Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

Murai, Kazuhiro; Kodama, Tatsuhiko; Shimoda, Akiyoshi; Fukuoka, Masayoshi; Fukutomi, Keisuke; Shigeno, Satoshi; Shiode, Yuto; Motooka, Daisuke; Higuchi, Yuichiro; Miyakawa, Kei; Suemizu, Hiroshi; Ryo, Akihide; Tahata, Yuki; Makino, Yuki; Yamada, Ryotaro; Sakamori, Ryotaro; Tatsumi, Tomohide; Takehara, Tetsuo

doi:10.1002/hep4.2014

Cited by 7 publications

(6 citation statements)

References 37 publications

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“…For KAT2B western, NE-PER Nuclear and Cytoplasmic extraction reagents (ThermoFisher Scientific) were used to enrich for nuclear proteins. PVDF membranes were incubated overnight with 1/1000 Cas9 (#14697, Cell Signalling) 18 , 1/2000 B-Actin (#ab8227, Abcam) 19 , 1/10,000 GAPDH (Abcam, ab128915) 20 , 1/100 KAT2B (#sc-13124, Santa Cruz Biotechnology) 21 , followed by 1 h incubation with HRP-conjugated secondary antibodies. Membranes were developed using SuperSignal West Pico PLUS Chemiluminescent substrate (ThermoFisher Scientific) and bands detected using iBright CL1000 (Invitrogen).…”

Section: Crispr-cas9 Screenmentioning

confidence: 99%

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

Pavlou

Foskolou

Patikas

et al. 2023

Sci Rep

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Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.

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Section: Crispr-cas9 Screenmentioning

confidence: 99%

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

Pavlou

Foskolou

Patikas

et al. 2023

Sci Rep

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“…The inhibition of the nonhomologous end joining (NHEJ)-mediated DNA repair machinery promoted the effect of CRISPR targeting HBV cccDNA. The combination of olaparib and CRISPR may present a therapeutic strategy for HBV infection [51].…”

Section: Olaparibmentioning

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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“…Even if the choice of the repair pathway following CRISPR/Cas DSBs still remains an ill-understood mechanism, it is reasonable to think that NHEJ would be preferentially activated in non-dividing cells, such as hepatocytes. In this respect, the manipulation of the NHEJ system yielded discordant results regarding the capacity for improving cccDNA degradation following CRISPR/Cas9 [108,109], proving the difficulty of modulating or controlling the effects of these complex and redundant DNA damagerepair pathways. The use of different cell culture models with respect to differentiation status and proliferative capacity has, therefore, to be taken into account when comparing results across studies.…”

Section: Permanent Modifications In Cccdna Sequencesmentioning

confidence: 99%

Gene Editing Technologies to Target HBV cccDNA

Martínez

Smekalova

Combe

et al. 2022

Viruses

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Hepatitis B virus (HBV) remains a significant cause of mortality and morbidity worldwide, since chronic HBV infection is associated with elevated risk of cirrhosis and hepatocellular carcinoma. Current licensed therapies against HBV efficiently suppress viral replication; however, they do not have significant effects on the intrahepatic covalently closed circular DNA (cccDNA) of the viral minichromosome responsible for viral persistence. Thus, life-long treatment is required to avoid viral rebound. There is a significant need for novel therapies that can reduce, silence or eradicate cccDNA, thus preventing HBV reemergence after treatment withdrawal. In this review, we discuss the latest developments and applications of gene editing and related approaches for directly targeting HBV DNA and, more specifically, cccDNA in infected hepatocytes.

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Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

Cited by 7 publications

References 37 publications

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

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

Gene Editing Technologies to Target HBV cccDNA

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