CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: 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 tissue reservoirs. 350LB EFFICIENT DELETION OF CCR5 PROVIDES COMPLETE PROTECTION AGAINST HIV IN XENOGRAFT MICE Daniel Claiborne 1 , Christian L. Boutwell 1 , Zachary Detwiler 2 , Tao Chen 1 , Radiana Trifonova 1 , David T. Scadden 3 , Tony W. Ho 2 , Todd M. Allen 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 CRISPR Therapeutics, Cambridge, MA, USA, 3 Harvard University, Cambridge, MA, USA Background: Hematopoietic stem cell transplant (HSCT) with CCR5d32/d32 defective stem cells has resulted in long-term remission of HIV infection in three patients (“Berlin”, “Dusseldorf”, and “Oxford”) that received allogeneic HSCT for co-occurring malignancies. However, the scarcity of HLA-matched, CCR5d32 homozygous stem cell donors represents a significant hurdle to more widespread adoption of HSCT for treatment of HIV infection. The ability to effectively delete CCR5 in autologous, mobilized, CD34+ hematopoietic stem progenitor cells (mobHSPCs) would overcome this hurdle and provide a path toward an autologous HSCT cure for HIV infection. Methods: Guides were screened for editing efficiency by CRISPR/Cas9 ribonucleoprotein (RNP) nucleofection of primary human CD4+ T cells. Edited CD4+ T cells were then stimulated and challenged with R5-tropic HIV. A dual guide approach engendered the highest level of CCR5 editing and complete protection from high titer HIV challenge in vitro and was selected for HSCPC editing and transplant. Results: Dual guides achieved a 92% CCR5 editing frequency in mobHSPCs from an anonymous HIV-negative donor (guide 1: 70%; guide 2: 58%; total: 92%). After transplant into NSG mice, CCR5edit HSPCs displayed slightly delayed but otherwise normal hematopoiesis resulting in human immune cell reconstitution with frequencies of human monocytes, B cells, and T cells comparable to the control sham (GFP guide) edited mice. High frequency CCR5 editing was detected in descendant monocytes, B cells, and T cells (median 89%), and the frequency of circulating T cells expressing CCR5 on the cell surface was <0.25% compared to 57% in the sham edited controls. Importantly, CCR5edit mice were completely refractory to challenge with an ID100 of a CCR5-tropic HIV (0/5 CCR5edit mice infected) that infected 8/8 control mice. CCR5edit mice further resisted a challenge dose of 50x ID100. In contrast, subsequent intraperitoneal challenge of a CCR5edit mouse with a CXCR4-tropic HIV strain resulted in robust infection and plasma viremia confirming CCR5-specific protection. Conclusion: These data demonstrate that high frequency CRISPR/Cas9- mediated editing of CCR5 in human HSPCs is achievable and is sufficient to prevent infection during multiple, high dose exposures to a highly pathogenic strain of HIV. These experiments provide the basis to explore the prevention of systemic HIV rebound in an autologous transplant setting to help guide future clinical approaches to achieve a functional cure. 351 ANTAGONISM OF PPARG FOR TH17 MUCOSAL IMMUNITY RESTORATION AND HIV-RESERVOIR PURGING Delphine Planas 1 , Yuwei Zhang 1 , Augustine Fert 1 , Julia M. Ruiz 1 , Jean-Phillipe Goulet 2 , Laurence Raymond Marchand 1 , Huicheng Chen 1 , Tomas Raul Wiche Salinas 1 , Annie Gosselin 1 , Wilfried-Wenceslas Bazie 3 , Caroline Gilbert 3 , Eric A. Cohen 4 , Jean-Pierre Routy 5 , Nicolas Chomont 1 , Petronela Ancuta 1 1 Centre de Recherche du CHUM, Montreal, QC, Canada, 2 Caprion Biosciences, Montreal, QC, Canada, 3 Laval University, Quebec City, QC, Canada, 4 Université de Montréal, Montreal, QC, Canada, 5 McGill University Health Centre, Glen site, Montreal, QC, Canada Background: The Th17-polarized CCR6+RORγt+CD4+T-cells are key players in mucosal homeostasis. These cells are preferential targets for HIV/SIV infection at mucosal sites and their depletion/functional alteration persist despite viral-suppressive antiretroviral therapy (ART) in people living with HIV (PLWH). Moreover, Th17 cells carrying replication-competent HIV persist during long-term ART. Therefore, novel Th17-targeted HIV remission/cure strategies are needed. Considering that PPARγ represses RORγt, Th17-specific master regulator and HIV transcription, we hypothesized that PPARγ pharmacological inhibition will enhance Th17-effector functions and facilitate HIV reactivation from latency.

Methods: PBMC from ART-treated PLWH (n=14; CD4 counts >300 cells/µl, plasma viral load <40 HIV-RNA copies/ml) and HIV- (n=8) were used to isolate total/CCR6+/CCR6- memory CD4+T-cells by magnetic and flow cytometry sorting. Cells from HIV- were stimulated via CD3/CD28 for 3 days, exposed to transmitted founder HIVTHRO and cultured in the presence/absence of the PPARγ antagonist T0070907 for 12 days. Short/long-term viral outgrowth assays (VOA) were performed with cells from ART-treated PLWH in the presence/ absence of T0070907 and/or antiretroviral drugs. Cell-associated (CA)/free HIV RNA/DNA and HIV-p24 levels were quantified by real-time PCR, ELISA, and flow cytometry. Transcriptional profiling was performed using the Illumina RNA Sequencing technology. Results were validated by flow cytometry, ELISA and miR29 antagomir. Results: While PPARγ antagonist increased IL-17A and CA HIV RNA levels in cells of ART-treated PLWH, viral outgrowth was unexpectedly inhibited. To define the mechanism of action, RNA-sequencing/functional validations were performed. PPARγ inhibition in CCR6+CD4+T-cells up-regulated transcripts linked to Th17 polarization (RORγt, STAT3, BCL6 IL-17A/F, IL-21), HIV transcription (CDK9, HTATIP2) and restriction (Caveolin-1, TRIM22, TRIM5α, BST2, miR29), and down-regulated transcripts encoding key HIV-dependency factors (CCR5, furin). Moreover, T0070907 increased the antiviral IL-21/miR29 axis. MiR29 antagomir increased HIV replication in the absence but not in presence of T0070907, pointing to miR29-independent antiviral mechanisms. Conclusion: These results provide the rationale for considering PPARγ antagonism as a novel strategy towards Th17-mediated mucosal immunity restoration and HIV-reservoir purging.

Poster Abstracts

352 A JAK1 INHIBITOR SUPPRESSES HIV-1-DRIVEN ABERRANT HOST GENE TRANSCRIPTION Yang-Hui Yeh 1 , Katharine Jenike 2 , Rachela Calvi 1 , Jennifer Chiarella 1 , Ya-Chi Ho 1 1 Yale University, New Haven, CT, USA, 2 Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: More than 50% of the latent reservoir are maintained through clonal expansion. While ART effectively blocks new rounds of infection, HIV-1 promoter remains intact, drives HIV-1 expression and aberrant cancer-related gene expression, and contributes to HIV-1 integration site-related clonal expansion. New therapeutic approaches targeting the clonal expansion of HIV-1-infected cells is required to reduce the size of the latent reservoir. We hypothesize that suppressing HIV-1 transcription can disrupt HIV-1-driven clonal expansion of the infected cells. Methods: We first developed a dual-reporter cell line model and screened a library of 1,430 FDA-approved small molecule compounds to identify HIV-1- suppressing agents. Second, we examined the effect of candidate HIV-1- suppressing agents on HIV-1 transcription and HIV-1-driven aberrant host gene

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