelong treatment with nucleotides/nucleoside antiviral agents is effective at suppressing HBV replication, however, adherence to daily therapy can be challenging. This review discusses recent advances in the development of long-acting formulations for HBV treatment and prevention, which could potentially improve adherence. Promising new compounds that target distinct steps of the virus life cycle are summarized. In addition to treatments that suppress viral replication, curative strategies are focused on the elimination of covalently closed circular DNA and the inactivation of the integrated viral DNA from infected hepatocytes. We highlight promising long-acting antivirals and genome editing strategies for the elimination or deactivation of persistent viral DNA products in development.
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Affiliation(s)
- Mojisola Ogunnaike
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Srijanee Das
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Samiksha S. Raut
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
| | - Ashrafi Sultana
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
| | - Mohammad Ullah Nayan
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
| | - Murali Ganesan
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Benson J. Edagwa
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
| | - Natalia A. Osna
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
- Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Larisa Y. Poluektova
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA; (M.O.); (S.D.); (S.S.R.); (A.S.); (M.U.N.); (M.G.)
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13
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Ali A, Zafar MM, Farooq Z, Ahmed SR, Ijaz A, Anwar Z, Abbas H, Tariq MS, Tariq H, Mustafa M, Bajwa MH, Shaukat F, Razzaq A, Maozhi R. Breakthrough in CRISPR/Cas system: Current and future directions and challenges. Biotechnol J 2023; 18:e2200642. [PMID: 37166088 DOI: 10.1002/biot.202200642] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2022] [Revised: 05/04/2023] [Accepted: 05/05/2023] [Indexed: 05/12/2023]
Abstract
Targeted genome editing (GE) technology has brought a significant revolution in fictional genomic research and given hope to plant scientists to develop desirable varieties. This technology involves inducing site-specific DNA perturbations that can be repaired through DNA repair pathways. GE products currently include CRISPR-associated nuclease DNA breaks, prime editors generated DNA flaps, single nucleotide-modifications, transposases, and recombinases. The discovery of double-strand breaks, site-specific nucleases (SSNs), and repair mechanisms paved the way for targeted GE, and the first-generation GE tools, ZFNs and TALENs, were successfully utilized in plant GE. However, CRISPR-Cas has now become the preferred tool for GE due to its speed, reliability, and cost-effectiveness. Plant functional genomics has benefited significantly from the widespread use of CRISPR technology for advancements and developments. This review highlights the progress made in CRISPR technology, including multiplex editing, base editing (BE), and prime editing (PE), as well as the challenges and potential delivery mechanisms.
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Affiliation(s)
- Ahmad Ali
- National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China
| | | | - Zunaira Farooq
- National Center for Soybean Improvement, Key Laboratory of Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, China
| | - Syed Riaz Ahmed
- Nuclear Institute for Agriculture and Biology College (NIAB-C), Pakistan Institute of Engineering and Applied Science (PIEAS), Nilore, Pakistan
| | - Aqsa Ijaz
- Nuclear Institute for Agriculture and Biology College (NIAB-C), Pakistan Institute of Engineering and Applied Science (PIEAS), Nilore, Pakistan
| | - Zunaira Anwar
- Nuclear Institute for Agriculture and Biology College (NIAB-C), Pakistan Institute of Engineering and Applied Science (PIEAS), Nilore, Pakistan
| | - Huma Abbas
- Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan
| | - Muhammad Sayyam Tariq
- Nuclear Institute for Agriculture and Biology College (NIAB-C), Pakistan Institute of Engineering and Applied Science (PIEAS), Nilore, Pakistan
| | - Hala Tariq
- Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Faisalabad, Pakistan
| | - Mahwish Mustafa
- Center of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan
| | | | - Fiza Shaukat
- Center of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan
| | - Abdul Razzaq
- Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, China
- Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan
| | - Ren Maozhi
- Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, China
- Institute of, Urban Agriculture, Chinese Academy of Agriculture Science, Chengdu, China
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14
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Nevola R, Beccia D, Rosato V, Ruocco R, Mastrocinque D, Villani A, Perillo P, Imbriani S, Delle Femine A, Criscuolo L, Alfano M, La Montagna M, Russo A, Marfella R, Cozzolino D, Sasso FC, Rinaldi L, Marrone A, Adinolfi LE, Claar E. HBV Infection and Host Interactions: The Role in Viral Persistence and Oncogenesis. Int J Mol Sci 2023; 24:7651. [PMID: 37108816 PMCID: PMC10145402 DOI: 10.3390/ijms24087651] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Revised: 04/14/2023] [Accepted: 04/19/2023] [Indexed: 04/29/2023] Open
Abstract
Hepatitis B virus (HBV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Despite the advent of vaccines and potent antiviral agents able to suppress viral replication, recovery from chronic HBV infection is still an extremely difficult goal to achieve. Complex interactions between virus and host are responsible for HBV persistence and the risk of oncogenesis. Through multiple pathways, HBV is able to silence both innate and adaptive immunological responses and become out of control. Furthermore, the integration of the viral genome into that of the host and the production of covalently closed circular DNA (cccDNA) represent reservoirs of viral persistence and account for the difficult eradication of the infection. An adequate knowledge of the virus-host interaction mechanisms responsible for viral persistence and the risk of hepatocarcinogenesis is necessary for the development of functional cures for chronic HBV infection. The purpose of this review is, therefore, to analyze how interactions between HBV and host concur in the mechanisms of infection, persistence, and oncogenesis and what are the implications and the therapeutic perspectives that follow.
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Affiliation(s)
- Riccardo Nevola
- Liver Unit, Ospedale Evangelico Betania, 80147 Naples, Italy; (V.R.); (D.M.); (P.P.); (E.C.)
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Domenico Beccia
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Valerio Rosato
- Liver Unit, Ospedale Evangelico Betania, 80147 Naples, Italy; (V.R.); (D.M.); (P.P.); (E.C.)
| | - Rachele Ruocco
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Davide Mastrocinque
- Liver Unit, Ospedale Evangelico Betania, 80147 Naples, Italy; (V.R.); (D.M.); (P.P.); (E.C.)
| | - Angela Villani
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Pasquale Perillo
- Liver Unit, Ospedale Evangelico Betania, 80147 Naples, Italy; (V.R.); (D.M.); (P.P.); (E.C.)
| | - Simona Imbriani
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Augusto Delle Femine
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Livio Criscuolo
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Maria Alfano
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Marco La Montagna
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Antonio Russo
- Department of Mental Health and Public Medicine, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy;
| | - Raffaele Marfella
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Domenico Cozzolino
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Ferdinando Carlo Sasso
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Luca Rinaldi
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Aldo Marrone
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Luigi Elio Adinolfi
- Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy; (D.B.); (R.R.); (A.V.); (S.I.); (A.D.F.); (L.C.); (M.A.); (M.L.M.); (R.M.); (D.C.); (F.C.S.); (L.R.); (A.M.); (L.E.A.)
| | - Ernesto Claar
- Liver Unit, Ospedale Evangelico Betania, 80147 Naples, Italy; (V.R.); (D.M.); (P.P.); (E.C.)
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15
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Akram F, Waheed HM, Shah FI, Haq IU, Nasir N, Akhtar MT, Farooq Gohar U. Burgeoning therapeutic strategies to curb the contemporary surging viral infections. Microb Pathog 2023; 179:106088. [PMID: 37004965 DOI: 10.1016/j.micpath.2023.106088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2022] [Revised: 03/23/2023] [Accepted: 03/24/2023] [Indexed: 04/03/2023]
Abstract
Significant efforts and initiatives were already made in the health care systems, however in the last few years; our world is facing emergences of viral infections which potentially leading to considerable challenges in terms of higher morbidity, mortality, increased and considerable financial loads on the affected populations. Over ten major epidemics or pandemics have been recorded in the twenty-first century, the ongoing coronavirus pandemic being one of them. Viruses being distinct obligate pathogens largely dependent on living beings are considered as one of the prominent causes of death globally. Although effective vaccines and antivirals have led to the eradication of imperative viral pathogens, the emergences of new viral infections as well as novel drug-resistant strains have necessitated the implementation of ingenious and efficient therapeutic approaches to treat viral outbreaks in the future. Nature being a constant source of tremendous therapeutical resources has inspired us to develop multi-target antiviral drugs, overcoming the challenges and limitations faced by pharmaceutical industry. Recent breakthroughs in the understanding of the cellular and molecular mechanisms of viral reproduction have laid the groundwork for potential treatment approaches including antiviral gene therapy relying on the application of precisely engineered nucleic acids for disabling pathogen replication. The development of RNA interference and advancements in genome manipulating tools have proven to be especially significant in this regard. In this review, we discussed mode of actions and pathophysiological events associated with the viral infections; followed by distributions, and advancement made towards the detection strategies for timely diagnosis. In the later section, current approaches to cope up the viral pathogens and their key limitations have also been elaborated. Lastly, we also explored some novel and potential targets to treat such infections, where attentions were made on next generation gene editing technologies.
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Martinez MG, Smekalova E, Combe E, Gregoire F, Zoulim F, Testoni B. Gene Editing Technologies to Target HBV cccDNA. Viruses 2022; 14:v14122654. [PMID: 36560658 PMCID: PMC9787400 DOI: 10.3390/v14122654] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Revised: 11/21/2022] [Accepted: 11/22/2022] [Indexed: 11/29/2022] Open
Abstract
Hepatitis B virus (HBV) remains a significant cause of mortality and morbidity worldwide, since chronic HBV infection is associated with elevated risk of cirrhosis and hepatocellular carcinoma. Current licensed therapies against HBV efficiently suppress viral replication; however, they do not have significant effects on the intrahepatic covalently closed circular DNA (cccDNA) of the viral minichromosome responsible for viral persistence. Thus, life-long treatment is required to avoid viral rebound. There is a significant need for novel therapies that can reduce, silence or eradicate cccDNA, thus preventing HBV reemergence after treatment withdrawal. In this review, we discuss the latest developments and applications of gene editing and related approaches for directly targeting HBV DNA and, more specifically, cccDNA in infected hepatocytes.
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Affiliation(s)
| | | | - Emmanuel Combe
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), 69008 Lyon, France
| | | | - Fabien Zoulim
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), 69008 Lyon, France
- Hospices Civils de Lyon (HCL), 69002 Lyon, France
- Université Claude-Bernard Lyon 1 (UCBL1), 69008 Lyon, France
| | - Barbara Testoni
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), 69008 Lyon, France
- Université Claude-Bernard Lyon 1 (UCBL1), 69008 Lyon, France
- Correspondence:
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17
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CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements. Cells 2022; 11:cells11223615. [PMID: 36429042 PMCID: PMC9688409 DOI: 10.3390/cells11223615] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 11/10/2022] [Accepted: 11/13/2022] [Indexed: 11/18/2022] Open
Abstract
This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity.
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18
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Watanabe T, Hayashi S, Tanaka Y. Drug Discovery Study Aimed at a Functional Cure for HBV. Viruses 2022; 14:1393. [PMID: 35891374 PMCID: PMC9321005 DOI: 10.3390/v14071393] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 06/19/2022] [Accepted: 06/23/2022] [Indexed: 02/04/2023] Open
Abstract
Hepatitis B virus (HBV) causes acute and, most importantly, chronic hepatitis B worldwide. Antiviral treatments have been developed to reduce viral loads but few patients with chronic hepatitis B (CHB) achieve a functional cure. The development of new therapeutic agents is desirable. Recently, many novel agents have been developed, including drugs targeting HBV-DNA and HBV-RNA. This review provides an overview of the developmental status of these drugs, especially direct acting antiviral agents (DAAs). Serological biomarkers of HBV infection are essential for predicting the clinical course of CHB. It is also important to determine the amount and activity of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. Hepatitis B core-associated antigen (HBcrAg) is a new HBV marker that has an important role in reflecting cccDNA in CHB, because it is associated with hepatic cccDNA, as well as serum HBV DNA. The highly sensitive HBcrAg (iTACT-HBcrAg) assay could be a very sensitive HBV activation marker and an alternative to HBV DNA testing for monitoring reactivation. Many of the drugs currently in clinical trials have shown efficacy in reducing hepatitis B surface antigen (HBsAg) levels. Combination therapies with DAAs and boost immune response are also under development; finding the best combinations will be important for therapeutic development.
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Affiliation(s)
| | | | - Yasuhito Tanaka
- Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan; (T.W.); (S.H.)
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19
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Wang Y, Li Y, Zai W, Hu K, Zhu Y, Deng Q, Wu M, Li Y, Chen J, Yuan Z. HBV covalently closed circular DNA minichromosomes in distinct epigenetic transcriptional states differ in their vulnerability to damage. Hepatology 2022; 75:1275-1288. [PMID: 34779008 DOI: 10.1002/hep.32245] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2021] [Revised: 10/20/2021] [Accepted: 11/10/2021] [Indexed: 12/19/2022]
Abstract
BACKGROUND AND AIMS HBV covalently closed circular DNA (cccDNA) is a major obstacle for a cure of chronic hepatitis B. Accumulating evidence suggests that epigenetic modifications regulate the transcriptional activity of cccDNA minichromosomes. However, it remains unclear how the epigenetic state of cccDNA affects its stability. APPROACHES AND RESULTS By using HBV infection cell models and in vitro and in vivo recombinant cccDNA (rcccDNA) and HBVcircle models, the reduction rate of HBV cccDNA and the efficacy of apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A)-mediated and CRISPR/CRISPR-associated 9 (Cas9)-mediated cccDNA targeting were compared between cccDNAs with distinct transcriptional activities. Interferon-α treatment and hepatitis B x protein (HBx) deletion were applied as two strategies for cccDNA repression. Chromatin immunoprecipitation and micrococcal nuclease assays were performed to determine the epigenetic pattern of cccDNA. HBV cccDNA levels remained stable in nondividing hepatocytes; however, they were significantly reduced during cell division, and the reduction rate was similar between cccDNAs in transcriptionally active and transcriptionally repressed states. Strikingly, HBV rcccDNA without HBx expression exhibited a significantly longer persistence in mice. The cccDNA with low transcriptional activity exhibited an epigenetically inactive pattern and was more difficult to access by APOBEC3A and engineered CRISPR-Cas9. The epigenetic regulator activating cccDNA increased its vulnerability to APOBEC3A. CONCLUSIONS HBV cccDNA minichromosomes in distinct epigenetic transcriptional states showed a similar reduction rate during cell division but significantly differed in their accessibility and vulnerability to targeted nucleases and antiviral agents. Epigenetic sensitization of cccDNA makes it more susceptible to damage and may potentially contribute to an HBV cure.
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Affiliation(s)
- Yang Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Yumeng Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Wenjing Zai
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Kongying Hu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Yuanfei Zhu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Qiang Deng
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
- Research Unit of Cure of Chronic Hepatitis B Virus InfectionChinese Academy of Medical SciencesShanghaiChina
- Shanghai Frontiers Science Center of Pathogenic Microbes and InfectionShanghaiChina
| | - Min Wu
- Shanghai Public Health Clinical CenterFudan UniversityShanghaiChina
| | - Yaming Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Jieliang Chen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
- Research Unit of Cure of Chronic Hepatitis B Virus InfectionChinese Academy of Medical SciencesShanghaiChina
- Shanghai Frontiers Science Center of Pathogenic Microbes and InfectionShanghaiChina
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
- Research Unit of Cure of Chronic Hepatitis B Virus InfectionChinese Academy of Medical SciencesShanghaiChina
- Shanghai Frontiers Science Center of Pathogenic Microbes and InfectionShanghaiChina
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20
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Li G, Li X, Zhuang S, Wang L, Zhu Y, Chen Y, Sun W, Wu Z, Zhou Z, Chen J, Huang X, Wang J, Li D, Li W, Wang H, Wei W. Gene editing and its applications in biomedicine. SCIENCE CHINA. LIFE SCIENCES 2022; 65:660-700. [PMID: 35235150 PMCID: PMC8889061 DOI: 10.1007/s11427-021-2057-0] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Accepted: 12/06/2021] [Indexed: 02/06/2023]
Abstract
The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.
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Affiliation(s)
- Guanglei Li
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Xiangyang Li
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Songkuan Zhuang
- Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, 518035, China
| | - Liren Wang
- Shanghai Frontiers Science Research Base of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Yifan Zhu
- Shanghai Frontiers Science Research Base of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Yangcan Chen
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China
| | - Wen Sun
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China
| | - Zeguang Wu
- Biomedical Pioneering Innovation Center, Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, 100871, China
| | - Zhuo Zhou
- Biomedical Pioneering Innovation Center, Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, 100871, China
| | - Jia Chen
- Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
| | - Xingxu Huang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
| | - Jin Wang
- Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, 518035, China.
| | - Dali Li
- Shanghai Frontiers Science Research Base of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China.
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.
- Bejing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China.
- HIT Center for Life Sciences, Harbin Institute of Technology, Harbin, 150001, China.
| | - Haoyi Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.
| | - Wensheng Wei
- Biomedical Pioneering Innovation Center, Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, 100871, China.
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21
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Xiao MH, Lin YF, Xie PP, Chen HX, Deng JW, Zhang W, Zhao N, Xie C, Meng Y, Liu X, Zhuang SM, Zhu Y, Fang JH. Downregulation of a mitochondrial micropeptide, MPM, promotes hepatoma metastasis by enhancing mitochondrial complex I activity. Mol Ther 2022; 30:714-725. [PMID: 34478872 PMCID: PMC8821931 DOI: 10.1016/j.ymthe.2021.08.032] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2021] [Revised: 07/25/2021] [Accepted: 08/25/2021] [Indexed: 02/07/2023] Open
Abstract
We and others have shown that MPM (micropeptide in mitochondria) regulates myogenic differentiation and muscle development. However, the roles of MPM in cancer development remain unknown. Here we revealed that MPM was downregulated significantly in human hepatocellular carcinoma (HCC) tissues and its decrease was associated with increased metastasis potential and HCC recurrence. Gain- and loss-of-function investigations disclosed that in vitro migration/invasion and in vivo liver/lung metastasis of hepatoma cells were repressed by restoring MPM expression and increased by silencing MPM. Mechanism investigations revealed that MPM interacted with NDUFA7. Mitochondrial complex I activity was inhibited by overexpressing MPM and enhanced by siMPM, and this effect of siMPM was attenuated by knocking down NDUFA7. The NAD+/NADH ratio, which was regulated by complex I, was reduced by MPM but increased by siMPM. Treatment with the NAD+ precursor nicotinamide abrogated the inhibitory effect of MPM on hepatoma cell migration. Further investigations showed that miR-17-5p bound to MPM and inhibited MPM expression. miR-17-5p upregulation was associated with MPM downregulation in HCC tissues. These findings indicate that a decrease in MPM expression may promote hepatoma metastasis by increasing mitochondrial complex I activity and the NAD+/NADH ratio.
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Affiliation(s)
- Man-Huan Xiao
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Yi-Fang Lin
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Peng-Peng Xie
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Hua-Xing Chen
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Jun-Wen Deng
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Wei Zhang
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Na Zhao
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Chen Xie
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Yu Meng
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China
| | - Xingguo Liu
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 510530, China
| | - Shi-Mei Zhuang
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China,Corresponding author: Shi-Mei Zhuang, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China.
| | - Ying Zhu
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China,Corresponding author: Ying Zhu, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China.
| | - Jian-Hong Fang
- MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China,Corresponding author: Jian-Hong Fang, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P.R. China.
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22
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Watanabe T, Inoue T, Tanaka Y. Hepatitis B Core-Related Antigen and New Therapies for Hepatitis B. Microorganisms 2021; 9:2083. [PMID: 34683404 PMCID: PMC8537336 DOI: 10.3390/microorganisms9102083] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2021] [Revised: 09/29/2021] [Accepted: 09/30/2021] [Indexed: 02/06/2023] Open
Abstract
The hepatitis B core-related antigen (HBcrAg) is an unprecedented novel HBV biomarker that plays an essential role in reflecting covalently closed circular DNA (cccDNA) in chronic hepatitis B (CHB) because its levels correlate with intrahepatic cccDNA and serum HBV DNA. In this review, we describe the clinical application of serum HBcrAg in CHB patients, with a particular focus on new therapies targeting intrahepatic HBV replication. (1) HBcrAg can be detected in clinical cases where serum HBV DNA is undetectable during anti-HBV therapy. (2) A highly sensitive HBcrAg assay (iTACT-HBcrAg) may be useful for monitoring HBV reactivation, as an alternative to HBV DNA. (3) Decreased HBcrAg levels have been significantly associated with promising outcomes in CHB patients, reducing the risk of progression or recurrence of hepatocellular carcinoma. Additionally, we focus on and discuss several drugs in development that target HBV replication, and monitoring HBcrAg may be useful for determining the therapeutic efficacies of such novel drugs. In conclusion, HBcrAg, especially when measured by the recently developed iTACT-HBcrAg assay, may be the most appropriate surrogate marker, over other HBV biomarkers, to predict disease progression and treatment response in CHB patients.
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Affiliation(s)
- Takehisa Watanabe
- Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan;
| | - Takako Inoue
- Department of Clinical Laboratory Medicine, Nagoya City University Hospital, Nagoya 467-8602, Japan;
| | - Yasuhito Tanaka
- Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan;
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23
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Bartoli A, Gabrielli F, Tassi A, Cursaro C, Pinelli A, Andreone P. Treatments for HBV: A Glimpse into the Future. Viruses 2021; 13:1767. [PMID: 34578347 PMCID: PMC8473442 DOI: 10.3390/v13091767] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 08/17/2021] [Accepted: 08/24/2021] [Indexed: 12/16/2022] Open
Abstract
The hepatitis B virus is responsible for most of the chronic liver disease and liver cancer worldwide. As actual therapeutic strategies have had little success in eradicating the virus from hepatocytes, and as lifelong treatment is often required, new drugs targeting the various phases of the hepatitis B virus (HBV) lifecycle are currently under investigation. In this review, we provide an overview of potential future treatments for HBV.
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Affiliation(s)
- Alessandra Bartoli
- Department of Medical and Surgical Sciences, Division of Internal Medicine, Maternal-Infantile and Adult, University of Modena and Reggio Emilia, 41126 Modena, Italy; (A.B.); (F.G.); (A.T.); (C.C.); (A.P.)
- Postgraduate School of Allergy and Clinical Immunology, University of Modena and Reggio Emilia, 41126 Modena, Italy
| | - Filippo Gabrielli
- Department of Medical and Surgical Sciences, Division of Internal Medicine, Maternal-Infantile and Adult, University of Modena and Reggio Emilia, 41126 Modena, Italy; (A.B.); (F.G.); (A.T.); (C.C.); (A.P.)
- Postgraduate School of Internal Medicine, University of Modena and Reggio Emilia, 41126 Modena, Italy
| | - Andrea Tassi
- Department of Medical and Surgical Sciences, Division of Internal Medicine, Maternal-Infantile and Adult, University of Modena and Reggio Emilia, 41126 Modena, Italy; (A.B.); (F.G.); (A.T.); (C.C.); (A.P.)
- Postgraduate School of Internal Medicine, University of Modena and Reggio Emilia, 41126 Modena, Italy
| | - Carmela Cursaro
- Department of Medical and Surgical Sciences, Division of Internal Medicine, Maternal-Infantile and Adult, University of Modena and Reggio Emilia, 41126 Modena, Italy; (A.B.); (F.G.); (A.T.); (C.C.); (A.P.)
| | - Ambra Pinelli
- Department of Medical and Surgical Sciences, Division of Internal Medicine, Maternal-Infantile and Adult, University of Modena and Reggio Emilia, 41126 Modena, Italy; (A.B.); (F.G.); (A.T.); (C.C.); (A.P.)
- Postgraduate School of Allergy and Clinical Immunology, University of Modena and Reggio Emilia, 41126 Modena, Italy
| | - Pietro Andreone
- Department of Medical and Surgical Sciences, Division of Internal Medicine, Maternal-Infantile and Adult, University of Modena and Reggio Emilia, 41126 Modena, Italy; (A.B.); (F.G.); (A.T.); (C.C.); (A.P.)
- Postgraduate School of Allergy and Clinical Immunology, University of Modena and Reggio Emilia, 41126 Modena, Italy
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24
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Ghosh S, Chakraborty A, Banerjee S. Persistence of Hepatitis B Virus Infection: A Multi-Faceted Player for Hepatocarcinogenesis. Front Microbiol 2021; 12:678537. [PMID: 34526974 PMCID: PMC8435854 DOI: 10.3389/fmicb.2021.678537] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Accepted: 07/06/2021] [Indexed: 12/14/2022] Open
Abstract
Hepatitis B virus (HBV) infection has a multi-dimensional effect on the host, which not only alters the dynamics of immune response but also persists in the hepatocytes to predispose oncogenic factors. The virus exists in multiple forms of which the nuclear localized covalently closed circular DNA (cccDNA) is the most stable and the primary reason for viral persistence even after clearance of surface antigen and viral DNA. The second reason is the existence of pregenomic RNA (pgRNA) containing virion particles. On the other hand, the integration of the viral genome in the host chromosome also leads to persistent production of viral proteins along with the chromosomal instabilities. The interferon treatment or administration of nucleot(s)ide analogs leads to reduction in the viral DNA load, but the pgRNA and surface antigen clearance are a slow process and complete loss of serological HBsAg is rare. The prolonged exposure of immune cells to the viral antigens, particularly HBs antigen, in the blood circulation results in T-cell exhaustion, which disrupts immune clearance of the virus and virus-infected cells. In addition, it predisposes immune-tolerant microenvironment, which facilitates the tumor progression. Thus cccDNA, pgRNA, and HBsAg along with the viral DNA could be the therapeutic targets in the early disease stages that may improve the quality of life of chronic hepatitis B patients by impeding the progression of the disease toward hepatocellular carcinoma.
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Affiliation(s)
| | | | - Soma Banerjee
- Centre for Liver Research, School of Digestive and Liver Diseases, Institute of Post Graduate Medical Education and Research, Kolkata, India
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25
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Recent developments with advancing gene therapy to treat chronic infection with hepatitis B virus. Curr Opin HIV AIDS 2021; 15:200-207. [PMID: 32141890 DOI: 10.1097/coh.0000000000000623] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
PURPOSE OF REVIEW The available vaccine and therapies against hepatitis B virus (HBV) rarely eliminate chronic infection with the virus. High mortality resulting from complicating cirrhosis and hepatocellular carcinoma makes improving anti-HBV therapy an important priority. Recent advances with using gene therapy to counter HBV have potential and are the focus of this review. RECENT FINDINGS The stable replication-competent HBV intermediate comprising covalently closed circular DNA (cccDNA) is the template for expression of all viral genes. Inactivating cccDNA has thus been a focus of research aimed at achieving cure for HBV infection. Many studies have reported profound inhibition of replication of the virus using silencing and editing techniques. Therapeutic gene silencing with synthetic short interfering RNA is now in clinical trials. Ability to mutate and permanently inactivate cccDNA with engineered gene editors, such as those derived from CRISPR/Cas or TALENs, is particularly appealing but has not yet reached clinical evaluation. SUMMARY Gene silencing and gene editing potentially provide the means to cure HBV infection. However, achieving efficient delivery of therapeutic sequences, ensuring their specificity of action and progress with other antiviral strategies are likely to determine utility of gene therapy for chronic HBV infection.
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Tsounis EP, Tourkochristou E, Mouzaki A, Triantos C. Toward a new era of hepatitis B virus therapeutics: The pursuit of a functional cure. World J Gastroenterol 2021; 27:2727-2757. [PMID: 34135551 PMCID: PMC8173382 DOI: 10.3748/wjg.v27.i21.2727] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Revised: 02/23/2021] [Accepted: 04/13/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) infection, although preventable by vaccination, remains a global health problem and a major cause of chronic liver disease. Although current treatment strategies suppress viral replication very efficiently, the optimal endpoint of hepatitis B surface antigen (HBsAg) clearance is rarely achieved. Moreover, the thorny problems of persistent chromatin-like covalently closed circular DNA and the presence of integrated HBV DNA in the host genome are ignored. Therefore, the scientific community has focused on developing innovative therapeutic approaches to achieve a functional cure of HBV, defined as undetectable HBV DNA and HBsAg loss over a limited treatment period. A deeper understanding of the HBV life cycle has led to the introduction of novel direct-acting antivirals that exert their function through multiple mechanisms, including inhibition of viral entry, transcriptional silencing, epigenetic manipulation, interference with capsid assembly, and disruption of HBsAg release. In parallel, another category of new drugs aims to restore dysregulated immune function in chronic hepatitis B accompanied by lethargic cellular and humoral responses. Stimulation of innate immunity by pattern-recognition receptor agonists leads to upregulation of antiviral cytokine expression and appears to contribute to HBV containment. Immune checkpoint inhibitors and adoptive transfer of genetically engineered T cells are breakthrough technologies currently being explored that may elicit potent HBV-specific T-cell responses. In addition, several clinical trials are attempting to clarify the role of therapeutic vaccination in this setting. Ultimately, it is increasingly recognized that elimination of HBV requires a treatment regimen based on a combination of multiple drugs. This review describes the rationale for progressive therapeutic interventions and discusses the latest findings in the field of HBV therapeutics.
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Affiliation(s)
- Efthymios P Tsounis
- Division of Gastroenterology, Department of Internal Medicine, Medical School, University of Patras, Patras 26504, Greece
| | - Evanthia Tourkochristou
- Division of Gastroenterology, Department of Internal Medicine, Medical School, University of Patras, Patras 26504, Greece
| | - Athanasia Mouzaki
- Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Patras 26504, Greece
| | - Christos Triantos
- Division of Gastroenterology, Department of Internal Medicine, Medical School, University of Patras, Patras 26504, Greece
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27
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Prifti GM, Moianos D, Giannakopoulou E, Pardali V, Tavis JE, Zoidis G. Recent Advances in Hepatitis B Treatment. Pharmaceuticals (Basel) 2021; 14:417. [PMID: 34062711 PMCID: PMC8147224 DOI: 10.3390/ph14050417] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 04/22/2021] [Accepted: 04/23/2021] [Indexed: 01/10/2023] Open
Abstract
Hepatitis B virus infection affects over 250 million chronic carriers, causing more than 800,000 deaths annually, although a safe and effective vaccine is available. Currently used antiviral agents, pegylated interferon and nucleos(t)ide analogues, have major drawbacks and fail to completely eradicate the virus from infected cells. Thus, achieving a "functional cure" of the infection remains a real challenge. Recent findings concerning the viral replication cycle have led to development of novel therapeutic approaches including viral entry inhibitors, epigenetic control of cccDNA, immune modulators, RNA interference techniques, ribonuclease H inhibitors, and capsid assembly modulators. Promising preclinical results have been obtained, and the leading molecules under development have entered clinical evaluation. This review summarizes the key steps of the HBV life cycle, examines the currently approved anti-HBV drugs, and analyzes novel HBV treatment regimens.
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Affiliation(s)
- Georgia-Myrto Prifti
- Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; (G.-M.P.); (D.M.); (E.G.); (V.P.)
| | - Dimitrios Moianos
- Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; (G.-M.P.); (D.M.); (E.G.); (V.P.)
| | - Erofili Giannakopoulou
- Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; (G.-M.P.); (D.M.); (E.G.); (V.P.)
| | - Vasiliki Pardali
- Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; (G.-M.P.); (D.M.); (E.G.); (V.P.)
| | - John E. Tavis
- Molecular Microbiology and Immunology, Saint Louis University, Saint Louis, MO 63104, USA;
| | - Grigoris Zoidis
- Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; (G.-M.P.); (D.M.); (E.G.); (V.P.)
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Ely A, Singh P, Smith TS, Arbuthnot P. In vitro transcribed mRNA for expression of designer nucleases: Advantages as a novel therapeutic for the management of chronic HBV infection. Adv Drug Deliv Rev 2021; 168:134-146. [PMID: 32485207 DOI: 10.1016/j.addr.2020.05.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Revised: 05/14/2020] [Accepted: 05/27/2020] [Indexed: 02/06/2023]
Abstract
Chronic infection with the hepatitis B virus (HBV) remains a significant worldwide medical problem. While diseases caused by HIV infection, tuberculosis and malaria are on the decline, new cases of chronic hepatitis B are on the rise. Because often fatal complications of cirrhosis and hepatocellular carcinoma are associated with chronic hepatitis B, the need for a cure is as urgent as ever. Currently licensed therapeutics fail to eradicate the virus and this is attributable to persistence of the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Elimination or inactivation of the viral cccDNA is thus a goal of research aimed at hepatitis B cure. The ability to engineer nucleases that are capable of specific cleavage of a DNA sequence now provides the means to disable cccDNA permanently. The scientific literature is replete with many examples of using designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) to inactivate HBV. However, important concerns about safety, dose control and efficient delivery need to be addressed before the technology is employed in a clinical setting. Use of in vitro transcribed mRNA to express therapeutic gene editors goes some way to overcoming these concerns. The labile nature of RNA limits off-target effects and enables dose control. Compatibility with hepatotropic non-viral vectors is convenient for the large scale preparation that will be required for advancing gene editing as a mode of curing chronic hepatitis B.
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Lee HW, Lee JS, Ahn SH. Hepatitis B Virus Cure: Targets and Future Therapies. Int J Mol Sci 2020; 22:ijms22010213. [PMID: 33379331 PMCID: PMC7795643 DOI: 10.3390/ijms22010213] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2020] [Revised: 12/19/2020] [Accepted: 12/21/2020] [Indexed: 02/06/2023] Open
Abstract
Chronic hepatitis B virus (HBV) infection is a major global health problem. It can cause progressive liver fibrosis leading to cirrhosis with end-stage liver disease, and a markedly increased risk of hepatocellular carcinoma. In the last two decades, substantial progress has been made in the treatment of chronic hepatitis, B. However, HBV is often reactivated after stopping nucloes(t)ide analogues because antivirals alone do not directly target covalently closed circular DNA, which is the template for all viral RNAs. Therefore, although currently available antiviral therapies achieve suppression of HBV replication in the majority of patients, hepatitis B surface antigen (HBsAg) loss and seroconversion is rarely achieved despite long-term antiviral treatment (HBsAg loss of less than 10% in 5 years). Various clinical trials of agents that interrupt the HBV life cycle in hepatocytes have been conducted. Potential treatment strategies and new agents are emerging as HBV cure. A combination of current and new anti-HBV agents may increase the rate of HBsAg seroclearance; thus, optimized regimens must be validated. Here, we review the newly investigated therapeutic compounds and the results of preclinical and/or clinical trials.
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Affiliation(s)
- Hye Won Lee
- Department of Internal Medicine, College of Medicine, Yonsei University, Seoul 03722, Korea; (H.W.L.); (J.S.L.)
- Institute of Gastroenterology, College of Medicine, Yonsei University, Seoul 03722, Korea
- Yonsei Liver Center, Severance Hospital, Seoul 03722, Korea
| | - Jae Seung Lee
- Department of Internal Medicine, College of Medicine, Yonsei University, Seoul 03722, Korea; (H.W.L.); (J.S.L.)
- Institute of Gastroenterology, College of Medicine, Yonsei University, Seoul 03722, Korea
- Yonsei Liver Center, Severance Hospital, Seoul 03722, Korea
| | - Sang Hoon Ahn
- Department of Internal Medicine, College of Medicine, Yonsei University, Seoul 03722, Korea; (H.W.L.); (J.S.L.)
- Institute of Gastroenterology, College of Medicine, Yonsei University, Seoul 03722, Korea
- Yonsei Liver Center, Severance Hospital, Seoul 03722, Korea
- Correspondence: ; Tel.: +82-2-2228-1936; Fax: +82-2-393-6884
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Suzuki Y, Onuma H, Sato R, Sato Y, Hashiba A, Maeki M, Tokeshi M, Kayesh MEH, Kohara M, Tsukiyama-Kohara K, Harashima H. Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition. J Control Release 2020; 330:61-71. [PMID: 33333121 DOI: 10.1016/j.jconrel.2020.12.013] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Revised: 12/02/2020] [Accepted: 12/10/2020] [Indexed: 12/25/2022]
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.
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Affiliation(s)
- Yuichi Suzuki
- Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
| | - Haruno Onuma
- Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
| | - Risa Sato
- Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
| | - Yusuke Sato
- Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan.
| | - Akari Hashiba
- Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
| | - Masatoshi Maeki
- Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, Hokkaido, Japan; JST PRESTO, Saitama, Japan
| | - Manabu Tokeshi
- Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, Hokkaido, Japan; Innovative Research Center for Preventive Medical Engineering, Nagoya University, Nagoya, Japan; Institute of Nano-Life Systems, Institutes of Innovation for Future Society, Nagoya University, Nagoya, Japan
| | - Mohammad Enamul Hoque Kayesh
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Department of Microbiology and Public Health, Patuakhali Science and Technology University, Patuakhali, Bangladesh
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Kyoko Tsukiyama-Kohara
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Hideyoshi Harashima
- Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan.
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CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2020; 20:258-275. [PMID: 33473359 PMCID: PMC7803634 DOI: 10.1016/j.omtm.2020.11.014] [Citation(s) in RCA: 54] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/17/2020] [Accepted: 11/18/2020] [Indexed: 02/07/2023]
Abstract
Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV+ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.
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Alexopoulou A, Vasilieva L, Karayiannis P. New Approaches to the Treatment of Chronic Hepatitis B. J Clin Med 2020; 9:jcm9103187. [PMID: 33019573 PMCID: PMC7601587 DOI: 10.3390/jcm9103187] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Revised: 09/26/2020] [Accepted: 09/28/2020] [Indexed: 02/07/2023] Open
Abstract
The currently recommended treatment for chronic hepatitis B virus (HBV) infection achieves only viral suppression whilst on therapy, but rarely hepatitis B surface antigen (HBsAg) loss. The ultimate therapeutic endpoint is the combination of HBsAg loss, inhibition of new hepatocyte infection, elimination of the covalently closed circular DNA (cccDNA) pool, and restoration of immune function in order to achieve virus control. This review concentrates on new antiviral drugs that target different stages of the HBV life cycle (direct acting antivirals) and others that enhance both innate and adaptive immunity against HBV (immunotherapy). Drugs that block HBV hepatocyte entry, compounds that silence or deplete the cccDNA pool, others that affect core assembly, agents that degrade RNase-H, interfering RNA molecules, and nucleic acid polymers are likely interventions in the viral life cycle. In the immunotherapy category, molecules that activate the innate immune response such as Toll-like-receptors, Retinoic acid Inducible Gene-1 (RIG-1) and stimulator of interferon genes (STING) agonists or checkpoint inhibitors, and modulation of the adaptive immunity by therapeutic vaccines, vector-based vaccines, or adoptive transfer of genetically-engineered T cells aim towards the restoration of T cell function. Future therapeutic trends would likely be a combination of one or more of the aforementioned drugs that target the viral life cycle and at least one immunomodulator.
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Affiliation(s)
- Alexandra Alexopoulou
- Department of Medicine, Medical School, National & Kapodistrian University of Athens, Hippokration General Hospital, 11527 Athens, Greece;
- Correspondence: ; Tel.: +30-2132-088-178; Fax: +30-2107-706-871
| | - Larisa Vasilieva
- Department of Medicine, Medical School, National & Kapodistrian University of Athens, Hippokration General Hospital, 11527 Athens, Greece;
| | - Peter Karayiannis
- Department of Basic and Clinical Sciences, Medical School, University of Nicosia, Engomi, CY-1700 Nicosia, Cyprus;
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Ailioaie LM, Litscher G. Curcumin and Photobiomodulation in Chronic Viral Hepatitis and Hepatocellular Carcinoma. Int J Mol Sci 2020; 21:7150. [PMID: 32998270 PMCID: PMC7582680 DOI: 10.3390/ijms21197150] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Revised: 09/24/2020] [Accepted: 09/26/2020] [Indexed: 12/13/2022] Open
Abstract
Immune modulation is a very modern medical field for targeting viral infections. In the race to develop the best immune modulator against viruses, curcumin, as a natural product, is inexpensive, without side effects, and can stimulate very well certain areas of the human immune system. As a bright yellow component of turmeric spice, curcumin has been the subject of thousands of scientific and clinical studies in recent decades to prove its powerful antioxidant properties and anticancer effects. Curcumin has been shown to influence inter- and intracellular signaling pathways, with direct effects on gene expression of the antioxidant proteins and those that regulate the immunity. Experimental studies have shown that curcumin modulates several enzyme systems, reduces nitrosative stress, increases the antioxidant capacity, and decreases the lipid peroxidation, protecting against fatty liver pathogenesis and fibrotic changes. Hepatitis B virus (HBV) affects millions of people worldwide, having sometimes a dramatic evolution to chronic aggressive infection, cirrhosis, and hepatocellular carcinoma. All up-to-date treatments are limited, there is still a gap in the scientific knowledge, and a sterilization cure may not yet be possible with the removal of both covalently closed circular DNA (cccDNA) and the embedded HBV DNA. With a maximum light absorption at 420 nm, the cytotoxicity of curcumin as photosensitizer could be expanded by the intravenous blue laser blood irradiation (IVBLBI) or photobiomodulation in patients with chronic hepatitis B infection, Hepatitis B e-antigen (HBeAg)-positive, noncirrhotic, but nonresponsive to classical therapy. Photobiomodulation increases DNA repair by the biosynthesis of complex molecules with antioxidant properties, the outset of repairing enzyme systems and new phospholipids for regenerating the cell membranes. UltraBioavailable Curcumin and blue laser photobiomodulation could suppress the virus and control better the disease by reducing inflammation/fibrosis and stopping the progression of chronic hepatitis, reversing fibrosis, and diminishing the progression of cirrhosis, and decreasing the incidence of hepatocellular carcinoma. Photodynamic therapy with blue light and curcumin opens new avenues for the effective prevention and cure of chronic liver infections and hepatocellular carcinoma. Blue laser light and UltraBioavailable Curcumin could be a new valuable alternative for medical applications in chronic B viral hepatitis and hepatocarcinoma, saving millions of lives.
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MESH Headings
- Antineoplastic Agents, Phytogenic/therapeutic use
- Antioxidants/therapeutic use
- Carcinoma, Hepatocellular/drug therapy
- Carcinoma, Hepatocellular/etiology
- Carcinoma, Hepatocellular/radiotherapy
- Carcinoma, Hepatocellular/virology
- Curcumin/therapeutic use
- DNA Repair/radiation effects
- DNA, Circular/antagonists & inhibitors
- DNA, Circular/genetics
- DNA, Circular/metabolism
- DNA, Viral/antagonists & inhibitors
- DNA, Viral/genetics
- DNA, Viral/metabolism
- Hepatitis B e Antigens/genetics
- Hepatitis B e Antigens/immunology
- Hepatitis B virus/drug effects
- Hepatitis B virus/growth & development
- Hepatitis B virus/pathogenicity
- Hepatitis B virus/radiation effects
- Hepatitis B, Chronic/complications
- Hepatitis B, Chronic/drug therapy
- Hepatitis B, Chronic/radiotherapy
- Hepatitis B, Chronic/virology
- Humans
- Immunologic Factors/therapeutic use
- Liver/drug effects
- Liver/immunology
- Liver/pathology
- Liver/radiation effects
- Liver Cirrhosis/drug therapy
- Liver Cirrhosis/etiology
- Liver Cirrhosis/radiotherapy
- Liver Cirrhosis/virology
- Liver Neoplasms/drug therapy
- Liver Neoplasms/etiology
- Liver Neoplasms/radiotherapy
- Liver Neoplasms/virology
- Low-Level Light Therapy/methods
- Photosensitizing Agents/therapeutic use
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Affiliation(s)
- Laura Marinela Ailioaie
- Department of Medical Physics, Alexandru Ioan Cuza University, 11 Carol I Boulevard, 700506 Iasi, Romania;
- Ultramedical & Laser Clinic, 83 Arcu Street, 700135 Iasi, Romania
| | - Gerhard Litscher
- Research Unit of Biomedical Engineering in Anesthesia and Intensive Care Medicine, Research Unit for Complementary and Integrative Laser Medicine, and Traditional Chinese Medicine (TCM) Research Center Graz, Medical University of Graz, Auenbruggerplatz 39, 8036 Graz, Austria
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34
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Rybicka M, Bielawski KP. Recent Advances in Understanding, Diagnosing, and Treating Hepatitis B Virus Infection. Microorganisms 2020; 8:E1416. [PMID: 32942584 PMCID: PMC7565763 DOI: 10.3390/microorganisms8091416] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Revised: 09/08/2020] [Accepted: 09/11/2020] [Indexed: 02/06/2023] Open
Abstract
Chronic hepatitis B virus (HBV) infection affects 292 million people worldwide and is associated with a broad range of clinical manifestations including cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Despite the availability of an effective vaccine HBV still causes nearly 900,000 deaths every year. Current treatment options keep HBV under control, but they do not offer a cure as they cannot completely clear HBV from infected hepatocytes. The recent development of reliable cell culture systems allowed for a better understanding of the host and viral mechanisms affecting HBV replication and persistence. Recent advances into the understanding of HBV biology, new potential diagnostic markers of hepatitis B infection, as well as novel antivirals targeting different steps in the HBV replication cycle are summarized in this review article.
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Affiliation(s)
- Magda Rybicka
- Department of Molecular Diagnostics, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland;
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35
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Duraisamy GS, Bhosale D, Lipenská I, Huvarova I, Růžek D, Windisch MP, Miller AD. Advanced Therapeutics, Vaccinations, and Precision Medicine in the Treatment and Management of Chronic Hepatitis B Viral Infections; Where Are We and Where Are We Going? Viruses 2020; 12:v12090998. [PMID: 32906840 PMCID: PMC7552065 DOI: 10.3390/v12090998] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Revised: 08/31/2020] [Accepted: 09/02/2020] [Indexed: 02/06/2023] Open
Abstract
The management of chronic hepatitis B virus (CHB) infection is an area of massive unmet clinical need worldwide. In spite of the development of powerful nucleoside/nucleotide analogue (NUC) drugs, and the widespread use of immune stimulators such as interferon-alpha (IFNα) or PEGylated interferon-alpha (PEG-IFNα), substantial improvements in CHB standards of care are still required. We believe that the future for CHB treatment now rests with advanced therapeutics, vaccination, and precision medicine, if all are to bring under control this most resilient of virus infections. In spite of a plethora of active drug treatments, anti-viral vaccinations and diagnostic techniques, the management of CHB infection remains unresolved. The reason for this is the very complexity of the virus replication cycle itself, giving rise to multiple potential targets for therapeutic intervention some of which remain very intractable indeed. Our review is focused on discussing the potential impact that advanced therapeutics, vaccinations and precision medicine could have on the future management of CHB infection. We demonstrate that advanced therapeutic approaches for the treatment of CHB, in the form of gene and immune therapies, together with modern vaccination strategies, are now emerging rapidly to tackle the limitations of current therapeutic approaches to CHB treatment in clinic. In addition, precision medicine approaches are now gathering pace too, starting with personalized medicine. On the basis of this, we argue that the time has now come to accelerate the design and creation of precision therapeutic approaches (PTAs) for CHB treatment that are based on advanced diagnostic tools and nanomedicine, and which could maximize CHB disease detection, treatment, and monitoring in ways that could genuinely eliminate CHB infection altogether.
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Affiliation(s)
- Ganesh Selvaraj Duraisamy
- Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic; (G.S.D.); (D.B.); (I.L.); (I.H.); (D.R.)
| | - Dattatry Bhosale
- Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic; (G.S.D.); (D.B.); (I.L.); (I.H.); (D.R.)
| | - Ivana Lipenská
- Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic; (G.S.D.); (D.B.); (I.L.); (I.H.); (D.R.)
| | - Ivana Huvarova
- Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic; (G.S.D.); (D.B.); (I.L.); (I.H.); (D.R.)
| | - Daniel Růžek
- Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic; (G.S.D.); (D.B.); (I.L.); (I.H.); (D.R.)
- Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branisovska 31, CZ-37005 České Budějovice, Czech Republic
| | - Marc P. Windisch
- Applied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Korea;
- Division of Bio-Medical Science and Technology, University of Science and Technology, Daejeon 305-350, Korea
| | - Andrew D. Miller
- Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic; (G.S.D.); (D.B.); (I.L.); (I.H.); (D.R.)
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemědělská 1, Černá Pole, CZ-61300 Brno, Czech Republic
- KP Therapeutics (Europe) s.r.o., Purkyňova 649/127, CZ-61200 Brno, Czech Republic
- Correspondence:
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36
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Abstract
Hepatitis B virus (HBV) infection causes chronic hepatitis and has long term complications. Individuals ever infected with HBV are at risk of viral reactivation under certain circumstances. This review summarizes studies on HBV persistence and reactivation with a focus on the definitions and mechanisms. Emphasis is placed on the interplay between HBV replication and host immunity as this interplay determines the patterns of persistence following viral acquisition. Chronic infections exhibit as overt persistence when a defective immune response fails to control the viral replication. The HBV genome persists despite an immune response in the form of covalently closed circular DNA (cccDNA) and integrated DNA, rendering an occult state of viral persistence in individuals whose infection appears to have been resolved. We have described HBV reactivation that occurs because of changes in the virus or the immune system. This review aims to raise the awareness of HBV reactivation and to understand how HBV persists, and discusses the risks of HBV reactivation in a variety of clinical settings.
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Affiliation(s)
- Yu Shi
- The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital of School of Medicine, Zhejiang University, Hangzhou, China
- Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China
- National Clinical Research Center for Infectious Diseases, China
| | - Min Zheng
- The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital of School of Medicine, Zhejiang University, Hangzhou, China
- Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China
- National Clinical Research Center for Infectious Diseases, China
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37
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Younis MA, Khalil IA, Harashima H. Gene Therapy for Hepatocellular Carcinoma: Highlighting the Journey from Theory to Clinical Applications. ADVANCED THERAPEUTICS 2020. [DOI: 10.1002/adtp.202000087] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Affiliation(s)
- Mahmoud A. Younis
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences Hokkaido University Kita‐12, Nishi‐6, Kita‐ku Sapporo 060‐0812 Japan
- Faculty of Pharmacy Assiut University Assiut 71526 Egypt
| | - Ikramy A. Khalil
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences Hokkaido University Kita‐12, Nishi‐6, Kita‐ku Sapporo 060‐0812 Japan
- Faculty of Pharmacy Assiut University Assiut 71526 Egypt
| | - Hideyoshi Harashima
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences Hokkaido University Kita‐12, Nishi‐6, Kita‐ku Sapporo 060‐0812 Japan
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38
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Xia Y, Guo H. Hepatitis B virus cccDNA: Formation, regulation and therapeutic potential. Antiviral Res 2020; 180:104824. [PMID: 32450266 PMCID: PMC7387223 DOI: 10.1016/j.antiviral.2020.104824] [Citation(s) in RCA: 119] [Impact Index Per Article: 23.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 05/03/2020] [Accepted: 05/18/2020] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) infection remains a major public health concern worldwide with about 257 million individuals chronically infected. Current therapies can effectively control HBV replication and slow down disease progress, but cannot cure HBV infection. Upon infection, HBV establishes a pool of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. The cccDNA exists as a minichromosome and resists to antivirals, thus a therapeutic eradication of cccDNA from the infected cells remains unattainable. In this review, we summarize the state of knowledge on the mechanisms underlying cccDNA formation and regulation, and discuss the possible strategies that may contribute to the eradication of HBV through targeting cccDNA.
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Affiliation(s)
- Yuchen Xia
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, China.
| | - Haitao Guo
- UPMC Hillman Cancer Center, Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA.
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39
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Screening and Evaluation of Novel Compounds against Hepatitis B Virus Polymerase Using Highly Purified Reverse Transcriptase Domain. Viruses 2020; 12:v12080840. [PMID: 32752057 PMCID: PMC7472185 DOI: 10.3390/v12080840] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2020] [Revised: 07/28/2020] [Accepted: 07/30/2020] [Indexed: 12/14/2022] Open
Abstract
Hepatitis B virus (HBV) polymerase seems to be very hard to express and purify sufficiently, which has long hampered the generation of anti-HBV drugs based on the nature of the polymerase. To date, there has been no useful system developed for drug screening against HBV polymerase. In this study, we successfully obtained a highly purified reverse transcriptase (RT) domain of the polymerase, which has a template/primer and substrate binding activity, and established a novel high-throughput screening (HTS) system using purified RT protein for finding novel polymerase inhibitors. To examine whether the assay system provides reliable results, we tested the small scale screening using pharmacologically active compounds. As a result, the pilot screening identified already-known anti-viral polymerase agents. Then, we screened 20,000 chemical compounds and newly identified four hits. Several of these compounds inhibited not only the HBV RT substrate and/ template/primer binding activity, but also Moloney murine leukemia virus RT activity, which has an elongation activity. Finally, these candidates did show to be effective even in the cell-based assay. Our screening system provides a useful tool for searching candidate inhibitors against HBV.
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40
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D-Pinitol treatment induced the apoptosis in human leukemia MOLT-4 cells by improved apoptotic signaling pathway. Saudi J Biol Sci 2020; 27:2134-2138. [PMID: 32714040 PMCID: PMC7376127 DOI: 10.1016/j.sjbs.2020.06.034] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Revised: 05/26/2020] [Accepted: 06/18/2020] [Indexed: 01/13/2023] Open
Abstract
Cancer is still remain as a global burden with the 18.1 million and 9.6 million new cases and mortlities, respectively estimated globally. Leukemia may arise at all ages varied from the infants to elders. In this exploration, we planned to evaluate the antiproliferative effect of D-pinitol on human leukemia MOLT-4 cells. Anticancer potential of D-pinitol was examined using MTT assay. Reactive oxygen species (ROS) generation was studied by fluorescence microscopic method using DCFH-DA staining. Apoptotic morphological alterations were determined by dual staining (acridine orange and ethidium bromide). Western blot and ELISA methods were employed to study apoptotic protein expression. D-pinitol treatment significantly induced cytotoxicity in human leukemia MOLT-4 cells. We observed that D-pinitol induces the generation of ROS in MOLT-4 cells. Further, we noticed that D-pinitol significantly induced apoptosis in a dosage dependent manner. Moreover, western blot and ELISA based analysis revealed that D-pinitol elevated the Bax, Caspase-3, Caspase-9 and attenuated the Bcl-2 expression in leukemic cancer cell. Our findings suggest that D-pinitol treatment induces the apoptosis in human leukemic cells by generating intracellular ROS and modulating apoptotic protein expression.
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Wu X, Ma W, Mei C, Chen X, Yao Y, Liu Y, Qin X, Yuan Y. Description of CRISPR/Cas9 development and its prospect in hepatocellular carcinoma treatment. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2020; 39:97. [PMID: 32487115 PMCID: PMC7268395 DOI: 10.1186/s13046-020-01603-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Accepted: 05/25/2020] [Indexed: 12/24/2022]
Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignancies today. Patients suffer from HCC since its high malignancy and limited treatment means. With the development of genetic research, new therapeutic strategy comes up in the way of gene editing. Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) was discovered as an immune sequence in bacteria and archaea. After artificial transformation and follow-up research, it is widely used as a gene editing tool. In this review, the development of CRISPR/Cas9 is summarized in retrospect. Through the evaluation of novel research in HCC, it is concluded that CRISPR/Cas9 would promote cancer research and provide a new tool for genetic treatment in prospect.
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Affiliation(s)
- Xiaoling Wu
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China
| | - Weijie Ma
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China
| | - Chengjie Mei
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China
| | - Xi Chen
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China
| | - Ye Yao
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China
| | - Yingyi Liu
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China
| | - Xian Qin
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China
| | - Yufeng Yuan
- Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei Province, China.
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Spyrou E, Smith CI, Ghany MG. Hepatitis B: Current Status of Therapy and Future Therapies. Gastroenterol Clin North Am 2020; 49:215-238. [PMID: 32389360 PMCID: PMC7444867 DOI: 10.1016/j.gtc.2020.01.003] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Despite the availability of a protective vaccine for over 3 decades, the number of persons with chronic hepatitis B virus (HBV) infection remains high. These persons are at risk for cirrhosis and hepatocellular carcinoma. Current treatment is effective at inhibiting viral replication and reducing complications of chronic HBV infection, but is not curative. There is a need for novel, finite therapy that can cure chronic HBV infection. Several agents are in early-phase development and can be broadly viewed as agents that target the virus directly or indirectly or the host immune response. This article highlights key developments in antiviral/immunomodulatory therapy, the rationale for these approaches, and possible therapeutic regimens.
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Affiliation(s)
- Elias Spyrou
- MedStar Georgetown Transplant Institute, MedStar Georgetown University Hospital, Washington, DC, USA,Nazih Zuhdi Transplant Institute, INTEGRIS Baptist Medical Center, Oklahoma City, OK, USA
| | - Coleman I. Smith
- MedStar Georgetown Transplant Institute, MedStar Georgetown University Hospital, Washington, DC, USA
| | - Marc G. Ghany
- Liver Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
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Poilil Surendran S, George Thomas R, Moon MJ, Park R, Kim DH, Kim KH, Jeong YY. Effect of hepato-toxins in the acceleration of hepatic fibrosis in hepatitis B mice. PLoS One 2020; 15:e0232619. [PMID: 32428024 PMCID: PMC7237019 DOI: 10.1371/journal.pone.0232619] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2020] [Accepted: 04/17/2020] [Indexed: 11/30/2022] Open
Abstract
Chronic liver diseases such as hepatitis B viral (HBV) infection and liver fibrosis have been a major health problem worldwide. However, less research has been conducted owing to the lack of animal models. The key purpose of this study was to determine the effects of different hepatotoxins in HBV-affected liver. In this study, we successfully generated a combined liver fibrosis model by administering HBV 1.2 plasmid and thioacetamide/ethanol (TAA/EtOH). To our knowledge, this is the first study in which an increase in the liver fibrosis level is observed by the intraperitoneal administration of TAA and EtOH in drinking water after the hydrodynamic transfection of the HBV 1.2 plasmid in C3H/HeN mice. The HBV+TAA/EtOH group exhibited higher level of hepatic fibrosis than that of the control groups. The hepatic stellate cell activation in the TAA- and EtOH-administered groups was demonstrated by the elevation in the level of fibrotic markers. In addition, high levels of collagen content and histopathological results were also used to confirm the prominent fibrotic levels. We established a novel HBV mice model by hydrodynamic injection-based HBV transfection in C3H/HeN mice. C3H/HeN mice were reported to have a higher HBV persistence level than that of the C57BL/6 mouse model. All the results showed an increased fibrosis level in the HBV mice treated with TAA and EtOH; hence, this model would be useful to understand the effect of hepatotoxins on the high risk of fibrosis after HBV infection. The acceleration of liver fibrosis can occur with prolonged administration as well as the high dosage of hepatotoxins in mice.
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Affiliation(s)
- Suchithra Poilil Surendran
- Department of Biomedical Sciences, Biomolecular Theranostics (BiT) Lab, Chonnam National University Medical School, Hwasun, Korea
- Department of Radiology, Biomolecular Theranostics (BiT) Lab, Chonnam National University Medical School, Hwasun, Korea
| | - Reju George Thomas
- Department of Biomedical Sciences, Biomolecular Theranostics (BiT) Lab, Chonnam National University Medical School, Hwasun, Korea
- Department of Radiology, Biomolecular Theranostics (BiT) Lab, Chonnam National University Medical School, Hwasun, Korea
| | - Myeong Ju Moon
- Department of Radiology, Biomolecular Theranostics (BiT) Lab, Chonnam National University Medical School, Hwasun, Korea
| | - Rayoung Park
- Department of Radiology, Biomolecular Theranostics (BiT) Lab, Chonnam National University Medical School, Hwasun, Korea
| | - Doo Hyun Kim
- Department of Pharmacology and Center for Cancer Research and Diagnostic Medicine, Konkuk University School of Medicine, Seoul, South Korea
| | - Kyun Hwan Kim
- Department of Pharmacology and Center for Cancer Research and Diagnostic Medicine, Konkuk University School of Medicine, Seoul, South Korea
| | - Yong Yeon Jeong
- Department of Radiology, Biomolecular Theranostics (BiT) Lab, Chonnam National University Medical School, Hwasun, Korea
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Yang YC, Chen YH, Kao JH, Ching C, Liu IJ, Wang CC, Tsai CH, Wu FY, Liu CJ, Chen PJ, Chen DS, Yang HC. Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing. MOLECULAR THERAPY-NUCLEIC ACIDS 2020; 20:480-490. [PMID: 32278307 PMCID: PMC7150432 DOI: 10.1016/j.omtn.2020.03.005] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated "base editors" (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.
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Affiliation(s)
- Yu-Chan Yang
- Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Yu-Hsiang Chen
- Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Jia-Horng Kao
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Chi Ching
- Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - I-Jung Liu
- Department of Nursing, Cardinal Tien Junior College of Healthcare and Management, New Taipei City, Taiwan
| | - Chih-Chiang Wang
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Cheng-Hsueh Tsai
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Fang-Yi Wu
- Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Chun-Jen Liu
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Pei-Jer Chen
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Ding-Shinn Chen
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Hung-Chih Yang
- Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan.
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Abstract
Chronic hepatitis B (CHB) is a widespread global infection and a leading cause of hepatocellular carcinoma and liver failure. Current approaches to treat CHB involve the suppression of viral replication with either interferon or nucleos(t)ide analog therapy, but neither of these approaches can reliably induce viral eradication, immunologic control or long-lived viral suppression in the absence of continued therapy. In this update, we explore the major obstacles of CHB cure and review new therapeutic strategies and drug candidates.
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Affiliation(s)
- Lydia Tang
- Division of Clinical Care & Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
- Program in Oncology, University of Maryland Marlene & Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD 21201, USA
| | - Shyam Kottilil
- Division of Clinical Care & Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
- Program in Oncology, University of Maryland Marlene & Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD 21201, USA
| | - Eleanor Wilson
- Division of Clinical Care & Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
- Program in Oncology, University of Maryland Marlene & Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD 21201, USA
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Wang J, Huang H, Liu Y, Chen R, Yan Y, Shi S, Xi J, Zou J, Yu G, Feng X, Lu F. HBV Genome and Life Cycle. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1179:17-37. [PMID: 31741332 DOI: 10.1007/978-981-13-9151-4_2] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Chronic hepatitis B virus (HBV) infection remains to be a serious threat to public health and is associated with many liver diseases including chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. Although nucleos(t)ide analogues (NA) and pegylated interferon-α (Peg-IFNα) have been confirmed to be efficient in inhibiting HBV replication, it is difficult to eradicate HBV and achieve the clinical cure of CHB. Therefore, long-term therapy has been recommended to CHB treatment under the current antiviral therapy. In this context, the new antiviral therapy targeting one or multiple critical steps of viral life cycle may be an alternative approach in future. In the last decade, the functional receptor [sodium-taurocholate cotransporting polypeptide (NTCP)] of HBV entry into hepatocytes has been discovered, and the immature nucleocapsids containing the non- or partially reverse-transcribed pregenomic RNA, the nucleocapsids containing double-strand linear DNA (dslDNA), and the empty particles devoid of any HBV nucleic acid have been found to be released into circulation, which have supplemented the life cycle of HBV. The understanding of HBV life cycle may offer a new instruction for searching the potential antiviral targets, and the new viral markers used to monitor the efficacy of antiviral therapy for CHB patients in the future.
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Affiliation(s)
- Jie Wang
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Hongxin Huang
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Yongzhen Liu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Ran Chen
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Ying Yan
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Shu Shi
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Jingyuan Xi
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Jun Zou
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Guangxin Yu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Xiaoyu Feng
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Fengmin Lu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China.
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Paximadis M, Perez Patrigeon S, Rajasuriar R, Tatoud R, Scully E, Arbuthnot P. Hepatitis B and HIV-1 2019 IAS Cure Forum: lessons and benefits from interdisciplinary research. J Virus Erad 2019; 5:234-244. [PMID: 31754448 PMCID: PMC6844409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
Affiliation(s)
- M Paximadis
- Centre for HIV and STIs, Cell Biology,
National Institute for Communicable Diseases and University of the Witwatersrand,
Johannesburg,
South Africa
| | - S Perez Patrigeon
- Division of Infectious Diseases,
Queen's University,
Kingston,
Ontario,
Canada
| | - R Rajasuriar
- Department of Medicine, Faculty of Medicine, University of Malaya and the Centre of Excellence for Research in AIDS (CERiA),
University of Malaya,
Malaysia
| | - R Tatoud
- International AIDS Society,
Geneva,
Switzerland,Corresponding author: Roger Tadoud
Avenue de France 23,
1202 Geneva,
Switzerland
| | - E Scully
- Division of Infectious Diseases, Department of Internal Medicine,
Johns Hopkins University,
Baltimore,
MD,
USA
| | - P Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science,
University of the Witwatersrand,
Johannesburg,
South Africa
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Paximadis M, Perez Patrigeon S, Rajasuriar R, Tatoud R, Scully E, Arbuthnot P. Hepatitis B and HIV-1 2019 IAS Cure Forum: lessons and benefits from interdisciplinary research. J Virus Erad 2019. [DOI: 10.1016/s2055-6640(20)30027-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
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Yeh ML, Huang JF, Dai CY, Yu ML, Chuang WL. Pharmacokinetics and pharmacodynamics of pegylated interferon for the treatment of hepatitis B. Expert Opin Drug Metab Toxicol 2019; 15:779-785. [PMID: 31593639 DOI: 10.1080/17425255.2019.1678584] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2019] [Accepted: 10/07/2019] [Indexed: 02/06/2023]
Abstract
Introduction: Interferon (IFN) had both antiviral and immunomodulatory effects, and was one of the approved treatments for hepatitis B virus (HBV). Herein, we reviewed the pharmacokinetics and pharmacodynamics of pegylated IFN-α (PegIFN-α) for the treatment of HBV. Areas covered: The steady-state serum levels of PegIFN-α were reached within 5 to 8 weeks, and the week 48 mean trough concentrations were approximately 2-fold higher than week 1. There was also no difference of the pharmacokinetics in male or female, healthy volunteers or patients with hepatitis B or C infection. PegIFN-α did not affect the metabolism of the cytochrome P450 (CYP) isozymes, except inhibition of CYP1A2. There was also no pharmacokinetic interaction between PegIFN-α and HBV nucleot(s)ide analogues (NUCs). Forty-eight weeks of PegIFN-α achieved 32% of HBeAg seroconversion, 32-43% of HBV DNA suppression, 41-59% of ALT normalization, and 3% of HBsAg seroconversion rate with a post-treatment durable response up to 80% in the initial responders. Expert opinion: On-treatment HBsAg titer guided the treatment of HBV with PegIFN-α. The recommendation of PegIFN-α and NUC combination or switch remained controversial. New immunotherapeutic agents are now in development. Although, PegIFN-α should continue to play a role in the treatment of HBV.
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Affiliation(s)
- Ming-Lun Yeh
- Hepatitis Center and Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital , Kaohsiung , Taiwan
- School of Medicine and Hepatitis Research Center, College of Medicine, Kaohsiung Medical University , Kaohsiung , Taiwan
| | - Jee-Fu Huang
- Hepatitis Center and Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital , Kaohsiung , Taiwan
- School of Medicine and Hepatitis Research Center, College of Medicine, Kaohsiung Medical University , Kaohsiung , Taiwan
- Center for Cancer Research and Center for Liquid Biopsy, Kaohsiung Medical University , Kaohsiung , Taiwan
| | - Chia-Yen Dai
- Hepatitis Center and Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital , Kaohsiung , Taiwan
- School of Medicine and Hepatitis Research Center, College of Medicine, Kaohsiung Medical University , Kaohsiung , Taiwan
- Center for Cancer Research and Center for Liquid Biopsy, Kaohsiung Medical University , Kaohsiung , Taiwan
| | - Ming-Lung Yu
- Hepatitis Center and Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital , Kaohsiung , Taiwan
- School of Medicine and Hepatitis Research Center, College of Medicine, Kaohsiung Medical University , Kaohsiung , Taiwan
- Center for Cancer Research and Center for Liquid Biopsy, Kaohsiung Medical University , Kaohsiung , Taiwan
- Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B) and Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University , Hsin-Chu , Taiwan
- Institute of Biomedical Sciences, National Sun Yat-Sen University , Kaohsiung , Taiwan
| | - Wan-Long Chuang
- Hepatitis Center and Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital , Kaohsiung , Taiwan
- School of Medicine and Hepatitis Research Center, College of Medicine, Kaohsiung Medical University , Kaohsiung , Taiwan
- Center for Cancer Research and Center for Liquid Biopsy, Kaohsiung Medical University , Kaohsiung , Taiwan
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Potential Application of TALENs against Murine Cytomegalovirus Latent Infections. Viruses 2019; 11:v11050414. [PMID: 31058862 PMCID: PMC6563206 DOI: 10.3390/v11050414] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2019] [Revised: 04/24/2019] [Accepted: 04/30/2019] [Indexed: 01/01/2023] Open
Abstract
Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring diseases. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids (MCMV1–2, 3–4, and 5–6) were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3–4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene (ie-1) DNA in five organs of mice, when compared with the controls. The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.
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