Gene Editing Approaches against Viral Infections and Strategy to Prevent Occurrence of Viral Escape

White, Martyn K.; Hu, Wenhui; Khalili, Kamel

doi:10.1371/journal.ppat.1005953

Cited by 18 publications

(19 citation statements)

References 89 publications

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“…This is based both on known species restrictions for HIV-1 infection and long-term establishment of tissue reservoirs of infection. Human hematopoietic stem cells (HSC) reconstituted NOD.Cg-Prkdc scid Il2rgt m1Wjl /SzJ (NSG) mice produce human T cells, that are broadly susceptible to HIV-1 infection 23–30 . The model permits evaluation of long-term viral infection in blood and tissues and ART-induced HIV-1 latency.…”

Section: Resultsmentioning

confidence: 99%

Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice

et al. 2019

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Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.

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Section: Resultsmentioning

confidence: 99%

Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice

et al. 2019

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“…As described previously, 10 , 12 the use of two gRNA configurations to excise large fragments of HIV-1 DNA mitigates the emergence of mutant virus with a small nucleotide indel mutation that becomes immune from CRISPR/Cas9. 16 , 18 It is also important to note that the use of two or more gRNAs, in addition to the excision of the intervening DNA fragments, more frequently introduces an indel mutation at each of the target sites and leads to the inactivation of the target gene. 16 During the past several years, we have been able to optimize our editing strategy, and developing pairs of gRNAs significantly enhanced the frequency of viral DNA excision by a multiplex of gRNAs.…”

Section: Resultsmentioning

confidence: 99%

“… 15 , 16 This concern, however, can be avoided by targeting multiple sites within the viral genome to excise large segments of viral DNA, resulting in a permanent inactivation of viral replication in the infected cells. 17 , 18 …”

Section: Introductionmentioning

confidence: 99%

Removal of HIV DNA by CRISPR from Patient Blood Engrafts in Humanized Mice

Bella

Kaminski

Mancuso

et al. 2018

Molecular Therapy - Nucleic Acids

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We used NOD/SCID mice, also known as NRG, to assess the ability of lentivirus-mediated intravenous delivery of CRISPR in editing the HIV-1 genome from the circulating PBMC engrafts, some of which homed within several animal solid tissues. Lentivirus-mediated delivery of a multiplex of guide RNAs accompanied by Cas9 endonuclease led to the excision of the targeted region of the viral genome positioned within the HIV-1 LTR from the in-vitro-infected human peripheral blood mononuclear cells (PBMCs) embedded in the spleens of NRG mice. Similarly, the treatment of NRG mice harboring PBMC engrafts derived from HIV-1-positive patients with the therapeutic lentivirus eliminated the presence of the viral DNA fragment in the blood, as well as in the spleen, lung, and liver, of the engrafted animals. Sanger sequence analysis of the viral DNA after treatment with the lentiviral vectors expressing Cas9 and gRNAs verified the editing and removal of the proviral DNA fragment from the viral genome at the predicted sites. This proof-of-concept study, for the first time, demonstrates successful excision of the HIV-1 proviral DNA from patient immune cell engrafts in humanized mice upon treatment with lentivirus-expressing CRISPR and causes a decline in the level of replication-competent virus.

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“…The occurrence of viral escape from CRISPR Cas9‐mediated inhibition may limit the use of Cas9/sgRNA in virus control or therapy . Combinations of two strong sgRNAs targeting different regions of the viral genome, or RNAi and CRISPR Cas9 can completely abrogate viral replication and prevent viral escape . Therefore, research into the combination of 2 strong sgRNAs targeting different regions of the PRV genome is ongoing.…”

Section: Resultsmentioning

confidence: 99%

Subculturing cells have no effect on CRISPR/Cas9‐mediated cleavage of UL30 gene in pseudorabies virus

Ren

Peng

Ouyang

et al. 2018

Anim Models and Exp Med

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CRISPR /Cas9‐mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA . Unexpectedly, we found previously that pseudorabies virus ( PRV ) could escape from CRISPR /Cas9‐mediated inhibition. In order to elucidate whether the escape of PRV from Cas9‐mediated inhibition was due to cell deficiencies, such as genetic instability of sg RNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sg RNA ‐expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9‐mediated cleavage of PRV . Different passages of PX 459‐ PRV cells can stably express sg RNA to facilitate Cas9/sg RNA cleavage on the UL 30 gene of PRV , resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

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Gene Editing Approaches against Viral Infections and Strategy to Prevent Occurrence of Viral Escape

Cited by 18 publications

References 89 publications

Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice

Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice

Removal of HIV DNA by CRISPR from Patient Blood Engrafts in Humanized Mice

Subculturing cells have no effect on CRISPR/Cas9‐mediated cleavage of UL30 gene in pseudorabies virus

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