CRISPR-Cas9 after LASER ART definitively eliminates HIV in infected humanized mice


  • Daniela Ovadia — Agenzia Zoe
  • Medical News
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Key messages

  • In HIV-1 infected humanised mice, treatment with sequential long-acting slow-effective release antiretroviral therapy (LASER ART) and CRISPR-Cas9 is able to obtain viral clearance in latent infectious reservoirs.
  • The virus is not detected in blood and tissues in a third of the dual treated animals, whereas HIV-1 is readily detected in mice which receive only one treatment.
  • Results provide proof-of-concept that permanent viral elimination is possible and suggest that to achieve optimal viral editing by CRISPR-Cas9, maximal viral restriction must be first established.

 

ART restricts HIV-1 infection but, as known, fails to eliminate integrated copies of proviral DNA from the host genome, and viral reactivation occurs when therapy is discontinued. LASER ART facilitates sustained inhibition of viral replication. Indeed, with effective drug concentrations in blood and tissues, viral reservoirs can be maintained for weeks thanks to long-acting hydrophobic lipophilic antiretroviral nanoparticles. However, LASER ART alone cannot rid the infected host of latent HIV-1. The authors examined whether this treatment, combined with a CRISP-Cas9 based gene-editing technology, can foster viral elimination.

Human haematopoietic stem cells reconstituted NSG mice (n=33), able to produce human T cells susceptible to HIV infection, were infected with HIV-1 for 2 weeks and viral infection was confirmed in 4 sacrificed animals. The remaining 29 were divided into 4 groups: untreated group (n=6, HIV-1 control); single intravenous injection of AAV9-CRISPR-Cas9 (n=6) 9 weeks after viral infection; intramuscular injection of LASER ART (n=10) 2 weeks after viral infection; LASER ART treatment followed by AAV9-CRISPR-Cas9 (n=7) 3 weeks after last LASER ART.

Evaluation of plasma viral load indicated that after administration of AAV9-CRISPR-Cas9, 2 of 7 mice showed no evidence for viral rebound at 14 weeks.

Viral DNA and RNA were not detected in the plasma, lymphoid tissue, bone marrow or brain (nested and digital-droplet PCR and RNAscope tests) of either mouse.

No CRISPR-Cas9 mediated off-target effects were detected.

HIV-1 was readily detected following sole LASER ART or CRISPR-Cas9 treatment.

Authors validated the eradication of HIV-1 infection, with two independent replicate experiments in separate sets of mice. Altogether, results revealed that a third or more of the animals that received sequential LASER ART and CRISPR-Cas9 therapy became virus free.

Finally, human immunocytes from dual treated mice were used in adoptive transfer in virus-free animals, which failed to produce infectious progeny virus.

Data support the idea that maximal viral restriction must be first established prior to excision to achieve optimal viral editing by CRISPR-Cas9.

Although further studies are needed, these proof-of-concept results offer readily defined and realistic pathways toward strategies for HIV-1 elimination.