Silencing integrated SIV proviral DNA with TAR‐specific CRISPR tools

Smith, Lisa M.; Hodara, Vida; Parodi, Laura M.; Callery, Jessica; Giavedoni, Luis D.

doi:10.1111/jmp.12494

Cited by 2 publications

(1 citation statement)

References 28 publications

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“…These positive results prompted a clinical trial, which is currently ongoing (NCT05144386). Several studies have evaluated the applicability of catalytically inactive Cas9 to reactivate or silence HIV and SIV transcription [114,[121][122][123][124][125][126][127][128], but all these studies are still in the pre-clinical stage.…”

Section: Genetic and Epigenetic Modulation To Impact Hiv Transcriptionmentioning

confidence: 99%

Targeting Viral Transcription for HIV Cure Strategies

Izquierdo-Pujol,

Puertas,

Martinez-Picado

et al. 2024

Microorganisms

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Combination antiretroviral therapy (ART) suppresses viral replication to undetectable levels, reduces mortality and morbidity, and improves the quality of life of people living with HIV (PWH). However, ART cannot cure HIV infection because it is unable to eliminate latently infected cells. HIV latency may be regulated by different HIV transcription mechanisms, such as blocks to initiation, elongation, and post-transcriptional processes. Several latency-reversing (LRA) and -promoting agents (LPA) have been investigated in clinical trials aiming to eliminate or reduce the HIV reservoir. However, none of these trials has shown a conclusive impact on the HIV reservoir. Here, we review the cellular and viral factors that regulate HIV-1 transcription, the potential pharmacological targets and genetic and epigenetic editing techniques that have been or might be evaluated to disrupt HIV-1 latency, the role of miRNA in post-transcriptional regulation of HIV-1, and the differences between the mechanisms regulating HIV-1 and HIV-2 expression.

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Section: Genetic and Epigenetic Modulation To Impact Hiv Transcriptionmentioning

confidence: 99%

Targeting Viral Transcription for HIV Cure Strategies

Izquierdo-Pujol,

Puertas,

Martinez-Picado

et al. 2024

Microorganisms

View full text Add to dashboard Cite

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Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA

Smith

Ladner

Hodara

et al. 2021

J Virol

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Human and simian immunodeficiency virus infections establish lifelong reservoir of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactive integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells co-transfected with SIV provirus and plasmids coding for RGNus identified SIV LTR, TAR, and RSS regions as the most effective at virus suppression; RGNi targeting these same regions inhibited virus production significantly. Multiplex plasmids that co-expressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations making these tools a desirable prospect for moving into animal studies. Importance: Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease, or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole genome sequencing demonstrated effective SIV sequence mutation and inactivation, and absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.

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Silencing integrated SIV proviral DNA with TAR‐specific CRISPR tools

Cited by 2 publications

References 28 publications

Targeting Viral Transcription for HIV Cure Strategies

Targeting Viral Transcription for HIV Cure Strategies

Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA

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