Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9

Kostyushev, Dmitry; Kostyusheva, Anastasiya; Brezgin, Sergey; Ponomareva, N. I.; Zakirova, Natalia F.; Egorshina, Aleksandra; Yanvarev, Dmitry V.; Bayurova, Ekaterina; Sudina, Anna; Goptar, Irina A.; Nikiforova, A. V.; Dunaeva, E. A.; Лисица, Т. С.; Abramov, I. V.; Frolova, Anastasiia; Lukashev, Alexander N.; Gordeychuk, Ilya; Zamyatnin, Andrey A.; Ivanov, Alexander R.; Chulanov, Vladimir

doi:10.1016/j.omtn.2023.02.001

Cited by 17 publications

(5 citation statements)

References 45 publications

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“…Because cccDNA formation in this system can only result from nuclear import of capsids, this demonstrates that the intracellular recycling pathway of rcDNA-containing capsids is a driver of cccDNA replenishment [ 35 ]. Recently, Kostyushev et al found that CRISPR-Cas9-mediated inactivation of HBV cccDNA is not sufficient for curing infection, because HBV replication rapidly rebounds in their system due to formation of HBV cccDNA from recycling rcDNA [ 55 ].…”

Section: Hbv Genome Recycling and Cccdna Replenishmentmentioning

confidence: 99%

Dynamics of Hepatitis B Virus Covalently Closed Circular DNA: A Mini-Review

Huang

2023

Microorganisms

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Eradication of cccDNA is an ideal goal of chronic hepatitis B (CHB) therapy. Understanding the changes in the cccDNA pool during therapy provides a basis for developing CHB treatment strategies. On the other hand, the shift in the balance of the cccDNA pool following therapies allowed researchers to investigate the dynamics of cccDNA. Central to the description of cccDNA dynamics is a parameter called cccDNA half-life. CccDNA half-life is not an intrinsic property of cccDNA molecules, but a description of an observed phenomenon characterized by cccDNA pool decline. Since cccDNA has to be in the nuclei of host cells to function, the half-life of cccDNA is determined by the state and destiny of the host cells. The major factors that drive cccDNA decay include noncytopathic effects and hepatocyte turnover (death and division). In some cases, the determining factor is not the half-life of cccDNA itself, but rather the half-life of the hepatocyte. The main purpose of this review is to analyze the major factors affecting cccDNA half-life and determine the areas requiring further study. In addition, the discrepancy in cccDNA half-life between short-term and long-term nucleot(s)ide analog (NUC) therapy was reported. Hypotheses were proposed to explain the multi-phasic decline of cccDNA during NUC therapy, and a framework based on cccDNA dynamics was suggested for the consideration of various anti-HBV strategies.

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Section: Hbv Genome Recycling and Cccdna Replenishmentmentioning

confidence: 99%

Dynamics of Hepatitis B Virus Covalently Closed Circular DNA: A Mini-Review

Huang

2023

Microorganisms

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“…HBV replication rapidly rebounds after CRISPR-Cas9 ribonucleoprotein (RNP) degradation, and the HBV cccDNA pool is reestablished due to de novo formation of HBV cccDNA from rcDNA. However, depleting HBV rcDNA in infected cells with lamivudine prevents cccDNA re-establishment and promotes the resolution of HBV infection via a single dose of CRISPR-Cas9 RNPs [114]. This suggests that blocking HBV cccDNA replenishment and re-establishment from HBV rcDNA conversion is critical for completely eliminating HBV from the infected cells.…”

Section: Discussionmentioning

confidence: 99%

Current Status and Challenges in Anti-Hepatitis B Virus Agents Based on Inactivation/Inhibition or Elimination of Hepatitis B Virus Covalently Closed Circular DNA

Zhuang,

Chen,

Chen

et al. 2023

Viruses

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There has been over half a century since the discovery of hepatitis B virus (HBV) to now, but approximately 300 million patients with chronic hepatitis B (CHB) still live in the world, resulting in about one million deaths every year. Although currently approved antivirals (e.g., nucleoside analogues) are effective at reducing HBV replication, they have almost no impact on the existing HBV covalently closed circular DNA (cccDNA) reservoir. HBV cccDNA is a critical obstacle to the complete elimination of the virus via antiviral therapy. The true cure of HBV infection requires the eradication of viral cccDNA from HBV-infected cells; thus, the development of new agents directly or indirectly targeting HBV cccDNA is urgently needed due to the limitations of current available drugs against HBV infection. In this regard, it is the major focus of current anti-HBV research worldwide via different mechanisms to either inactivate/inhibit (functional cure) or eliminate (complete cure) HBV cccDNA. Therefore, this review discussed and summarized recent advances and challenges in efforts to inactivate/silence or eliminate viral cccDNA using anti-HBV agents from different sources, such as small molecules (including epigenetic drugs) and polypeptides/proteins, and siRNA or gene-editing approaches targeting/attenuating HBV cccDNA via different mechanisms, as well as future directions that may be considered in efforts to truly cure chronic HBV infection. In conclusion, no breakthrough has been made yet in attenuating HBV cccDNA, although a number of candidates have advanced into the phase of clinical trials. Furthermore, the overwhelming majority of the candidates function to indirectly target HBV cccDNA. No outstanding candidate directly targets HBV cccDNA. Relatively speaking, CCC_R08 and nitazoxanide may be some of the most promising agents to clear HBV infection in small molecule compounds. Additionally, CRISPR-Cas9 systems can directly target HBV cccDNA for decay and demonstrate significant anti-HBV activity. Consequently, gene-editing approaches targeting HBV cccDNA may be one of the most promising means to achieve the core goal of anti-HBV therapeutic strategies. In short, more basic studies on HBV infection need to be carried out to overcome these challenges.

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“…The methylation of cccDNA can produce heterochromatic patches, reducing the accessibility of chromatin to the targeted CRISPR/Cas9. The experimental data indicates that cccDNA methylation can significantly decrease the enzymatic activity of Cas9, but this effect is overcome by increasing the ratio of the CRISPR/Cas9 complex to the target cccDNA [ 46 ].…”

Section: Hepatitis B Virusmentioning

confidence: 99%

CRISPR/Cas9 as a New Antiviral Strategy for Treating Hepatitis Viral Infections

Bartosh,

Dome,

Zhukova

et al. 2023

IJMS

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Hepatitis is an inflammatory liver disease primarily caused by hepatitis A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV) viruses. The chronic forms of hepatitis resulting from HBV and HCV infections can progress to cirrhosis or hepatocellular carcinoma (HCC), while acute hepatitis can lead to acute liver failure, sometimes resulting in fatality. Viral hepatitis was responsible for over 1 million reported deaths annually. The treatment of hepatitis caused by viral infections currently involves the use of interferon-α (IFN-α), nucleoside inhibitors, and reverse transcriptase inhibitors (for HBV). However, these methods do not always lead to a complete cure for viral infections, and chronic forms of the disease pose significant treatment challenges. These facts underscore the urgent need to explore novel drug developments for the treatment of viral hepatitis. The discovery of the CRISPR/Cas9 system and the subsequent development of various modifications of this system have represented a groundbreaking advance in the quest for innovative strategies in the treatment of viral infections. This technology enables the targeted disruption of specific regions of the genome of infectious agents or the direct manipulation of cellular factors involved in viral replication by introducing a double-strand DNA break, which is targeted by guide RNA (spacer). This review provides a comprehensive summary of our current knowledge regarding the application of the CRISPR/Cas system in the regulation of viral infections caused by HAV, HBV, and HCV. It also highlights new strategies for drug development aimed at addressing both acute and chronic forms of viral hepatitis.

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Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9

Cited by 17 publications

References 45 publications

Dynamics of Hepatitis B Virus Covalently Closed Circular DNA: A Mini-Review

Dynamics of Hepatitis B Virus Covalently Closed Circular DNA: A Mini-Review

Current Status and Challenges in Anti-Hepatitis B Virus Agents Based on Inactivation/Inhibition or Elimination of Hepatitis B Virus Covalently Closed Circular DNA

CRISPR/Cas9 as a New Antiviral Strategy for Treating Hepatitis Viral Infections

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