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Lei Z, Wang L, Gao H, Guo S, Kang X, Yuan J, Lv Z, Jiang Y, Yi J, Chen Z, Wang G. Mechanisms underlying the compromised clinical efficacy of interferon in clearing HBV. Virol J 2024; 21:314. [PMID: 39633459 PMCID: PMC11619119 DOI: 10.1186/s12985-024-02589-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2024] [Accepted: 11/26/2024] [Indexed: 12/07/2024] Open
Abstract
Hepatitis B virus (HBV) is a hepatotropic DNA virus that can cause acute or chronic hepatitis, representing a significant global health concern. By 2019, approximately 296 million individuals were chronically infected with HBV, with 1.5 million new cases annually and 820,000 deaths due to HBV-related cirrhosis and liver cancer. Current treatments for chronic hepatitis B include nucleotide analogs (NAs) and interferons (IFNs), particularly IFN-α. NAs, such as entecavir and tenofovir, inhibit viral reverse transcription, while IFN-α exerts antiviral effects by directly suppressing viral replication, modulating viral genome epigenetics, degrading cccDNA, and activating immune responses. Despite its potential, IFN-α shows limited clinical efficacy, partly due to HBV's interference with the IFN signaling pathway. HBV encodes proteins like HBc, Pol, HBsAg, and HBx that disrupt IFN-α function. For example, HBV Pol inhibits STAT1 phosphorylation, HBsAg suppresses STAT3 phosphorylation, and HBx interferes with IFN-α efficacy through multiple mechanisms. Additionally, HBV downregulates key genes in the IFN signaling pathway, further diminishing IFN-α's antiviral effects. Understanding these interactions is crucial for improving IFN-α-based therapies. Future research may focus on overcoming HBV resistance by targeting viral proteins or optimizing IFN-α delivery. In summary, HBV's ability to resist IFN-α limits its therapeutic effectiveness, highlighting the need for new strategies to enhance treatment outcomes.
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Affiliation(s)
- Zhuoyan Lei
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Luye Wang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Hanlin Gao
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Shubian Guo
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Xinjian Kang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Jiajun Yuan
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Ziying Lv
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Yuxin Jiang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Jinping Yi
- Department of Clinical Laboratory, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Zhi Chen
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University, Hangzhou, China
| | - Gang Wang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China.
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Zhang W, Chen J, Sun W, Xie N, Tian F, Ruan Q, Song J. The impact of hepatitis B surface antigen seroconversion on the durability of functional cure induced by pegylated interferon alpha treatment. Virol J 2024; 21:243. [PMID: 39363288 PMCID: PMC11448035 DOI: 10.1186/s12985-024-02522-8] [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: 07/09/2024] [Accepted: 09/27/2024] [Indexed: 10/05/2024] Open
Abstract
BACKGROUND Hepatitis B surface antigen (HBsAg) loss is regarded as a pivotal criterion for assessing functional cure in patients diagnosed chronic hepatitis B (CHB). We conducted the research to investigate the real-world performance of HBsAg seroconversion in sustaining HBsAg loss. METHODS This retrospective analysis confirmed 295 patients who attained HBsAg loss through combination therapy involving nucleos(t)ide analogues (NAs) and pegylated interferon alpha (peg-IFNα). Employing Kaplan-Meier estimates method to conduct survival analysis. The forest plot was used to visualize the results of multivariate Cox regression, and selected variables were included in the nomogram. RESULTS HBsAg seroreversion was observed in 45 patients during follow-up periods, with a lower recurrence risk in patients with HBsAg seroconversion at the end of peg-IFNα therapy (EOT) (10.3% vs 37.3% at 96-week, P < 0.0001). Moreover, the sustainability of hepatitis B surface antibody (anti-HBs) in participants continuing therapy after HBsAg seroconversion was superior to those discontinued prematurely (72.5% vs 54.5% at 96 weeks, P = 0.012). Additionally, the former group was also relatively less likely to experience HBsAg reversion during long-term observation (8.4% vs 14.3% at 96 weeks, P = 0.280). Hepatitis B envelope antigen (HBeAg) status, anti-HBs status and consolidation treatment screened by multivariable analysis were utilized to construct a predictive model for HBsAg reversion. The concordance index(C-index = 0.77) and calibration plots indicated satisfactory discrimination and consistency of nomogram. CONCLUSIONS HBsAg seroconversion was beneficial for sustaining functional cure in patients treated with peg-IFNα. Continuing consolidation therapy after HBsAg seroconversion also contributed to maintain HBsAg seroconversion and improve the durability of HBsAg loss. The nomogram illustrated its efficacy as a valuable instrument in showcasing survival probability of functional cure.
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Affiliation(s)
- Wencong Zhang
- Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China
| | - Jia Chen
- Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China
| | - Wenjin Sun
- Department of Infectious Diseases, Ezhou Central Hospital, Ezhou, China
| | - Nana Xie
- Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China
| | - Fangbing Tian
- Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China
| | - Qiurong Ruan
- Institute of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.
| | - Jianxin Song
- Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.
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Pondé RADA, Amorim GDSP. Elimination of the hepatitis B virus: A goal, a challenge. Med Res Rev 2024; 44:2015-2034. [PMID: 38528684 DOI: 10.1002/med.22030] [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: 10/06/2023] [Revised: 01/24/2024] [Accepted: 02/05/2024] [Indexed: 03/27/2024]
Abstract
The hepatitis B elimination is a goal proposed by the WHO to be achieved by 2030 through the adoption of synergistic measures for the prevention and chronic HBV infection treatment. Complete cure is characterized by the HBV elimination from the body and is the goal of the chronic hepatitis B treatment, which once achieved, will enable the hepatitis B elimination. This, today, has been a scientific challenge. The difficulty in achieving a complete cure is due to the indefinite maintenance of a covalently closed episomal circular DNA (cccDNA) reservoir and the maintenance and persistence of an insufficient and dysfunctional immune response in chronically infected patients. Among the measures adopted to eliminate hepatitis B, two have the potential to directly interfere with the virus cycle, but with limited effect on HBV control. These are conventional vaccines-blocking transmission and antiviral therapy-inhibiting replication. Vaccines, despite their effectiveness in protecting against horizontal transmission and preventing mother-to-child vertical transmission, have no effect on chronic infection or potential to eliminate the virus. Treatment with antivirals suppresses viral replication, but has no curative effect, as it has no action against cccDNA. Therapeutic vaccines comprise an additional approach in the chronic infection treatment, however, they have only a modest effect on the immune system, enhancing it temporarily. This manuscript aims to address (1) the cccDNA persistence in the hepatocyte nucleus and the immune response dysfunction in chronically infected individuals as two primary factors that have hampered the treatment and HBV elimination from the human body; (2) the limitations of antiviral therapy and therapeutic vaccines, as strategies to control hepatitis B; and (3) the possibly promising therapeutic approaches for the complete cure and elimination of hepatitis B.
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Affiliation(s)
- Robério Amorim de Almeida Pondé
- Secretaria de Estado da Saúde-SES, Superintendência de Vigilância em Saúde-SUVISA/GO, Gerência de Vigilância Epidemiológica de Doenças Transmissíveis-GVEDT/Coordenação de Análises e Pesquisas-CAP, Goiânia, Goiás, Brazil
- Department of Microbiology, Laboratory of Human Virology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Goiás, Brazil
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Tang L, Remiszewski S, Snedeker A, Chiang LW, Shenk T. An allosteric inhibitor of sirtuin 2 blocks hepatitis B virus covalently closed circular DNA establishment and its transcriptional activity. Antiviral Res 2024; 226:105888. [PMID: 38641024 DOI: 10.1016/j.antiviral.2024.105888] [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: 11/29/2023] [Revised: 04/10/2024] [Accepted: 04/12/2024] [Indexed: 04/21/2024]
Abstract
296 million people worldwide are predisposed to developing severe end-stage liver diseases due to chronic hepatitis B virus (HBV) infection. HBV forms covalently closed circular DNA (cccDNA) molecules that persist as episomal DNA in the nucleus of infected hepatocytes and drive viral replication. Occasionally, the HBV genome becomes integrated into host chromosomal DNA, a process that is believed to significantly contribute to circulating HBsAg levels and HCC development. Neither cccDNA accumulation nor expression from integrated HBV DNA are directly targeted by current antiviral treatments. In this study, we investigated the antiviral properties of a newly described allosteric modulator, FLS-359, that targets sirtuin 2 (SIRT2), an NAD+-dependent deacylase. Our results demonstrate that SIRT2 modulation by FLS-359 and by other tool compounds inhibits cccDNA synthesis following de novo infection of primary human hepatocytes and HepG2 (C3A)-NTCP cells, and FLS-359 substantially reduces cccDNA recycling in HepAD38 cells. While pre-existing cccDNA is not eradicated by short-term treatment with FLS-359, its transcriptional activity is substantially impaired, likely through inhibition of viral promoter activities. Consistent with the inhibition of viral transcription, HBsAg production by HepG2.2.15 cells, which contain integrated HBV genomes, is also suppressed by FLS-359. Our study provides further insights on SIRT2 regulation of HBV infection and supports the development of potent SIRT2 inhibitors as HBV antivirals.
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Affiliation(s)
- Liudi Tang
- Evrys Bio, LLC, Pennsylvania Biotechnology Center, Doylestown, PA, 18902, USA; Baruch S. Blumberg Institute, Doylestown, PA, 18902, USA.
| | - Stacy Remiszewski
- Evrys Bio, LLC, Pennsylvania Biotechnology Center, Doylestown, PA, 18902, USA
| | | | - Lillian W Chiang
- Evrys Bio, LLC, Pennsylvania Biotechnology Center, Doylestown, PA, 18902, USA
| | - Thomas Shenk
- Evrys Bio, LLC, Pennsylvania Biotechnology Center, Doylestown, PA, 18902, USA; Department of Molecular Biology, Princeton University, Princeton, NJ, 08540, USA
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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: 0] [Impact Index Per Article: 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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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: 5] [Impact Index Per Article: 2.5] [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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7
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Colón-Thillet R, Stone D, Loprieno MA, Klouser L, Roychoudhury P, Santo TK, Xie H, Stensland L, Upham SL, Pepper G, Huang ML, Aubert M, Jerome KR. Liver-Humanized NSG-PiZ Mice Support the Study of Chronic Hepatitis B Virus Infection and Antiviral Therapies. Microbiol Spectr 2023; 11:e0517622. [PMID: 37199630 PMCID: PMC10269919 DOI: 10.1128/spectrum.05176-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2023] [Accepted: 04/27/2023] [Indexed: 05/19/2023] Open
Abstract
Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic infection is established. Only humans and great apes are fully permissive to HBV infection, and this species restriction has impacted HBV research by limiting the utility of small animal models. To combat HBV species restrictions and enable more in vivo studies, liver-humanized mouse models have been developed that are permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV. HBV selectively replicates in human hepatocytes within chimeric livers, and HBV-positive (HBV+) mice secrete infectious virions and hepatitis B surface antigen (HBsAg) into blood while also harboring covalently closed circular DNA (cccDNA). HBV+ mice develop chronic infections lasting at least 169 days, which should enable the study of new curative therapies targeting chronic HBV, and respond to entecavir therapy. Furthermore, HBV+ human hepatocytes in NSG-PiZ mice can be transduced by AAV3b and AAV.LK03 vectors, which should enable the study of gene therapies that target HBV. In summary, our data demonstrate that liver-humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing chronic hepatitis B (CHB) models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy. IMPORTANCE Liver-humanized mouse models have become the gold standard for the in vivo study of hepatitis B virus (HBV), yet their complexity and cost have prohibited widespread use of existing models in research. Here, we show that the NSG-PiZ liver-humanized mouse model, which is relatively inexpensive and simple to establish, can support chronic HBV infection. Infected mice are fully permissive to hepatitis B, supporting both active replication and spread, and can be used to study novel antiviral therapies. This model is a viable and cost-effective alternative to other liver-humanized mouse models that are used to study HBV.
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Affiliation(s)
- Rossana Colón-Thillet
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
| | - Daniel Stone
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
| | - Michelle A. Loprieno
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
| | - Lindsay Klouser
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
| | - Pavitra Roychoudhury
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Tracy K. Santo
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Hong Xie
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Laurence Stensland
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Sarah L. Upham
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Gregory Pepper
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Meei-Li Huang
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Martine Aubert
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
| | - Keith R. Jerome
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
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Salama II, Sami SM, Salama SI, Abdel-Latif GA, Shaaban FA, Fouad WA, Abdelmohsen AM, Raslan HM. Current and novel modalities for management of chronic hepatitis B infection. World J Hepatol 2023; 15:585-608. [PMID: 37305370 PMCID: PMC10251278 DOI: 10.4254/wjh.v15.i5.585] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/19/2022] [Revised: 03/13/2023] [Accepted: 04/12/2023] [Indexed: 05/24/2023] Open
Abstract
Over 296 million people are estimated to have chronic hepatitis B viral infection (CHB), and it poses unique challenges for elimination. CHB is the result of hepatitis B virus (HBV)-specific immune tolerance and the presence of covalently closed circular DNA as mini chromosome inside the nucleus and the integrated HBV. Serum hepatitis B core-related antigen is the best surrogate marker for intrahepatic covalently closed circular DNA. Functional HBV "cure" is the durable loss of hepatitis B surface antigen (HBsAg), with or without HBsAg seroconversion and undetectable serum HBV DNA after completing a course of treatment. The currently approved therapies are nucleos(t)ide analogues, interferon-alpha, and pegylated-interferon. With these therapies, functional cure can be achieved in < 10% of CHB patients. Any variation to HBV or the host immune system that disrupts the interaction between them can lead to reactivation of HBV. Novel therapies may allow efficient control of CHB. They include direct acting antivirals and immunomodulators. Reduction of the viral antigen load is a crucial factor for success of immune-based therapies. Immunomodulatory therapy may lead to modulation of the host immune system. It may enhance/restore innate immunity against HBV (as toll-like-receptors and cytosolic retinoic acid inducible gene I agonist). Others may induce adaptive immunity as checkpoint inhibitors, therapeutic HBV vaccines including protein (HBsAg/preS and hepatitis B core antigen), monoclonal or bispecific antibodies and genetically engineered T cells to generate chimeric antigen receptor-T or T-cell receptor-T cells and HBV-specific T cells to restore T cell function to efficiently clear HBV. Combined therapy may successfully overcome immune tolerance and lead to HBV control and cure. Immunotherapeutic approaches carry the risk of overshooting immune responses causing uncontrolled liver damage. The safety of any new curative therapies should be measured in relation to the excellent safety of currently approved nucleos(t)ide analogues. Development of novel antiviral and immune modulatory therapies should be associated with new diagnostic assays used to evaluate the effectiveness or to predict response.
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Affiliation(s)
- Iman Ibrahim Salama
- Department of Community Medicine Research, National Research Centre, Giza 12411, Dokki, Egypt.
| | - Samia M Sami
- Department of Child Health, National Research Centre, Giza 12411, Dokki, Egypt
| | - Somaia I Salama
- Department of Community Medicine Research, National Research Centre, Giza 12411, Dokki, Egypt
| | - Ghada A Abdel-Latif
- Department of Community Medicine Research, National Research Centre, Giza 12411, Dokki, Egypt
| | - Fatma A Shaaban
- Department of Child Health, National Research Centre, Giza 12411, Dokki, Egypt
| | - Walaa A Fouad
- Department of Community Medicine Research, National Research Centre, Giza 12411, Dokki, Egypt
| | - Aida M Abdelmohsen
- Department of Community Medicine Research, National Research Centre, Giza 12411, Dokki, Egypt
| | - Hala M Raslan
- Department of Internal Medicine, National Research Centre, Giza 12411, Dokki, Egypt
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9
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Prospects for using CRISPR-Cas9 system in the treatment of human viral diseases. ACTA BIOMEDICA SCIENTIFICA 2023. [DOI: 10.29413/abs.2023-8.1.5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/14/2023] Open
Abstract
The aim. To analyze the possibility of using the genetic mechanisms of CRISPR-Cas9 technology in the prevention and treatment of certain viral diseases.Materials and methods. The search for publications was carried out in Russian and foreign literature using the following search engines: RSCI, Cyberleninka, eLibrary, PubMed, Cochrane Library, etc. A review of domestic and international scientific papers on the research topic was carried out using search keywords: CRISPR, genetic engineering, genome editing, Cas9, sgRNA.Results. A review of using CRISPR-Cas9 method (“genetic scissors”) as a gene therapy for some viral diseases was carried out, and its main advantages and disadvantages were revealed. An analysis of the data of scientific studies on genetic research methods over the past decade discovers the main aspects of CRISPR-Cas9 technology, modern classification and prospects for using this technology in clinical practice for the treatment and prevention of human viral diseases. The possibilities of creating a more versatile and stable version of the CRISPR-Cas9 technology are considered. Particular attention is paid to the technological difficulties and obstacles that scientists face when implementing this system for targeted use in clinical medicine.Conclusion. One of the rapidly developing areas in science giving promising prospects for modern healthcare is genetic engineering, especially in cases where scientific developments are applied in clinical practice. The discovery of “genetic scissors” technology has revolutionized all medicine. Wide opportunities for developing new treatment methods for many viral diseases and creating conditions for their early prevention opened up for the medical community. In the future, with the introduction of this technology into clinical practice, it will become possible to treat diseases that have not previously responded to ongoing therapy and were considered incurable.
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Hawsawi YM, Shams A, Theyab A, Siddiqui J, Barnawee M, Abdali WA, Marghalani NA, Alshelali NH, Al-Sayed R, Alzahrani O, Alqahtani A, Alsulaiman AM. The State-of-the-Art of Gene Editing and its Application to Viral Infections and Diseases Including COVID-19. Front Cell Infect Microbiol 2022; 12:869889. [PMID: 35782122 PMCID: PMC9241565 DOI: 10.3389/fcimb.2022.869889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2022] [Accepted: 05/09/2022] [Indexed: 11/26/2022] Open
Abstract
Gene therapy delivers a promising hope to cure many diseases and defects. The discovery of gene-editing technology fueled the world with valuable tools that have been employed in various domains of science, medicine, and biotechnology. Multiple means of gene editing have been established, including CRISPR/Cas, ZFNs, and TALENs. These strategies are believed to help understand the biological mechanisms of disease progression. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been designated the causative virus for coronavirus disease 2019 (COVID-19) that emerged at the end of 2019. This viral infection is a highly pathogenic and transmissible disease that caused a public health pandemic. As gene editing tools have shown great success in multiple scientific and medical areas, they could eventually contribute to discovering novel therapeutic and diagnostic strategies to battle the COVID-19 pandemic disease. This review aims to briefly highlight the history and some of the recent advancements of gene editing technologies. After that, we will describe various biological features of the CRISPR-Cas9 system and its diverse implications in treating different infectious diseases, both viral and non-viral. Finally, we will present current and future advancements in combating COVID-19 with a potential contribution of the CRISPR system as an antiviral modality in this battle.
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Affiliation(s)
- Yousef M. Hawsawi
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
- College of Medicine, Al-Faisal University, Riyadh, Saudi Arabia
| | - Anwar Shams
- Department of Pharmacology, College of Medicine, Taif University, Mecca, Saudi Arabia
| | - Abdulrahman Theyab
- College of Medicine, Al-Faisal University, Riyadh, Saudi Arabia
- Department of Laboratory & Blood Bank, Security Forces Hospital, Mecca, Saudi Arabia
| | - Jumana Siddiqui
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Mawada Barnawee
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Wed A. Abdali
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Nada A. Marghalani
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Nada H. Alshelali
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Rawan Al-Sayed
- Research Center, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia
| | - Othman Alzahrani
- Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Saudi Arabia
- Genome and Biotechnology Unit, Faculty of Science, University of Tabuk, Tabuk, Saudi Arabia
| | - Alanoud Alqahtani
- Bristol Medical School, Faculty of Health Sciences, University of Bristol, Bristol, United Kingdom
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11
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Bianca C, Sidhartha E, Tiribelli C, El-Khobar KE, Sukowati CHC. Role of hepatitis B virus in development of hepatocellular carcinoma: Focus on covalently closed circular DNA. World J Hepatol 2022; 14:866-884. [PMID: 35721287 PMCID: PMC9157711 DOI: 10.4254/wjh.v14.i5.866] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 01/31/2022] [Accepted: 04/25/2022] [Indexed: 02/06/2023] Open
Abstract
Chronic infection with hepatitis B virus (HBV) remains a major global health problem, especially in developing countries. It may lead to prolonged liver damage, fibrosis, cirrhosis, and hepatocellular carcinoma. Persistent chronic HBV infection is related to host immune response and the stability of the covalently closed circular DNA (cccDNA) in human hepatocytes. In addition to being essential for viral transcription and replication, cccDNA is also suspected to play a role in persistent HBV infections or hepatitis relapses since cccDNA is very stable in non-dividing human hepatocytes. Understanding the pathogenicity and oncogenicity of HBV components would be essential in the development of new diagnostic tools and treatment strategies. This review summarizes the role and molecular mechanisms of HBV cccDNA in hepatocyte transformation and hepatocarcinogenesis and current efforts to its detection and targeting.
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Affiliation(s)
- Claryssa Bianca
- Department of Biomedicine, Indonesia International Institute for Life Sciences, Jakarta 13210, Indonesia
| | - Elizabeth Sidhartha
- Department of Biomedicine, Indonesia International Institute for Life Sciences, Jakarta 13210, Indonesia
| | - Claudio Tiribelli
- Centro Studi Fegato, Fondazione Italiana Fegato ONLUS, Trieste 34149, Italy
| | - Korri Elvanita El-Khobar
- Eijkman Center for Molecular Biology, National Research and Innovation Agency (BRIN), Jakarta 10340, Indonesia
| | - Caecilia H C Sukowati
- Centro Studi Fegato, Fondazione Italiana Fegato ONLUS, Trieste 34149, Italy
- Eijkman Center for Molecular Biology, National Research and Innovation Agency (BRIN), Jakarta 10340, Indonesia.
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12
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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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13
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Cortese MF, Riveiro-Barciela M, Tabernero D, Rodriguez-Algarra F, Palom A, Sopena S, Rando-Segura A, Roade L, Kuchta A, Ferrer-Costa R, Quer J, Pacin B, Vila M, Casillas R, Garcia-Garcia S, Esteban R, Pumarola T, Buti M, Rodriguez-Frias F. Standardized Hepatitis B Virus RNA Quantification in Untreated and Treated Chronic Patients: a Promising Marker of Infection Follow-Up. Microbiol Spectr 2022; 10:e0214921. [PMID: 35377229 PMCID: PMC9045303 DOI: 10.1128/spectrum.02149-21] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Accepted: 03/11/2022] [Indexed: 02/07/2023] Open
Abstract
The measurement and interpretation of HBV DNA and RNA levels in HBV infected patients treated with antiviral therapy supports the objective of HBV disease management. Here, we quantified circulating HBV RNA through a standardized and sensitive assay in follow-up samples from both naive and treated patients as a marker of infection evolution. HBV DNA (HBV DNA for use in Cobas 6800/8800 Automated Roche Molecular Systems), RNA (Roche HBV RNA Investigational Assay for use in the Cobas 6800/8800; Roche), HBeAg and HBsAg (Elycsys HBsAg chemiluminescence immunoassay by Cobas 8000; Roche), and core-related antigen (Lumipulse G chemiluminescence assay; Fujirebio) levels were measured in cohorts of untreated or nucleos(t)ide treated, HBV-infected subjects in an outpatient hospital setting. HBV DNA levels in untreated people were 3.6 log10 higher than corresponding RNA levels and were stable over 5 years of observation. While only five of 52 treated patients had DNA levels below the lower limit of quantification (10 IU/mL) at the end of follow-up, 13 had HBV RNA levels persistently above this limit, including eight with undetectable DNA. In samples with undetectable core-related antigen we observed a median HBsAg titer 2.7-fold higher than in samples with undetectable RNA (adjusted P = 0.012). Detectable HBV RNA with undetectable HBV DNA was a negative predictor of HBsAg decrease to a level ≤100 IU/mL (P = 0.03). In naive patients the difference between HBV DNA and RNA was higher than previously reported. HBV RNA rapidly decreased during treatment. However, in some cases, it was detectable even after years of effective therapy, being a negative predictor of HBsAg decrease. The investigational RNA assay for use on the Cobas 6800/8800 instruments is a sensitive and standardized method that could be applied in general management of HBV infection. IMPORTANCE This study focused on the quantification of circulating HBV RNA by using a standardized and sensitive assay. Thanks to this system we observed a higher difference between circulating HBV DNA and RNA than previously reported. In treated patients, HBV RNA decreased together with DNA, although some patients presented detectable levels even after years of successful antiviral treatment, suggesting a persistent viral transcription. Of note, the detection of viral RNA when HBV DNA is undetectable was a negative predictor of HBsAg decrease to a level ≤100 IU/mL. This assay could be extremely helpful in HBV patients management to study viral transcription and to identify those treated patients that may achieve sustained viral suppression.
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Affiliation(s)
- Maria Francesca Cortese
- Clinical Biochemestry, Vall D'hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
| | - Mar Riveiro-Barciela
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
- Liver Unit, Internal Medicine Department, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - David Tabernero
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
| | - Francisco Rodriguez-Algarra
- The Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
| | - Adriana Palom
- Liver Unit, Internal Medicine Department, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Sara Sopena
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Ariadna Rando-Segura
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
- Virology Unit, Microbiology Department, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Luisa Roade
- Liver Unit, Internal Medicine Department, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Alison Kuchta
- Roche Molecular Systems, Inc., Pleasanton, California, USA
| | - Roser Ferrer-Costa
- Department of Biochemistry, Vall D'Hebron University Hospital, Barcelona, Spain
| | - Josep Quer
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
- Digestive and Liver Disease, Vall D'hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Beatriz Pacin
- Clinical Biochemestry, Vall D'hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
| | - Marta Vila
- Clinical Biochemestry, Vall D'hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Rosario Casillas
- Clinical Biochemestry, Vall D'hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Selene Garcia-Garcia
- Clinical Biochemestry, Vall D'hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
| | - Rafael Esteban
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
- Liver Unit, Internal Medicine Department, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Tomás Pumarola
- Virology Unit, Microbiology Department, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
- Universitat Autònoma de Barcelona, Bellaterra, Spain
| | - Maria Buti
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
- Liver Unit, Internal Medicine Department, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Francisco Rodriguez-Frias
- Clinical Biochemestry, Vall D'hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain
- Biochemistry and Microbiology, Liver Pathology Unit, Vall D'hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain
- Centro De Investigación Biomédica En Red, Enfermedades Hepáticas y Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid, Spain
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Martinez MG, Combe E, Inchauspe A, Mangeot PE, Delberghe E, Chapus F, Neveu G, Alam A, Carter K, Testoni B, Zoulim F. CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants. mBio 2022; 13:e0288821. [PMID: 35389262 PMCID: PMC9040760 DOI: 10.1128/mbio.02888-21] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Accepted: 02/25/2022] [Indexed: 12/18/2022] Open
Abstract
Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. IMPORTANCE Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.
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Affiliation(s)
| | - Emmanuel Combe
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France
| | - Aurore Inchauspe
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France
- Evotec, Lyon, France
| | - Philippe Emmanuel Mangeot
- Centre International de Recherche en Infectiologie, INSERM U1111, CNRS UMR-5308, INSERM and Ecole Normale Superieure de Lyon, Lyon, France
| | - Elodie Delberghe
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France
| | - Fleur Chapus
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France
| | | | | | | | - Barbara Testoni
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France
| | - Fabien Zoulim
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France
- University of Lyon, Université Claude-Bernard, Lyon, France
- Hospices Civils de Lyon, Lyon, France
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15
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Kim SW, Yoon JS, Lee M, Cho Y. Toward a complete cure for chronic hepatitis B: Novel therapeutic targets for hepatitis B virus. Clin Mol Hepatol 2022; 28:17-30. [PMID: 34281294 PMCID: PMC8755466 DOI: 10.3350/cmh.2021.0093] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Revised: 07/05/2021] [Accepted: 07/18/2021] [Indexed: 11/09/2022] Open
Abstract
Hepatitis B virus (HBV) affects approximately 250 million patients worldwide, resulting in the progression to cirrhosis and hepatocellular carcinoma, which are serious public health problems. Although universal vaccination programs exist, they are only prophylactic and not curative. In the HBV life cycle, HBV forms covalently closed circular DNA (cccDNA), which is the viral minichromosome, in the nuclei of human hepatocytes and makes it difficult to achieve a complete cure with the current nucleos(t)ide analogs and interferon therapies. Current antiviral therapies rarely eliminate cccDNA; therefore, lifelong antiviral treatment is necessary. Recent trials for antiviral treatment of chronic hepatitis B have been focused on establishing a functional cure, defined by either the loss of hepatitis B surface antigen, undetectable serum HBV DNA levels, and/or seroconversion to hepatitis B surface antibody. Novel therapeutic targets and molecules are in the pipeline for early clinical trials aiming to cure HBV infection. The ideal strategy for achieving a long-lasting functional or complete cure might be using combination therapies targeting different steps of the HBV life cycle and immunomodulators. This review summarizes the current knowledge about novel treatments and combination treatments for a complete HBV cure.
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Affiliation(s)
- Sun Woong Kim
- Department of Internal Medicine, CHA Gangnam Medical Center, CHA University School of Medicine, Seoul, Korea
| | - Jun Sik Yoon
- Department of Internal Medicine, Busan Paik Hospital, Inje University College of Medicine, Busan, Korea
| | - Minjong Lee
- Department of Internal Medicine, Ewha Womans University College of Medicine, Seoul, Korea
| | - Yuri Cho
- Center for Liver and Pancreatobiliary Cancer, National Cancer Center, Goyang, Korea
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16
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Batskikh S, Morozov S, Vinnitskaya E, Sbikina E, Borunova Z, Dorofeev A, Sandler Y, Saliev K, Kostyushev D, Brezgin S, Kostyusheva A, Chulanov V. May Previous Hepatitis B Virus Infection Be Involved in Etiology and Pathogenesis of Autoimmune Liver Diseases? Adv Ther 2022; 39:430-440. [PMID: 34762287 DOI: 10.1007/s12325-021-01983-5] [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: 09/29/2021] [Accepted: 10/29/2021] [Indexed: 02/07/2023]
Abstract
INTRODUCTION Viral infections, especially with hepatotropic viruses, may trigger autoimmune liver diseases (AILDs) and deteriorate their course. However, association of previous hepatitis B virus (HBV) infection (presence of anti-HBc with or without anti-HBs or HBV DNA in serum) with AILDs is poorly studied so far. The aim of the study was to assess the prevalence of previous hepatitis B virus infection markers and its clinical significance in patients with autoimmune liver diseases. METHODS The study was based on the data obtained from 234 consecutive HBsAg-negative patients with AILDs [81 with autoimmune hepatitis (AIH), 122 with primary biliary cholangitis (PBC) and 31 with primary sclerosing cholangitis (PSC)] and 131 subjects of the control group without liver diseases. Blood samples of the enrolled patients were tested for anti-HBc and HBV DNA. Samples of liver tissue were examined by standard morphologic protocol and, in anti-HBc positive subjects, for HBV DNA. We assessed estimated risks of AILDs according to anti-HBc positivity and association of anti-HBc positivity with stage of liver fibrosis. RESULTS Anti-HBc was detected in 14.5% participants in the control group vs 26.1% (p = 0.016) in patients with AILDs (including 27.1% subjects with PBC (p = 0.021 vs control group), in 29% of PSC and 23.5% in AIH. HBV DNA was detected in three patients with PBC and in one with AIH. Positive anti-HBc test result was associated with higher risk of AILDs-odds ratio (OR) = 2.078 [95% confidence interval (CI) 1.179-3.665], especially in PBC: OR (95% CI) 2.186 (1.165-4.101). Odds of advanced stage of liver fibrosis (F3-F4 by METAVIR) in anti-HBc-positive subjects with PBC were also higher compared to those who had no previous HBV infection: OR (95% CI) 2.614 (1.153-5.926). CONCLUSIONS Significant proportions of patients with AILDs are anti-HBc positive, and some of them have OBI. Among patients with AILDs, anti-HBc-positivity is most widespread in the PBC group and in subjects with advanced stage of liver fibrosis. Our data may support the idea of an important role of previous HBV infection in the etiology and pathogenesis of AILDs (namely PBC).
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Affiliation(s)
- Sergey Batskikh
- Moscow Clinical Scientific Center n.a. A.S. Loginov, Entuziastov Shosse, 86, 111123, Moscow, Russia
| | - Sergey Morozov
- Department Gastroenterology, Hepatology and Nutrition, Federal Research Center of Nutrition and Biotechnology, Kashirskoye Shosse, 21, 115446, Moscow, Russia.
| | - Elena Vinnitskaya
- Moscow Clinical Scientific Center n.a. A.S. Loginov, Entuziastov Shosse, 86, 111123, Moscow, Russia
| | - Evgeniya Sbikina
- Moscow Clinical Scientific Center n.a. A.S. Loginov, Entuziastov Shosse, 86, 111123, Moscow, Russia
| | - Zanna Borunova
- Moscow Clinical Scientific Center n.a. A.S. Loginov, Entuziastov Shosse, 86, 111123, Moscow, Russia
| | - Alexey Dorofeev
- Moscow Clinical Scientific Center n.a. A.S. Loginov, Entuziastov Shosse, 86, 111123, Moscow, Russia
| | - Yulia Sandler
- Moscow Clinical Scientific Center n.a. A.S. Loginov, Entuziastov Shosse, 86, 111123, Moscow, Russia
| | - Kirill Saliev
- Moscow Clinical Scientific Center n.a. A.S. Loginov, Entuziastov Shosse, 86, 111123, Moscow, Russia
| | - Dmitry Kostyushev
- National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, 127994, Moscow, Russia
- Division of Biotechnology, Scientific Center for Genetics and Life Sciences, Sirius University of Science and Technology, 354340, Sochi, Russia
| | - Sergey Brezgin
- National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, 127994, Moscow, Russia
- Division of Biotechnology, Scientific Center for Genetics and Life Sciences, Sirius University of Science and Technology, 354340, Sochi, Russia
| | - Anastasiya Kostyusheva
- National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, 127994, Moscow, Russia
| | - Vladimir Chulanov
- National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, 127994, Moscow, Russia
- Division of Biotechnology, Scientific Center for Genetics and Life Sciences, Sirius University of Science and Technology, 354340, Sochi, Russia
- Department of Infectious Diseases, Sechenov First Moscow State Medical University, 119146, Moscow, Russia
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17
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Mnyandu N, Limani SW, Arbuthnot P, Maepa MB. Advances in designing Adeno-associated viral vectors for development of anti-HBV gene therapeutics. Virol J 2021; 18:247. [PMID: 34903258 PMCID: PMC8670254 DOI: 10.1186/s12985-021-01715-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Accepted: 11/26/2021] [Indexed: 12/25/2022] Open
Abstract
Despite the five decades having passed since discovery of the hepatitis B virus (HBV), together with development of an effective anti-HBV vaccine, infection with the virus remains a serious public health problem and results in nearly 900,000 annual deaths worldwide. Current therapies do not eliminate the virus and viral replication typically reactivates after treatment withdrawal. Hence, current endeavours are aimed at developing novel therapies to achieve a functional cure. Nucleic acid-based therapeutic approaches are promising, with several candidates showing excellent potencies in preclinical and early stages of clinical development. However, this class of therapeutics is yet to become part of standard anti-HBV treatment regimens. Obstacles delaying development of gene-based therapies include lack of clinically relevant delivery methods and a paucity of good animal models for preclinical characterisation. Recent studies have demonstrated safety and efficiency of Adeno-associated viral vectors (AAVs) in gene therapy. However, AAVs do have flaws and this has prompted research aimed at improving design of novel and artificially synthesised AAVs. Main goals are to improve liver transduction efficiencies and avoiding immune clearance. Application of AAVs to model HBV replication in vivo is also useful for characterising anti-HBV gene therapeutics. This review summarises recent advances in AAV engineering and their contributions to progress with anti-HBV gene therapy development.
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Affiliation(s)
- Njabulo Mnyandu
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Shonisani Wendy Limani
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Mohube Betty Maepa
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
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18
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Alvarez-Astudillo F, Garrido D, Varas-Godoy M, Gutiérrez JL, Villanueva RA, Loyola A. The histone variant H3.3 regulates the transcription of the hepatitis B virus. Ann Hepatol 2021; 21:100261. [PMID: 33007428 DOI: 10.1016/j.aohep.2020.09.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Accepted: 09/17/2020] [Indexed: 02/04/2023]
Abstract
INTRODUCTION AND OBJECTIVES About 250 million people around the world are chronically infected with the hepatitis B virus (HBV). Those people are at risk of developing hepatocellular carcinoma. The HBV genome is organized as a minichromosome in the infected hepatocyte and is associated with histones and non-histone proteins. In recent years, many groups have investigated the transcriptional regulation of HBV mediated by post-translational modifications on the histones associated with the covalently closed circular DNA (cccDNA). Our aim is to investigate the role of the histone variant H3.3. MATERIALS AND METHODS An in vitro HBV replication model system based on the transfection of linear HBV genome monomeric molecules was used. We then either ectopically expressed or reduced the levels of H3.3, and of its histone chaperone HIRA. Viral intermediates were quantified and the level of H3K4me3 using Chromatin immunoprecipitation (ChIP) assay was measured. RESULTS Histone variant H3.3 ectopically expressed in cells assembles into the viral cccDNA, correlating with increasing levels of the active histone mark H3K4me3 and HBV transcription. The opposite results were found upon diminishing H3.3 levels. Furthermore, the assembly of H3.3 into the cccDNA is dependent on the histone chaperone HIRA. Diminishing HIRA levels causes a reduction in the HBV intermediates. CONCLUSIONS Histone variant H3.3 positively regulates HBV transcription. Importantly, the characterization of the viral chromatin dynamics might allow the discovery of new therapeutic targets to develop drugs for the treatment of chronically-infected HBV patients.
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Affiliation(s)
| | - Daniel Garrido
- Fundación Ciencia & Vida, Avenida Zañartu 1482, Ñuñoa, 7780272, Santiago, Chile
| | - Manuel Varas-Godoy
- Centro de Biología Celular y Biomedicina (CEBICEM), Facultad de Medicina y Ciencia, Universidad, San Sebastián, Santiago, 7510157, Chile
| | - José Leonardo Gutiérrez
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Barrio Universitario s/n, Concepción, 4070043, Chile
| | - Rodrigo A Villanueva
- Fundación Ciencia & Vida, Avenida Zañartu 1482, Ñuñoa, 7780272, Santiago, Chile.
| | - Alejandra Loyola
- Fundación Ciencia & Vida, Avenida Zañartu 1482, Ñuñoa, 7780272, Santiago, Chile; Universidad San Sebastián, Santiago, 7510157, Chile.
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19
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In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors. Viruses 2021; 13:v13112247. [PMID: 34835053 PMCID: PMC8618177 DOI: 10.3390/v13112247] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 10/30/2021] [Accepted: 11/04/2021] [Indexed: 12/23/2022] Open
Abstract
The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals’ potency.
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20
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Miao J, Gao P, Li Q, He K, Zhang L, Wang J, Huang L. Advances in Nanoparticle Drug Delivery Systems for Anti-Hepatitis B Virus Therapy: A Narrative Review. Int J Mol Sci 2021; 22:ijms222011227. [PMID: 34681886 PMCID: PMC8538950 DOI: 10.3390/ijms222011227] [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: 09/10/2021] [Revised: 09/26/2021] [Accepted: 10/11/2021] [Indexed: 12/11/2022] Open
Abstract
Chronic hepatitis B (CHB) is an infectious viral disease that is prevalent worldwide. Traditional nucleoside analogues, as well as the novel drug targets against hepatitis B virus (HBV), are associated with certain critical factors that influence the curative effect, such as biological stability and safety, effective drug delivery, and controlled release. Nanoparticle drug delivery systems have significant advantages and have provided a basis for the development of anti-HBV strategies. In this review, we aim to review the advances in nanoparticle drug delivery systems for anti-hepatitis B virus therapy by summarizing the relevant literature. First, we focus on the characteristics of nanoparticle drug delivery systems for anti-HBV therapy. Second, we discuss the nanoparticle delivery systems for anti-HBV nucleoside drugs, gene-based drugs, and vaccines. Lastly, we provide an overview of the prospects for nanoparticle-based anti-HBV agents.
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Affiliation(s)
- Jing Miao
- Department of Pharmacy, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China; (J.M.); (P.G.); (K.H.); (L.Z.)
- Zhejiang Provincial Key Laboratory for Drug Evaluation and Clinical Research, Hangzhou 310003, China
| | - Peng Gao
- Department of Pharmacy, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China; (J.M.); (P.G.); (K.H.); (L.Z.)
| | - Qian Li
- Department of Pharmacy, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China;
| | - Kaifeng He
- Department of Pharmacy, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China; (J.M.); (P.G.); (K.H.); (L.Z.)
| | - Liwen Zhang
- Department of Pharmacy, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China; (J.M.); (P.G.); (K.H.); (L.Z.)
| | - Junyan Wang
- Department of Pharmacy, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China; (J.M.); (P.G.); (K.H.); (L.Z.)
- Correspondence: (J.W.); (L.H.)
| | - Lingfei Huang
- Department of Pharmacy, The Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou 310052, China; (J.M.); (P.G.); (K.H.); (L.Z.)
- Correspondence: (J.W.); (L.H.)
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21
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Lv W, Li T, Wang S, Wang H, Li X, Zhang S, Wang L, Xu Y, Wei W. The Application of the CRISPR/Cas9 System in the Treatment of Hepatitis B Liver Cancer. Technol Cancer Res Treat 2021; 20:15330338211045206. [PMID: 34605326 PMCID: PMC8493308 DOI: 10.1177/15330338211045206] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was originally discovered in prokaryotes and functions as part of the adaptive immune system. The experimental research of many scholars, as well as scientific and technological advancements, has allowed prokaryote-derived CRISPR/Cas genome-editing systems to transform our ability to manipulate, detect, image, and annotate specific DNA and RNA sequences in the living cells of diverse species. Through modern genetic engineering editing technology and high-throughput gene sequencing, we can edit and splice covalently closed circular DNA to silence it, and correct the mutation and deletion of liver cancer genes to achieve precise in situ repair of defective genes and prohibit viral infection or replication. Such manipulations do not destroy the structure of the entire genome and facilitate the cure of diseases. In this review, we discussed the possibility that CRISPR/Cas could be used as a treatment for patients with liver cancer caused by hepatitis B virus infection, and reviewed the challenges incurred by this effective gene-editing technology.
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Affiliation(s)
- Wei Lv
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Tao Li
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Shanshan Wang
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Huihui Wang
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Xuemei Li
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Shubing Zhang
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Lianzi Wang
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Yuanhong Xu
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
| | - Wei Wei
- 36639The First Affiliated Hospital of Anhui Medical University, Hefei, China
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22
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Lin H, Li G, Peng X, Deng A, Ye L, Shi L, Wang T, He J. The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses. Front Cell Infect Microbiol 2021; 11:590989. [PMID: 34513721 PMCID: PMC8430244 DOI: 10.3389/fcimb.2021.590989] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 08/04/2021] [Indexed: 12/12/2022] Open
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a set of versatile gene-editing toolkit that perform diverse revolutionary functions in various fields of application such as agricultural practices, food industry, biotechnology, biomedicine, and clinical research. Specially, as a novel antiviral method of choice, CRISPR/Cas9 system has been extensively and effectively exploited to fight against human infectious viruses. Infectious diseases including human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papillomavirus (HPV), and other viruses are still global threats with persistent potential to probably cause pandemics. To facilitate virus removals, the CRISPR/Cas9 system has already been customized to confer new antiviral capabilities into host animals either by modifying host genome or by directly targeting viral inherent factors in the form of DNA. Although several limitations and difficulties still need to be conquered, this technology holds great promises in the treatment of human viral infectious diseases. In this review, we will first present a brief biological feature of CRISPR/Cas9 systems, which includes a description of CRISPR/Cas9 structure and composition; thereafter, we will focus on the investigations and applications that employ CRISPR/Cas9 system to combat several human infectious viruses and discuss challenges and future perspectives of using this new platform in the preclinical and clinical settings as an antiviral strategy.
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Affiliation(s)
- Huafeng Lin
- Changsha Hospital for Maternal and Child Health Care of Hunan Normal University, Changsha, China.,Institute of Food Safety and Nutrition, Jinan University, Guangzhou, China
| | - Gang Li
- Institute of Biomedicine and Department of Cell Biology, Jinan University, Guangzhou, China
| | - Xiangwen Peng
- Changsha Hospital for Maternal and Child Health Care of Hunan Normal University, Changsha, China
| | - Aimin Deng
- Changsha Hospital for Maternal and Child Health Care of Hunan Normal University, Changsha, China
| | - Lei Ye
- Institute of Food Safety and Nutrition, Jinan University, Guangzhou, China
| | - Lei Shi
- Institute of Food Safety and Nutrition, Jinan University, Guangzhou, China
| | - Tuanmei Wang
- Changsha Hospital for Maternal and Child Health Care of Hunan Normal University, Changsha, China
| | - Jun He
- Changsha Hospital for Maternal and Child Health Care of Hunan Normal University, Changsha, China
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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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Martinez MG, Boyd A, Combe E, Testoni B, Zoulim F. Covalently closed circular DNA: The ultimate therapeutic target for curing HBV infections. J Hepatol 2021; 75:706-717. [PMID: 34051332 DOI: 10.1016/j.jhep.2021.05.013] [Citation(s) in RCA: 95] [Impact Index Per Article: 23.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 05/03/2021] [Accepted: 05/07/2021] [Indexed: 12/16/2022]
Abstract
Current antiviral therapies, such as pegylated interferon-α and nucleos(t)ide analogues, effectively improve the quality of life of patients with chronic hepatitis B. However, they can only control the infection rather than curing infected hepatocytes. Complete HBV cure is hampered by the lack of therapies that can directly affect the viral minichromosome (in the form of covalently closed circular DNA [cccDNA]). Approaches currently under investigation in early clinical trials are aimed at achieving a functional cure, defined as the loss of HBsAg and undetectable HBV DNA levels in serum. However, achieving a complete HBV cure requires therapies that can directly target the cccDNA pool, either via degradation, lethal mutations or functional silencing. In this review, we discuss cutting-edge technologies that could lead to non-cytolytic direct cccDNA targeting and cure of infected hepatocytes.
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Affiliation(s)
| | - Anders Boyd
- Stichting HIV Monitoring, Amsterdam, the Netherlands; Department of Infectious Diseases, Research and Prevention, Public Health Service of Amsterdam, Amsterdam, the Netherlands
| | - Emmanuel Combe
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France
| | - Barbara Testoni
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France
| | - Fabien Zoulim
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France; University of Lyon, Université Claude- Bernard (UCBL), 69008 Lyon, France; Hospices Civils de Lyon (HCL), 69002 Lyon, France.
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25
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Ohsaki E, Suwanmanee Y, Ueda K. Chronic Hepatitis B Treatment Strategies Using Polymerase Inhibitor-Based Combination Therapy. Viruses 2021; 13:v13091691. [PMID: 34578273 PMCID: PMC8473100 DOI: 10.3390/v13091691] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2021] [Revised: 08/19/2021] [Accepted: 08/25/2021] [Indexed: 12/12/2022] Open
Abstract
Viral polymerase is an essential enzyme for the amplification of the viral genome and is one of the major targets of antiviral therapies. However, a serious concern to be solved in hepatitis B virus (HBV) infection is the difficulty of eliminating covalently closed circular (ccc) DNA. More recently, therapeutic strategies targeting various stages of the HBV lifecycle have been attempted. Although cccDNA-targeted therapies are attractive, there are still many problems to be overcome, and the development of novel polymerase inhibitors remains an important issue. Interferons and nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are the only therapeutic options currently available for HBV infection. Many studies have reported that the combination of interferons and NRTI causes the loss of hepatitis B surface antigen (HBsAg), which is suggestive of seroconversion. Although NRTIs do not directly target cccDNA, they can strongly reduce the serum viral DNA load and could suppress the recycling step of cccDNA formation, improve liver fibrosis/cirrhosis, and reduce the risk of hepatocellular carcinoma. Here, we review recent studies on combination therapies using polymerase inhibitors and discuss the future directions of therapeutic strategies for HBV infection.
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26
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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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27
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Smith T, Singh P, Chmielewski KO, Bloom K, Cathomen T, Arbuthnot P, Ely A. Improved Specificity and Safety of Anti-Hepatitis B Virus TALENs Using Obligate Heterodimeric FokI Nuclease Domains. Viruses 2021; 13:v13071344. [PMID: 34372550 PMCID: PMC8310341 DOI: 10.3390/v13071344] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Accepted: 07/02/2021] [Indexed: 01/04/2023] Open
Abstract
Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral core and surface open reading frames were modified with second- and third-generation FokI nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of FokI homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication.
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Affiliation(s)
- Tiffany Smith
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Prashika Singh
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Kay Ole Chmielewski
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg & Medical Faculty, University of Freiburg, 79106 Freiburg, Germany; (K.O.C.); (T.C.)
| | - Kristie Bloom
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg & Medical Faculty, University of Freiburg, 79106 Freiburg, Germany; (K.O.C.); (T.C.)
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Abdullah Ely
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
- Correspondence: ; Tel.: +27-(0)11-717-2561
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28
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Singh P, Kairuz D, Arbuthnot P, Bloom K. Silencing hepatitis B virus covalently closed circular DNA: The potential of an epigenetic therapy approach. World J Gastroenterol 2021; 27:3182-3207. [PMID: 34163105 PMCID: PMC8218364 DOI: 10.3748/wjg.v27.i23.3182] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 03/23/2021] [Accepted: 05/07/2021] [Indexed: 02/06/2023] Open
Abstract
Global prophylactic vaccination programmes have helped to curb new hepatitis B virus (HBV) infections. However, it is estimated that nearly 300 million people are chronically infected and have a high risk of developing hepatocellular carcinoma. As such, HBV remains a serious health priority and the development of novel curative therapeutics is urgently needed. Chronic HBV infection has been attributed to the persistence of the covalently closed circular DNA (cccDNA) which establishes itself as a minichromosome in the nucleus of hepatocytes. As the viral transcription intermediate, the cccDNA is responsible for producing new virions and perpetuating infection. HBV is dependent on various host factors for cccDNA formation and the minichromosome is amenable to epigenetic modifications. Two HBV proteins, X (HBx) and core (HBc) promote viral replication by modulating the cccDNA epigenome and regulating host cell responses. This includes viral and host gene expression, chromatin remodeling, DNA methylation, the antiviral immune response, apoptosis, and ubiquitination. Elimination of the cccDNA minichromosome would result in a sterilizing cure; however, this may be difficult to achieve. Epigenetic therapies could permanently silence the cccDNA minichromosome and promote a functional cure. This review explores the cccDNA epigenome, how host and viral factors influence transcription, and the recent epigenetic therapies and epigenome engineering approaches that have been described.
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Affiliation(s)
- Prashika Singh
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
| | - Dylan Kairuz
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
| | - Kristie Bloom
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
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Kovesdi I, Bakacs T. Therapeutic Exploitation of Viral Interference. Infect Disord Drug Targets 2021; 20:423-432. [PMID: 30950360 DOI: 10.2174/1871526519666190405140858] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2019] [Revised: 03/18/2019] [Accepted: 03/19/2019] [Indexed: 02/06/2023]
Abstract
Viral interference, originally, referred to a state of temporary immunity, is a state whereby infection with a virus limits replication or production of a second infecting virus. However, replication of a second virus could also be dominant over the first virus. In fact, dominance can alternate between the two viruses. Expression of type I interferon genes is many times upregulated in infected epithelial cells. Since the interferon system can control most, if not all, virus infections in the absence of adaptive immunity, it was proposed that viral induction of a nonspecific localized temporary state of immunity may provide a strategy to control viral infections. Clinical observations also support such a theory, which gave credence to the development of superinfection therapy (SIT). SIT is an innovative therapeutic approach where a non-pathogenic virus is used to infect patients harboring a pathogenic virus. For the functional cure of persistent viral infections and for the development of broad- spectrum antivirals against emerging viruses a paradigm shift was recently proposed. Instead of the virus, the therapy should be directed at the host. Such a host-directed-therapy (HDT) strategy could be the activation of endogenous innate immune response via toll-like receptors (TLRs). Superinfection therapy is such a host-directed-therapy, which has been validated in patients infected with two completely different viruses, the hepatitis B (DNA), and hepatitis C (RNA) viruses. SIT exerts post-infection interference via the constant presence of an attenuated non-pathogenic avian double- stranded (ds) RNA viral vector which boosts the endogenous innate (IFN) response. SIT could, therefore, be developed into a biological platform for a new "one drug, multiple bugs" broad-spectrum antiviral treatment approach.
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Affiliation(s)
- Imre Kovesdi
- ImiGene, Inc., Rockville, MD, USA,HepC, Inc., Budapest, Hungary
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Colón-Thillet R, Jerome KR, Stone D. Optimization of AAV vectors to target persistent viral reservoirs. Virol J 2021; 18:85. [PMID: 33892762 PMCID: PMC8067653 DOI: 10.1186/s12985-021-01555-7] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2021] [Accepted: 04/14/2021] [Indexed: 12/18/2022] Open
Abstract
Gene delivery of antiviral therapeutics to anatomical sites where viruses accumulate and persist is a promising approach for the next generation of antiviral therapies. Recombinant adeno-associated viruses (AAV) are one of the leading vectors for gene therapy applications that deliver gene-editing enzymes, antibodies, and RNA interference molecules to eliminate viral reservoirs that fuel persistent infections. As long-lived viral DNA within specific cellular reservoirs is responsible for persistent hepatitis B virus, Herpes simplex virus, and human immunodeficiency virus infections, the discovery of AAV vectors with strong tropism for hepatocytes, sensory neurons and T cells, respectively, is of particular interest. Identification of natural isolates from various tissues in humans and non-human primates has generated an extensive catalog of AAV vectors with diverse tropisms and transduction efficiencies, which has been further expanded through molecular genetic approaches. The AAV capsid protein, which forms the virions' outer shell, is the primary determinant of tissue tropism, transduction efficiency, and immunogenicity. Thus, over the past few decades, extensive efforts to optimize AAV vectors for gene therapy applications have focused on capsid engineering with approaches such as directed evolution and rational design. These approaches are being used to identify variants with improved transduction efficiencies, alternate tropisms, reduced sequestration in non-target organs, and reduced immunogenicity, and have produced AAV capsids that are currently under evaluation in pre-clinical and clinical trials. This review will summarize the most recent strategies to identify AAV vectors with enhanced tropism and transduction in cell types that harbor viral reservoirs.
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Affiliation(s)
- Rossana Colón-Thillet
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA, USA
| | - Keith R Jerome
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA, USA
- Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
| | - Daniel Stone
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA, USA.
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31
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Maepa MB, Bloom K, Ely A, Arbuthnot P. Hepatitis B virus: promising drug targets and therapeutic implications. Expert Opin Ther Targets 2021; 25:451-466. [PMID: 33843412 DOI: 10.1080/14728222.2021.1915990] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Introduction: Current therapy for infection with hepatitis B virus (HBV) rarely clears the virus, and viremia commonly resurges following treatment withdrawal. To prevent serious complications of the infection, research has been aimed at identifying new viral and host targets that can be exploited to inactivate HBV replication.Areas covered: This paper reviews the use of these new molecular targets to advance anti-HBV therapy. Emphasis is on appraising data from pre-clinical and early clinical studies described in journal articles published during the past 10 years and available from PubMed.Expert opinion: The wide range of viral and host factors that can be targeted to disable HBV is impressive and improved insight into HBV molecular biology continues to provide the basis for new drug design. In addition to candidate therapies that have direct or indirect actions on HBV covalently closed circular DNA (cccDNA), compounds that inhibit HBsAg secretion, viral entry, destabilize viral RNA and effect enhanced immune responses to HBV show promise. Preclinical and clinical evaluation of drug candidates, as well as investigating use of treatment combinations, are encouraging. The field is poised at an interesting stage and indications are that reliably achieving functional cure from HBV infection is a tangible goal.
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Affiliation(s)
- Mohube Betty Maepa
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
| | - Kristie Bloom
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
| | - Abdullah Ely
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
| | - Patrick Arbuthnot
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
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Wang G, Guan J, Khan NU, Li G, Shao J, Zhou Q, Xu L, Huang C, Deng J, Zhu H, Chen Z. Potential capacity of interferon-α to eliminate covalently closed circular DNA (cccDNA) in hepatocytes infected with hepatitis B virus. Gut Pathog 2021; 13:22. [PMID: 33845868 PMCID: PMC8040234 DOI: 10.1186/s13099-021-00421-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2021] [Accepted: 04/01/2021] [Indexed: 12/14/2022] Open
Abstract
Interferon-alpha (IFN-α) and nucleot(s)ide analogs (NAs) are first-line drugs for the treatment of chronic hepatitis B virus (HBV) infections. Generally, NAs target the reverse transcription of HBV pregenomic RNA, but they cannot eliminate covalently-closed-circular DNA (cccDNA). Although effective treatment with NAs can dramatically decrease HBV proteins and DNA loads, and even promote serological conversion, cccDNA persists in the nucleus of hepatocytes due to the lack of effective anti-cccDNA drugs. Of the medications currently available, only IFN-α can potentially target cccDNA. However, the clinical effects of eradicating cccDNA using IFN-α in the hepatocytes of patients with HBV are not proficient as well as expected and are not well understood. Herein, we review the anti-HBV mechanisms of IFN-α involving cccDNA modification as the most promising approaches to cure HBV infection. We expect to find indications of promising areas of research that require further study to eliminate cccDNA of HBV in patients.
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Affiliation(s)
- Gang Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Jun Guan
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Nazif U Khan
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Guojun Li
- Institute for Hepatology, Shenzhen Third People's Hospital, National Clinical Research Center for Infectious Disease, Shenzhen, 518112, Guangdong, China.,The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, 518112, Shenzhen, China
| | - Junwei Shao
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Qihui Zhou
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Lichen Xu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Chunhong Huang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Jingwen Deng
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Haihong Zhu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China
| | - Zhi Chen
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China.
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Sharma G, Sharma AR, Bhattacharya M, Lee SS, Chakraborty C. CRISPR-Cas9: A Preclinical and Clinical Perspective for the Treatment of Human Diseases. Mol Ther 2021; 29:571-586. [PMID: 33238136 PMCID: PMC7854284 DOI: 10.1016/j.ymthe.2020.09.028] [Citation(s) in RCA: 133] [Impact Index Per Article: 33.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Revised: 07/12/2020] [Accepted: 09/16/2020] [Indexed: 12/21/2022] Open
Abstract
At present, the idea of genome modification has revolutionized the modern therapeutic research era. Genome modification studies have traveled a long way from gene modifications in primary cells to genetic modifications in animals. The targeted genetic modification may result in the modulation (i.e., either upregulation or downregulation) of the predefined gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) is a promising genome-editing tool that has therapeutic potential against incurable genetic disorders by modifying their DNA sequences. In comparison with other genome-editing techniques, CRISPR-Cas9 is simple, efficient, and very specific. This enabled CRISPR-Cas9 genome-editing technology to enter into clinical trials against cancer. Besides therapeutic potential, the CRISPR-Cas9 tool can also be applied to generate genetically inhibited animal models for drug discovery and development. This comprehensive review paper discusses the origin of CRISPR-Cas9 systems and their therapeutic potential against various genetic disorders, including cancer, allergy, immunological disorders, Duchenne muscular dystrophy, cardiovascular disorders, neurological disorders, liver-related disorders, cystic fibrosis, blood-related disorders, eye-related disorders, and viral infection. Finally, we discuss the different challenges, safety concerns, and strategies that can be applied to overcome the obstacles during CRISPR-Cas9-mediated therapeutic approaches.
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Affiliation(s)
- Garima Sharma
- Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Ashish Ranjan Sharma
- Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon, Gangwon-Do 24252, Republic of Korea
| | - Manojit Bhattacharya
- Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon, Gangwon-Do 24252, Republic of Korea
| | - Sang-Soo Lee
- Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon, Gangwon-Do 24252, Republic of Korea.
| | - Chiranjib Chakraborty
- Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon, Gangwon-Do 24252, Republic of Korea; Department of Biotechnology, School of Life Science and Biotechnology, Adamas University, Barasat-Barrackpore Road, Kolkata, West Bengal 700126, India.
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Scott TA, Morris KV. Designer nucleases to treat malignant cancers driven by viral oncogenes. Virol J 2021; 18:18. [PMID: 33441159 PMCID: PMC7805041 DOI: 10.1186/s12985-021-01488-1] [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: 11/11/2020] [Accepted: 01/02/2021] [Indexed: 11/22/2022] Open
Abstract
Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.
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Affiliation(s)
- Tristan A Scott
- Center for Gene Therapy, City of Hope, Beckman Research Institute and Hematological Malignancy and Stem Cell Transplantation Institute at the City of Hope, 1500 E. Duarte Rd, Duarte, CA, 91010, USA.
| | - Kevin V Morris
- Center for Gene Therapy, City of Hope, Beckman Research Institute and Hematological Malignancy and Stem Cell Transplantation Institute at the City of Hope, 1500 E. Duarte Rd, Duarte, CA, 91010, USA
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35
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Bove G, Mehnert AK, Dao Thi VL. iPSCs for modeling hepatotropic pathogen infections. IPSCS FOR STUDYING INFECTIOUS DISEASES 2021:149-213. [DOI: 10.1016/b978-0-12-823808-0.00013-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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Gao YG, Huangfu SY, Patil S, Tang Q, Sun W, Li Y, Lu ZL, Qian A. [12]aneN 3-based multifunctional compounds as fluorescent probes and nucleic acids delivering agents. Drug Deliv 2020; 27:66-80. [PMID: 31858838 PMCID: PMC6968532 DOI: 10.1080/10717544.2019.1704943] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2019] [Revised: 12/03/2019] [Accepted: 12/11/2019] [Indexed: 12/31/2022] Open
Abstract
A series of multifunctional compounds (MFCs) 1a-1e based on 1,8-naphthalimide and [12]aneN3 building blocks were designed and synthesized. They were used as not only fluorescent probes for recognition of Cu2+ ions but also as non-viral gene vectors for DNA and RNA delivery. Furthermore, their complexes with Cu2+ (1-Cu) could also selectively stain lysosome in HeLa cells. In order to achieve high performance multifunctional materials, structure-performance relationship of MFCs 1a-1e was studied. It was found that MFCs 1a-1e exhibited highly selective fluorescence turn-off for Cu2+, without interference by other metal ions in aqueous solution. The fluorescence emission of 1a-1e was quenched by a factor of 10-fold, 47-fold, 6-fold, 64-fold, and 15-fold respectively in the presence of Cu2+ ions. Due to high sensitivity, good water solubility, and low cytotoxicity, MFCs 1a-1d were successfully applied in the recognition of Cu2+ and selectively staining lysosome in HeLa cells. Most importantly, MFCs 1a and 1b had excellent HeLa cell selectivity in RNA delivery, and their performances were far better than lipofectamine 2000 and 25 kDa PEI.
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Affiliation(s)
- Yong-Guang Gao
- Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China
| | - Shu-Yuan Huangfu
- Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Suryaji Patil
- Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Quan Tang
- Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China
| | - Wan Sun
- Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China
| | - Yu Li
- Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Zhong-Lin Lu
- Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China
| | - Airong Qian
- Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
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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: 40] [Impact Index Per Article: 8.0] [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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Ligat G, Goto K, Verrier E, Baumert TF. Targeting Viral cccDNA for Cure of Chronic Hepatitis B. CURRENT HEPATOLOGY REPORTS 2020; 19:235-244. [PMID: 36034467 PMCID: PMC7613435 DOI: 10.1007/s11901-020-00534-w] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Purpose of Review Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), is a major cause of advanced liver disease and hepatocellular carcinoma (HCC) worldwide. HBV replication is characterized by the synthesis of covalently closed circular (ccc) DNA which is not targeted by antiviral nucleos(t)ide analogues (NUCs) the key modality of standard of care. While HBV replication is successfully suppressed in treated patients, they remain at risk for developing HCC. While functional cure, characterized by loss of HBsAg, is the first goal of novel antiviral therapies, curative treatments eliminating cccDNA remain the ultimate goal. This review summarizes recent advances in the discovery and development of novel therapeutic strategies and their impact on cccDNA biology. Recent Findings Within the last decade, substantial progress has been made in the understanding of cccDNA biology including the discovery of host dependency factors, epigenetic regulation of cccDNA transcription and immune-mediated degradation. Several approaches targeting cccDNA either in a direct or indirect manner are currently at the stage of discovery, preclinical or early clinical development. Examples include genome-editing approaches, strategies targeting host dependency factors or epigenetic gene regulation, nucleocapsid modulators and immune-mediated degradation. Summary While direct-targeting cccDNA strategies are still largely at the preclinical stage of development, capsid assembly modulators and immune-based approaches have reached the clinical phase. Clinical trials are ongoing to assess their efficacy and safety in patients including their impact on viral cccDNA. Combination therapies provide additional opportunities to overcome current limitations of individual approaches.
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Affiliation(s)
- Gaëtan Ligat
- Université de Strasbourg, 67000 Strasbourg, France
- Institut de Recherche sur les Maladies Virales et Hépatiques, Inserm U1110, 3 Rue Koeberlé, 67000 Strasbourg, France
| | - Kaku Goto
- Université de Strasbourg, 67000 Strasbourg, France
- Institut de Recherche sur les Maladies Virales et Hépatiques, Inserm U1110, 3 Rue Koeberlé, 67000 Strasbourg, France
| | - Eloi Verrier
- Université de Strasbourg, 67000 Strasbourg, France
- Institut de Recherche sur les Maladies Virales et Hépatiques, Inserm U1110, 3 Rue Koeberlé, 67000 Strasbourg, France
| | - Thomas F. Baumert
- Université de Strasbourg, 67000 Strasbourg, France
- Institut de Recherche sur les Maladies Virales et Hépatiques, Inserm U1110, 3 Rue Koeberlé, 67000 Strasbourg, France
- Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Nouvel Hôpital Civil, 67000 Strasbourg, France
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Martinez MG, Villeret F, Testoni B, Zoulim F. Can we cure hepatitis B virus with novel direct-acting antivirals? Liver Int 2020; 40 Suppl 1:27-34. [PMID: 32077597 DOI: 10.1111/liv.14364] [Citation(s) in RCA: 52] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/27/2019] [Accepted: 12/27/2019] [Indexed: 12/12/2022]
Abstract
Current treatments against chronic hepatitis B (CHB) include pegylated interferon alpha (Peg-IFNα) and nucleos(t)ide analogs (NAs), the latter targeting the viral retrotranscriptase, thus inhibiting de novo viral production. Although these therapies control infection and improve the patient's quality of life, they do not cure HBV-infected hepatocytes. A complete HBV cure is currently not possible because of the presence of the stable DNA intermediate covalently closed circular DNA (cccDNA). Current efforts are focused on achieving a functional cure, defined by the loss of Hepatitis B surface antigen (HBsAg) and undetectable HBV DNA levels in serum, and on exploring novel targets and molecules that are in the pipeline for early clinical trials. The likelihood of achieving a long-lasting functional cure, with no rebound after therapy cessation, is higher using combination therapies targeting different steps in the hepatitis B virus (HBV) replication cycle. Novel treatments and their combinations are discussed for their potential to cure HBV infection, as well as exciting new technologies that could directly target cccDNA and cure without killing the infected cells.
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Affiliation(s)
- Maria G Martinez
- Cancer Research Center of Lyon (CRCL), Lyon, France.,INSERM, U1052, Lyon, France
| | - François Villeret
- Cancer Research Center of Lyon (CRCL), Lyon, France.,INSERM, U1052, Lyon, France
| | - Barbara Testoni
- Cancer Research Center of Lyon (CRCL), Lyon, France.,INSERM, U1052, Lyon, France
| | - Fabien Zoulim
- Cancer Research Center of Lyon (CRCL), Lyon, France.,INSERM, U1052, Lyon, France.,Hospices Civils de Lyon (HCL), Lyon, France.,University of Lyon, UMR_S1052, UCBL, Lyon, France.,Institut Universitaire de France (IUF), Paris, France
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40
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Foca A, Dhillon A, Lahlali T, Lucifora J, Salvetti A, Rivoire M, Lee A, Durantel D. Antiviral activity of PLK1-targeting siRNA delivered by lipid nanoparticles in HBV-infected hepatocytes. Antivir Ther 2020; 25:151-162. [PMID: 32496211 DOI: 10.3851/imp3361] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/25/2020] [Indexed: 10/24/2022]
Abstract
BACKGROUND A link between HBV and PLK1 was clearly evidenced in HBV-driven carcinogenesis, and we have also recently shown that PLK1 is a proviral factor in the early phases of HBV infection. Moreover, we have shown that BI-2536, a small molecule PLK1 inhibitor, was very efficient at inhibiting HBV DNA neosynthesis, notably by affecting nucleocapsid assembly as a result of the modulation of HBc phosphorylation. Yet, as small molecule kinase inhibitors often feature poor selectivity, a more specific and safer strategy to target PLK1 would be needed for a potential development against chronic HBV infections. METHODS Here, we analysed using both freshly isolated primary human hepatocytes and differentiated HepaRG, the anti-HBV properties of an LNP-encapsulated PLK1-targeting siRNA. Standard assays were used to monitor the effect of LNP siPLK1, or controls (LNP siHBV and LNP siNon-targeting), on HBV replication and cell viability. RESULTS A dose as low as 100 ng/ml of LNP-siPLK1 resulted in a >75% decrease in secreted HBV DNA (viral particles), which was comparable to that obtained with LNP siHBV or 10 µM of tenofovir (TFV), without affecting cell viability. Interestingly, and in contrast to that obtained with TFV, a strong inhibition of viral RNA and HBe/HBsAg secretions was also observed under LNP siPLK1 treatment. This correlated with a significant intracellular decrease of vRNA accumulation, which was independent of any change in cccDNA levels, thus suggesting a transcriptional or post-transcriptional modulation. Such an effect was not obtained with a biochemical approach of PLK1 inhibition, suggesting an enzymatic-independent role of PLK1. CONCLUSIONS This study emphasizes that a specific PLK1 inhibition could help in achieving an improved HBsAg loss in CHB patients, likely in combination with other HBsAg-targeting strategies.
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Affiliation(s)
- Adrien Foca
- Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France
- University of Lyon, Université Claude-Bernard (UCBL), UMR_S1052, UCBL, Lyon, France
| | | | - Thomas Lahlali
- Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France
- University of Lyon, Université Claude-Bernard (UCBL), UMR_S1052, UCBL, Lyon, France
| | - Julie Lucifora
- Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France
- University of Lyon, Université Claude-Bernard (UCBL), UMR_S1052, UCBL, Lyon, France
| | - Anna Salvetti
- Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France
- University of Lyon, Université Claude-Bernard (UCBL), UMR_S1052, UCBL, Lyon, France
| | | | - Amy Lee
- Arbutus Biopharma Corporation, Burnaby, BC, Canada
| | - David Durantel
- Cancer Research Center of Lyon (CRCL), INSERM U1052, Lyon, France
- University of Lyon, Université Claude-Bernard (UCBL), UMR_S1052, UCBL, Lyon, France
- Labex DEVweCAN, Lyon, France
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41
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Soriano V, Barreiro P, Cachay E, Kottilil S, Fernandez-Montero JV, de Mendoza C. Advances in hepatitis B therapeutics. Ther Adv Infect Dis 2020; 7:2049936120965027. [PMID: 33117536 PMCID: PMC7570774 DOI: 10.1177/2049936120965027] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Accepted: 09/18/2020] [Indexed: 12/18/2022] Open
Abstract
Despite the availability of both effective preventive vaccines and oral antivirals, over 250 million people are chronically infected with the hepatitis B virus (HBV). Globally, chronic hepatitis B is the leading cause of hepatocellular carcinoma, which represents the third cause of cancer mortality, accounting for nearly 1 million annual deaths. Current oral nucleos(t)ide therapy with tenofovir or entecavir suppresses serum HBV-DNA in most treated patients, but rarely is accompanied by HBsAg loss. Thus, treatment has to be given lifelong to prevent viral rebound. A broad spectrum of antivirals that block the HBV life cycle at different steps are in clinical development, including entry inhibitors, cccDNA disrupters/silencers, translation inhibitors, capsid assembly modulators, polymerase inhibitors and secretion inhibitors. Some of them exhibit higher potency than current oral nucleos(t)ides. Drugs in more advanced stages of clinical development are bulevirtide, JNJ-6379, ABI-H0731, ARO-HBV and REP-2139. To date, only treatment with ARO-HBV and with REP-2139 have resulted in HBsAg loss in a significant proportion of patients. Combination therapies using distinct antivirals and/or immune modulators are expected to maximize treatment benefits. The current goal is to achieve a 'functional cure', with sustained serum HBsAg after drug discontinuation. Ultimately, the goal of HBV therapy will be virus eradication, an achievement that would require the elimination of the cccDNA reservoir within infected hepatocytes.
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Affiliation(s)
- Vicente Soriano
- UNIR Health Sciences School and Medical Center, 28040 Madrid, Porto Velho, Madrid 76801-059, Spain
| | - Pablo Barreiro
- Infectious Diseases Department, La Paz University Hospital, Madrid, Spain
| | - Edward Cachay
- Infectious Diseases Unit, Owen Clinic, University of California, San Diego, CA, USA
| | - Shyamasundaran Kottilil
- Infectious Diseases Department, Institute of Human Virology, University of Maryland, Baltimore, MD, USA
| | | | - Carmen de Mendoza
- Puerta de Hierro University Hospital and Research Institute, Majadahonda, Spain
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42
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Bellizzi A, Ahye N, Jalagadugula G, Wollebo HS. A Broad Application of CRISPR Cas9 in Infectious Diseases of Central Nervous System. J Neuroimmune Pharmacol 2019; 14:578-594. [PMID: 31512166 PMCID: PMC6898781 DOI: 10.1007/s11481-019-09878-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Accepted: 08/26/2019] [Indexed: 12/16/2022]
Abstract
Virus-induced diseases or neurological complications are huge socio-economic burden to human health globally. The complexity of viral-mediated CNS pathology is exacerbated by reemergence of new pathogenic neurotropic viruses of high public relevance. Although the central nervous system is considered as an immune privileged organ and is mainly protected by barrier system, there are a vast majority of neurotropic viruses capable of gaining access and cause diseases. Despite continued growth of the patient population and a number of treatment strategies, there is no successful viral specific therapy available for viral induced CNS diseases. Therefore, there is an urgent need for a clear alternative treatment strategy that can effectively target neurotropic viruses of DNA or RNA genome. To address this need, rapidly growing gene editing technology based on CRISPR/Cas9, provides unprecedented control over viral genome editing and will be an effective, highly specific and versatile tool for targeting CNS viral infection. In this review, we discuss the application of this system to control CNS viral infection and associated neurological disorders and future prospects. Graphical Abstract CRISPR/Cas9 technology as agent control over CNS viral infection.
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Affiliation(s)
- Anna Bellizzi
- Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Room 756 MERB, 3500 N. Broad Street, Philadelphia, PA, 19140, USA
| | - Nicholas Ahye
- Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Room 756 MERB, 3500 N. Broad Street, Philadelphia, PA, 19140, USA
| | - Gauthami Jalagadugula
- Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Room 756 MERB, 3500 N. Broad Street, Philadelphia, PA, 19140, USA
| | - Hassen S Wollebo
- Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Room 756 MERB, 3500 N. Broad Street, Philadelphia, PA, 19140, USA.
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43
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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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44
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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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45
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Zhu A, Liao X, Li S, Zhao H, Chen L, Xu M, Duan X. HBV cccDNA and Its Potential as a Therapeutic Target. J Clin Transl Hepatol 2019; 7:258-262. [PMID: 31608218 PMCID: PMC6783673 DOI: 10.14218/jcth.2018.00054] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Revised: 04/02/2019] [Accepted: 07/10/2019] [Indexed: 12/13/2022] Open
Abstract
Chronic hepatitis B virus infection continues to be a major health burden worldwide. It can cause various degrees of liver damage and is strongly associated with the development of liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA in the nucleus of infected cells cannot be disabled by present therapies which may lead to HBV persistence and relapse. In this review, we summarized the current knowledge on hepatitis B virus covalently closed circular DNA and its potential role as a therapeutic target.
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Affiliation(s)
- Anjing Zhu
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Xinzhong Liao
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Shuang Li
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Hang Zhao
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Limin Chen
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Min Xu
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Xiaoqiong Duan
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
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46
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Bloom K, Kaldine H, Cathomen T, Mussolino C, Ely A, Arbuthnot P. Inhibition of replication of hepatitis B virus using transcriptional repressors that target the viral DNA. BMC Infect Dis 2019; 19:802. [PMID: 31510934 PMCID: PMC6739920 DOI: 10.1186/s12879-019-4436-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2019] [Accepted: 09/03/2019] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Chronic infection with hepatitis B virus (HBV) is a serious global health problem. Persistence of the virus occurs as a result of stability of the replication intermediate comprising covalently closed circular DNA (cccDNA). Development of drugs that are capable of disabling this cccDNA is vital. METHODS To investigate an epigenetic approach to inactivating viral DNA, we engineered transcriptional repressors that comprise an HBV DNA-binding domain of transcription activator like effectors (TALEs) and a fused Krüppel Associated Box (KRAB). These repressor TALEs (rTALEs) targeted the viral surface open reading frame and were placed under transcription control of constitutively active or liver-specific promoters. RESULTS Evaluation in cultured cells and following hydrodynamic injection of mice revealed that the rTALEs significantly inhibited production of markers of HBV replication without evidence of hepatotoxicity. Increased methylation of HBV DNA at CpG island II showed that the rTALEs caused intended epigenetic modification. CONCLUSIONS Epigenetic modification of HBV DNA is a new and effective means of inactivating the virus in vivo. The approach has therapeutic potential and avoids potentially problematic unintended mutagenesis of gene editing.
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Affiliation(s)
- Kristie Bloom
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Private Bag 3, WITS, Johannesburg, 2050, South Africa
| | - Haajira Kaldine
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Private Bag 3, WITS, Johannesburg, 2050, South Africa
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany
- Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany
| | - Abdullah Ely
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Private Bag 3, WITS, Johannesburg, 2050, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Private Bag 3, WITS, Johannesburg, 2050, South Africa.
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47
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Mohd-Ismail NK, Lim Z, Gunaratne J, Tan YJ. Mapping the Interactions of HBV cccDNA with Host Factors. Int J Mol Sci 2019; 20:ijms20174276. [PMID: 31480501 PMCID: PMC6747236 DOI: 10.3390/ijms20174276] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2019] [Revised: 08/26/2019] [Accepted: 08/27/2019] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) infection is a major health problem affecting about 300 million people globally. Although successful administration of a prophylactic vaccine has reduced new infections, a cure for chronic hepatitis B (CHB) is still unavailable. Current anti-HBV therapies slow down disease progression but are not curative as they cannot eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The cccDNA minichromosome persists in the nuclei of infected hepatocytes where it forms the template for all viral transcription. Interactions between host factors and cccDNA are crucial for its formation, stability, and transcriptional activity. Here, we summarize the reported interactions between HBV cccDNA and various host factors and their implications on HBV replication. While the virus hijacks certain cellular processes to complete its life cycle, there are also host factors that restrict HBV infection. Therefore, we review both positive and negative regulation of HBV cccDNA by host factors and the use of small molecule drugs or sequence-specific nucleases to target these interactions or cccDNA directly. We also discuss several reporter-based surrogate systems that mimic cccDNA biology which can be used for drug library screening of cccDNA-targeting compounds as well as identification of cccDNA-related targets.
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Affiliation(s)
- Nur K Mohd-Ismail
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore 117545, Singapore
| | - Zijie Lim
- Department of Medicine, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore 119228, Singapore
| | - Jayantha Gunaratne
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, Singapore
| | - Yee-Joo Tan
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore 117545, Singapore.
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673, Singapore.
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48
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Fanning GC, Zoulim F, Hou J, Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure. Nat Rev Drug Discov 2019; 18:827-844. [PMID: 31455905 DOI: 10.1038/s41573-019-0037-0] [Citation(s) in RCA: 390] [Impact Index Per Article: 65.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/09/2019] [Indexed: 02/06/2023]
Abstract
Chronic hepatitis B virus (HBV) infection is a common cause of liver disease globally, with a disproportionately high burden in South-East Asia. Vaccines and nucleoside or nucleotide drugs are available and reduce both new infection rates and the development of liver disease in HBV-positive persons who adhere to long-term suppressive treatment. Although there is still considerable value in optimizing access to virus-suppressing regimens, the scientific and medical communities have embarked on a concerted journey to identify new antiviral drugs and immune interventions aimed at curing infection. The mechanisms and drug targets being explored are diverse; however, the field universally recognizes the importance of addressing the persistence of episomal covalently closed circular DNA, the existence of integrated HBV DNA in the host genome and the large antigen load, particularly of hepatitis B surface antigen. Another major challenge is to reinvigorate the exhausted immune response within the liver microenvironment. Ultimately, combinations of new drugs will be required to cure infection. Here we critically review the recent literature that describes the rationale for curative therapies and the resulting compounds that are being tested in clinical trials for hepatitis B.
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Affiliation(s)
- Gregory C Fanning
- Janssen Pharmaceuticals, China Research & Development, Shanghai, China.
| | - Fabien Zoulim
- Cancer Research Centre of Lyon, INSERM U1052, Lyon University, Hospices Civils de Lyon, Lyon, France
| | - Jinlin Hou
- Department of Infectious Disease, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Antonio Bertoletti
- Emerging Infectious Diseases Program, Duke-NUS Medical School, Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
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49
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Nucleic acid vaccines for hepatitis B and C virus. INFECTION GENETICS AND EVOLUTION 2019; 75:103968. [PMID: 31325609 DOI: 10.1016/j.meegid.2019.103968] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/02/2019] [Revised: 05/25/2019] [Accepted: 07/15/2019] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections accounts for an important global health problem affecting over 250 million people all around the world. They can cause acute, transient and chronic infections in the human liver. Chronic infection of liver can lead to its failure or cancer. To deal with this problem, alternative approaches or strategies to inhibit these infections have already been started. DNA and mRNA-based vaccination will increase the efficacy and reduce toxicity in patients with Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections. Gene vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development, low-cost manufacture and safe administration. MRNA-based vaccination is a method to elicit potent antigen-specific humoral and cell-mediated immune responses with a superior safety profile compared with DNA vaccines. Exploring the intricacies of these pathways can potentially help the researchers to explore newer vaccines. In this study, DNA and mRNA-based vaccination are introduced as an approach to treat Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections. DNA and mRNA-based vaccines as one of the most successful therapeutics are introduced and the clinical outcomes of their exploitation are explained.
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50
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Martinez MG, Testoni B, Zoulim F. Biological basis for functional cure of chronic hepatitis B. J Viral Hepat 2019; 26:786-794. [PMID: 30803126 DOI: 10.1111/jvh.13090] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Accepted: 02/04/2019] [Indexed: 12/13/2022]
Abstract
Chronic hepatitis B (CHB) infection affects over 250 millon people worldwide and 800000 are expected to die yearly due to the development of hepatocellular carcinoma (HCC). Current antiviral therapies include nucleoside analogs (NAs) that target the viral retrotranscriptase inhibiting de novo viral production. Pegylated interferon (Peg-IFN) is also effective in reducing the viral DNA load in serum. However, both treatments remain limited to control the infection, aiming for viral suppression and improving the quality of life of the infected patients. Complete cure is not possible due to the presence of the stable DNA intermediate covalently closed circular DNA (cccDNA). Attempts to achieve a functional cure are thus ongoing and novel targets and molecules, together with different combination therapies are currently in the pipeline for early clinical trials. In this review we discuss novel treatments both targeting directly and indirectly cccDNA. As we gain knowledge in the Hepatitis B virus (HBV) transcriptional control, and newer technologies emerge that could potentially allow the destruction of cccDNA, exciting new possibilities for curative therapies are discussed.
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Affiliation(s)
- Maria G Martinez
- Cancer Research Center of Lyon (CRCL), Lyon, France.,INSERM U1052, Lyon, France
| | - Barbara Testoni
- Cancer Research Center of Lyon (CRCL), Lyon, France.,INSERM U1052, Lyon, France
| | - Fabien Zoulim
- Cancer Research Center of Lyon (CRCL), Lyon, France.,INSERM U1052, Lyon, France.,Hospices Civils de Lyon (HCL), Lyon, France.,University of Lyon, UMR_S1052, UCBL, Lyon, France.,Institut Universitaire de France (IUF), Paris, France
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