Gene editing and elimination of latent herpes simplex virus in vivo

Aubert, Martine; Strongin, Daniel E.; Roychoudhury, Pavitra; Loprieno, Michelle A.; Haick, Anoria K.; Klouser, Lindsay M.; Stensland, Laurence; Huang, Meei‐Li; Makhsous, Negar; Tait, Alexander N.; Feelixge, Harshana S. De Silva; Galetto, Román; Duchâteau, Philippe; Greninger, Alexander L.; Stone, Daniel; Jerome, Keith R.

doi:10.1038/s41467-020-17936-5

Cited by 62 publications

(86 citation statements)

References 51 publications

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“…This led to detectable excision of ~2.5 kb of viral DNA and to nearly complete abolition of viral release. These findings are aligned with reports that ~10% and 39-63% editing efficiencies from multiplex endonuclease treatments resulted in more than 90% knockdown in herpes simplex virus and HIV-1, respectively (23,38). Dysregulation of positive feedback loops, as in the case of tat/rev-directed CRISPR-Cas9, and the formation of dominant negative proteins encoded after gene editing may explain the disproportionately high efficacy in each of these cases.…”

Section: Discussionsupporting

confidence: 86%

Exonic Disruption Facilitates Antiviral CRISPR-Cas9 Activity for Multistrain HIV-1 Elimination

Herskovitz¹,

Hasan

Patel³

et al. 2021

Preprint

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A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high mutation rate of HIV-1 gives rise to numerous circulating strains with increased capacity for immune evasion and antiretroviral drug resistance. To facilitate viral elimination while accounting for this diversity, we propose genetic inactivation of proviral DNA with CRISPR-spCas9. We designed a library of “mosaic gRNAs” against a HIV-1 consensus sequence constructed from 4004 clinical strains, targeting the viral transcriptional regulator tat. Testing in 7 HIV-1 transmitted founder strains led, on average, to viral reductions of 82% with tandem TatD and TatE (TatDE) treatment. No off-target cleavages were recorded. Lentiviral transduction of TatDE attenuated latency reversal by 94% in HIV-infected, transcriptionally silent ACH2 T cells. In all, TatDE guide RNAs successfully disrupted 5 separate HIV-1 exons (tat1-2/rev1-2/gp41) providing a pathway for CRISPR-directed HIV-1 cure therapies.Significance StatementOver 38 million individuals worldwide are infected with HIV-1, which necessitates lifelong dependence on antiretroviral therapy (ART) to prevent viral replication that leads to AIDS. Efforts to rid hosts of HIV-1 are limited by the virus’ abilities to integrate proviral DNA in nuclei, mutate their genomes, and lay dormant for decades during ART treatment. We developed mosaic guide RNAs, TatD and TatE, for CRISPR-Cas9 that recognize the majority of known HIV-1 strains and inactivate 94% of proviral DNA in latently infected cells. Tandem TatDE-CRISPR inactivation of 5 viral exons (tat1-2, rev1-2, and gp41), which blocked HIV-1 replication for 28 days in CD4+ T cells without unwanted editing to the host genome, may serve as a viable strategy for HIV cure.

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Section: Discussionsupporting

confidence: 86%

Exonic Disruption Facilitates Antiviral CRISPR-Cas9 Activity for Multistrain HIV-1 Elimination

Herskovitz¹,

Hasan

Patel³

et al. 2021

Preprint

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“…In our own work with latent HSV infection in mice, we have observed that simultaneous targeting of two sites in the viral genome leads to elimination of >90% of latent HSV, while indel frequencies in the virus remaining after therapy are typically only 5%–10%. 76 In a surrogate in vitro assay in which an episomal template was cleaved by one to three independent sgRNAs, we did not see evidence of template degradation in either HepG2 or Huh7 cells ( Figure S9 ), but these HepG2 and Huh7 models do not contain cccDNA, and 72 h may not be a sufficient period of time for degradation to occur, so the significance of this finding is unclear. It therefore remains unclear whether degradation of Cas9-linearized cccDNA is responsible for the reduction in cccDNA levels.…”

Section: Discussionmentioning

confidence: 82%

CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

Stone

Long²,

Loprieno

et al. 2021

Molecular Therapy - Methods & Clinical Development

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Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR- Staphylococcus aureus ( Sa )Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV- Sa Cas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of Sa Cas9 delivery to HBV + human hepatocytes than those without gene editing. HBV-specific AAV- Sa Cas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.

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“…To investigate if Cas9/gRNA targeting affects HSV-1 infection in vivo, Jerome and colleagues used adenovirus associated virus (AAV)-based vectors to deliver Cas9 and gRNA expression cassettes in a mouse model of HSV-1 ocular infection [ 26 ]. SaCas9 and gRNAs targeting UL30 and UL54 were used.…”

Section: Development Of Crispr/cas9-based Technologies Against Hsvmentioning

confidence: 99%

“…SaCas9 and gRNAs targeting UL30 and UL54 were used. Several gRNAs were able to promote substantial levels of gene editing on HSV-1 genomes in latently-infected cultured neurons transduced by SaCas9/gRNA-expressing AAV vectors [ 26 ].…”

Section: Development Of Crispr/cas9-based Technologies Against Hsvmentioning

confidence: 99%

“…In mouse experiments, latent HSV-1 infection was established in mice through ocular infection, followed by delivery of AAV vectors carrying SaCas9 and gRNA expressions cassettes. Trigeminal ganglia (TG) neurons were isolated and analyzed [ 26 ]. Consistent with their results with single HE treatment, PCR quantification showed similar HSV-1 levels in the neurons of treated and control animals [ 26 , 27 ].…”

Section: Development Of Crispr/cas9-based Technologies Against Hsvmentioning

confidence: 99%

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Development of Genome Editing Approaches against Herpes Simplex Virus Infections

Zhang

Hsiao

Liu

2021

Viruses

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Herpes simplex virus 1 (HSV-1) is a herpesvirus that may cause cold sores or keratitis in healthy or immunocompetent individuals, but can lead to severe and potentially life-threatening complications in immune-immature individuals, such as neonates or immune-compromised patients. Like all other herpesviruses, HSV-1 can engage in lytic infection as well as establish latent infection. Current anti-HSV-1 therapies effectively block viral replication and infection. However, they have little effect on viral latency and cannot completely eliminate viral infection. These issues, along with the emergence of drug-resistant viral strains, pose a need to develop new compounds and novel strategies for the treatment of HSV-1 infection. Genome editing methods represent a promising approach against viral infection by modifying or destroying the genetic material of human viruses. These editing methods include homing endonucleases (HE) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) RNA-guided nuclease system. Recent studies have showed that both HE and CRISPR/Cas systems are effective in inhibiting HSV-1 infection in cultured cells in vitro and in mice in vivo. This review, which focuses on recently published progress, suggests that genome editing approaches could be used for eliminating HSV-1 latent and lytic infection and for treating HSV-1 associated diseases.

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Gene editing and elimination of latent herpes simplex virus in vivo

Cited by 62 publications

References 51 publications

Exonic Disruption Facilitates Antiviral CRISPR-Cas9 Activity for Multistrain HIV-1 Elimination

Exonic Disruption Facilitates Antiviral CRISPR-Cas9 Activity for Multistrain HIV-1 Elimination

CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

Development of Genome Editing Approaches against Herpes Simplex Virus Infections

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