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Editing of SIV in NonHuman Primates by CRISPR-CAS9 in Viral Reservoirs

March 11, 2020

by: Jennifer Gordon, Tricia H. Burdo, Pietro Mancuso, Chen Chen, Rafal Kaminski, Mark Lewis, Kamel Khalili, Temple University School of Medicine

Antiretroviral therapy (ART) suppresses but does not eliminate replication competent HIV proviral DNA from latently infected cells, thus resulting in viral reactivation upon ART cessation. Therefore, removal of HIV proviral DNA from infected individuals is needed. We have assessed a CRISPR-Cas9 based gene editing strategy for the elimination of SIV proviral DNA in the rhesus macaque model.

An all-in-one AAV9 gene therapy vector was constructed to deliver CRISPR-Cas9 plus two gRNAs targeting sequences within the 5' and 3' viral LTRs and the Gag gene to excise the intervening proviral DNA fragment. Ten adult Indian rhesus macaques were i.v. infected with SIVmac239 then treated daily with a drug regimen of tenofovir, emtricitabine and dolutegravir (5.1/50/2.5mg/kg daily s.q.). Animals were randomized into groups to receive low versus high dose of AAV9-CRISPR-Cas9 in a single i.v. infusion (low dose: 1.4x10^12 GC/kg n=4; high dose: 1.4x10^13 GC/kg n=3) as well as control SIV infected animals (n=3). Longitudinal blood samples and lymph node biopsies were collected, and animals were necropsied at 3 (n=8) or 6 months (n=2) after CRISPR treatment.

SIV-infected animals treated with AAV9-CRISPR-Cas9 at both high and low doses showed in vivo excision of viral DNA from serial blood samples. Results from Sanger sequencing confirmed the precise breakpoint of the viral DNA in samples in which excision was detected. Biodistribution of the AAV9-CRISPR-Cas9 vector was assessed by PCR to detect the presence of the Cas9 gene sequence. DNA scope was performed on lymph nodes and spleen to detect the AAV9-CRISPR-Cas9 viral vector and expression of the Cas9 gene. Broad excision of SIV proviral DNA was observed in blood, lymph nodes, and other tissues known to be viral reservoirs including spleen, gut, and brain. A dose response between low and high doses, as well as temporal distribution between 3 and 6 months, was observed for AAV9-CRISPR-Cas9 viral DNA in the blood.

Here we demonstrate broad SIV DNA excision in viral reservoirs leading to permanent inactivation of SIV proviral DNA in a one shot CRISPR molecule. We observed biodistribution of AAV9-CRISPR-Cas9 in the blood in a dose and time dependent manner for the elimination of SIV DNA. These findings support the utilization of AAV9-CRISPR-Cas9 as a potential therapeutic strategy for in vivo gene editing of HIV proviral DNA from latent issue reservoirs.

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