CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

402 A Phase I Clinical Trial of Autologous CD4 + T Cells ModifiedWith a Retroviral Vector Expressing the MazF Endoribonuclease in Patients With HIV-1 Jeffrey M. Jacobson 1 ; Hideto Chono 2 ; Meghan Metz 1 ; Gabriela Plesa 3 ; Julie Jadlowsky 3 ; Simon Lacey 3 ; Bruce Levine 3 ; HirofumiYoshioka 2 ; Junichi Minemo 2 ; Carl H. June 3 1 Drexel University College of Medicine, Philadelphia, PA, US; 2 Takara Bio Inc., Shiga, Japan; 3 University of Pennsylvania, Philadelphia, PA, US Background: The E.coli -derived MazF endoribonuclease specifically cleaves single stranded RNAs at ACA sequences. HIV-1 contains over 240 ACA sequences, making it especially sensitive to MazF activity. In this study, autologous CD4 + T cells are modified using a retroviral vector containing the mazF gene expressed under the control of the HIV LTR. MazF expression is thus activated by Tat conditionally during HIV replication. This study is designed to evaluate the safety and durability of MazF-modified CD4 + T (MazF-T) cells, and assess related antiviral effects. Methods: This is an exploratory Phase I, Open Label, Dual Cohort study evaluating safety, tolerability and immunogenicity of autologous CD4 + T cells expressing the MazF endoribonuclease gene in HIV+ subjects. Both cohorts consist of subjects on combination antiretroviral therapy (cART) with CD4 counts >350 cells/mm 3 and undetectable HIV-1 RNA levels. Subjects in cohort 1 remain on cART throughout the duration of the study. Subjects in cohort 2 participate in a 16 week analytical treatment interruption (ATI) beginning 2 weeks post T cell infusion. All subjects are infused with a single dose of MazF-T cells and are evaluated for persistence of modified cells, impact on CD4 count and effects on HIV viral load. Results: To date, 4 subjects in cohort 1 have each received a single infusion of 0.5-1x10 10 cells. There have been no SAE related to MazF-T. All 8 AE related to study drug have been grade 1 in severity. The CD4/CD8 ratio increased in 3 of 4 subjects post infusion, before stabilizing near baseline. Absolute CD4 count remained stable, or slightly increased as compared to baseline. As these subjects remained on cART, all viral loads remained undetectable. MazF DNA signal was detected by qPCR in all 4 subjects’ peripheral blood at the most recently available timepoint, including 2 subjects who completed the study at Day 180. Conclusions: These preliminary results suggest that autologous MazF-modified CD4 + T cells are safe and well-tolerated in aviremic HIV+ subjects and are able to persist out to 6 months post-infusion. Future results in subjects participating in an ATI will further elucidate the anti-HIV effects associated with MazF-T cells in the presence of viremia. 403 CRISPRs Are Able to Efficiently Target Latent HIV and Halt New Infections Robert Jan Lebbink; Dorien de Jong; FemkeWolters; Emmanuel J.Wiertz; Monique Nijhuis University Medical Center Utrecht, Utrecht, Netherlands Background: Currently available combination antiretroviral therapy (cART) can successfully control HIV replication. However, conventional treatment lacks the ability to clear the latent reservoir and stop viral production, which remains the major obstacle in achieving a cure. Novel strategies are required to permanently disrupt the HIV genome in the latently infected cells. In this study we have investigated the potential of the CRISPR-Cas9 RNA guided DNA endonucleases system to edit the HIV genome, prevent re-activation from latently infected cells and halt new infections. Methods: The CRISPR-Cas9 system is comprised of a Cas9 protein, which in combination with a guideRNA (gRNA), is able to cleave a complementary dsDNA sequence. gRNAs were designed to target HIV LTR, protease (PR), reverse transcriptase (RT), integrase (IN) and the structural matrix protein (MA). gRNAs against human B2M, HLA and CD45 were used as controls. The CRISPR-Cas9 systemwas cloned in a lentivirus vector and used to transduce SupT1 and Jurkat cells. The latter is latently infected with near full-length HIV and expresses GFP upon TNF α stimulation (J.Lat Full Length Clone 15.4). SupT1 cells are transduced with the lentiviral constructs and subsequently infected with HIV using three different MOIs and viral replication was monitored by HIV DNA quantification and HIV CA-p24 production. On and off targeting efficiency (three genes per CRISPR) of the different CRISPRs was assessed by deep sequence analysis. Results: Lentiviral transduction in SupT1 and Jurkat cells resulted in stable expression of the CRISPR-Cas9 system. Deep sequence analysis demonstrated efficient HIV genome editing (75-99%) and an off-target efficiency ranging between 1.7-0.4%. TNF α –induced HIV reactivation from latently infected T cells was significantly reduced after transduction with CRISPRs specifically targeting the LTR (empty vector 48 ± 2.98% GFP + cells vs LTR-6 15 ± 6.63% and LTR-4 26 ± 2.76% GFP + cells; p<0.05). Subsequently, we investigated the potential of CRISPRS targeting the LTR, PR, RT, IN and MA to inhibit viral replication. HIV DNA quantification demonstrated up to 40-fold reduction in intracellular HIV DNA and a significant reduction in virus production ranging from 91-99% inhibition (p <0.05). Conclusions: The newly discovered CRISPR-Cas9 system is able to target HIV efficiently in both primary infection and latency models and may provide a specific, efficacious prophylactic and therapeutic anti-viral approach.

Poster Abstracts

TUESDAY, FEBRUARY 24, 2015 Session P-F6 Poster Session

Poster Hall

2:30 pm– 4:00 pm HDAC Inhibitors 404 Histone Deacetylase (HDAC) and Histone Acetyltransferase (HAT) Inhibitors Have Opposing Effects on Cellular Susceptibility to HIV Infection Mark B. Lucera 2 ; Curtis Dobrowolski 1 ; Jonathan Karn 1 ; John C.Tilton 2 1 Case Western Reserve University, Cleveland Heights, OH, US; 2 Case Western Reserve University, Cleveland, OH, US

Background: Latency reversing agents (LRA), such as HDAC inhibitors, are being employed as part of a “shock-and-kill” approach to reactivate and eradicate latent proviruses. We recently reported that the HDAC inhibitor vorinostat significantly increases productive viral infection in vitro , raising potential concern for its use as a LRA in patients. This effect was also observed with other broad-spectrum HDAC inhibitors as well as the cytoplasmic HDAC6-specific inhibitor tubacin. Our work with HDAC inhibitors led us to hypothesize that treatment with opposing histone acetyltransferase (HAT) inhibitors would subsequently decrease HIV productive infection. Methods: Primary CD4+ T cells were treated with HDAC/HAT inhibitors and infected with combination reporter or replication competent viruses. Viral fusion and LTR-driven gene expression were measured by flow cytometry. 2-LTR circle transcripts were monitored by PCR and deep sequencing. Results: Here we report that the HDAC inhibitor vorinostat enhances post-entry infection efficiency 2-3 fold in both single cycle and replication competent experiments. This was found to be a cytoplasmic mechanism in which treatment increased kinetics and efficiency of reverse transcription (RT) and nuclear import of HIV DNA. Recapitulation of this effect with broad-spectrum HDAC inhibitors and cytoplasmic HDAC6 inhibitor tubacin suggest that enhancement of viral infection may be an undesirable effect common to this class of agents. Conversely, treatment with the histone acetyltransferase (HAT) inhibitors garcinol and curcumin was found to significantly reduce LTR-driven EGFP expression in both a time and dose-dependent manner. Additional work is being conducted to ascertain the mechanism of these inhibitors.

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CROI 2015