CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

assess mRNA expression, toxicity, and activation. PBMCs from people living with HIV (PLWH) on ART were treated with CD4-targeted Tat mRNA-LNPs and assayed for p24 expression. Results: A nontoxic Tat variant demonstrated similar transactivation activity when compared to wildtype variants and drove HIV expression in neighboring, nontransfected cells, indicating secretion of active Tat. Tat mRNA transfection of J-Lat 10.6 resulted in dose-dependent viral reactivation. Multiomic analysis of treated J-Lat 10.6 revealed significant changes in expression of four genes (|log 2 -fold change| > 0.25, p < 0.05): HIV-1 (0.37 log 2 -fold increase), MTRNR2L12 (0.33 log 2 -fold decrease), RPS10 (0.31 log 2 -fold decrease), and SNHG25 (0.26 log 2 -fold decrease). Minimal changes to ATAC accessibility of the proviral genome 2-3 days post-treatment were seen despite GFP signal in ~60% of cells. All J-Lat clones reactivated, albeit heterogeneously, upon treatment with Tat mRNA-LNPs or Romidepsin, but less so with other agents. Ibalizumab-targeting of Tat or GFP mRNA-LNPs resulted in efficient mRNA delivery and expression in rhesus and human CD4 T cells, with no observed cytotoxicity (Live/Dead, Annexin V), change in activation markers (CD69, CD25, CD38, HLA-DR), or widespread changes to gene expression in bulk RNAseq. CD4 targeted Tat mRNA-LNP treatment of PBMCs from three PLWH with inducible reservoirs resulted in p24 expression in both cellular pellet (0.05-0.5 pg/ml) and supernatant (0.15-0.2 pg/ml). Conclusion: CD4-targeted Tat mRNA-LNPs selectively drive viral gene expression without cytotoxicity. These findings show that viral transcriptional programs, such as latency, can be modified using cell-targeted mRNA gene therapy. Potent HIV Latency Reversal by Lipid Nanoparticles Encapsulating HIV Tat mRNA Bridget M Fisher , Paula M. Cevaal, Stanislav Kan, Abdalla Ali, Abigail Tan, Rory Shepherd, Youry Kim, Marvin Holz, Damian Purcell, Frank Caruso, Sharon R. Lewin, Michael Roche University of Melbourne, Melbourne, Australia Background: One approach to eliminating the HIV reservoir is to upregulate proviral transcription and protein production, to induce the clearance of latently infected cells. To date, latency reversal agents (LRAs) have demonstrated poor efficacy in reversing latency and exhibited off-target effects. This is likely due to targeting host pathways and transcription factors. Here we developed lipid nanoparticles (LNPs) encapsulating mRNA encoding for the HIV Trans-activator of Transcription (Tat) protein, and characterised their ability to reverse latency in a potent, HIV-specific manner. Methods: LNPs encapsulating mRNA encoding either Tat (Tat-LNP) or mCherry reporter protein (mCherry-LNP) were formulated through microfluidic mixing. Transfection efficiency was assessed in non-stimulated CD4+ T cells from HIV negative donors. HIV latency reversal was assessed in the latently infected J-Lat A2 cell line by measuring GFP induction, and ex vivo in CD4+ T cells isolated from people living with HIV (PLWH) on suppressive antiretroviral therapy (ART) using digital RT-PCR measuring various HIV transcripts. Cellular activation and viability were characterised ex vivo by assessing CD69, CD25 and HLA/DR expression by flow cytometry. Results: mCherry-LNPs potently transfected non-stimulated CD4+ T cells (75±3.6% mean±SEM mCherry+ cells). Tat-LNPs induced HIV reactivation in J-Lat A2 cells, with a fold increase of 68.2±10.3 (mean±SEM; EC 50 =5.3ng/mL) relative to untreated. In ex vivo CD4+ T cells from PLWH on suppressive ART (n=5), treatment with Tat-LNPs for 48-72 hours induced a 3.3-fold (median) increase in HIV transcription initiation (measured by TAR transcripts); 23.5-fold increase in elongated transcripts (Long-LTR); 36.1-fold increase in completed transcripts (polyadenylated transcripts); and 105.9-fold increase in multiply spliced transcripts (Tat-Rev), relative to untreated. Tat-LNPs outperformed the PMA/PHA positive control for elongation, polyadenylation and splicing. Treatment with Tat-LNPs did not cause T cell activation or toxicity (median 85.5% viability). Conclusion: Our mRNA encoding the HIV Tat protein can be encapsulated into LNPs and delivered to latently infected CD4+ T cells. Tat-LNPs reverse latency in a potent, HIV-specific manner, thereby overcoming the limitations of current LRAs which infrequently upregulate multiply-spliced transcripts and exhibit toxicities. These data warrant the further development of Tat-LNPs as a novel LRA

reservoir at week 12, BCL-2+KI67+ CD4+ TN cells in the total RUX group at week 5, CD127+ CD8+ TTD cells in the total RUX group at week 5, and CD25+ CD8+ TTM cells in the total RUX group at week 5. Conclusion: Ruxolitinib decays the HIV-1 reservoir and resets immune balance in PWH on ART. Based on observed reservoir decay (week 5 to 12), our model predicts a 99.99% decay in 2.83 years, should rate of decay remain constant. These data are foundational for further human trials with Jak 1/2 inhibitors such as ruxolitinib towards HIV-1 elimination. Short-Term BCL-2 Inhibition at ART Initiation Transiently Reduces SIV Reservoir in Rhesus Macaques Tomas Raul Wiche Salinas 1 , Justin L. Harper 1 , Kevin Nguyen 1 , James Auger 1 , Laurence Raymond Marchand 1 , Mackenzie Cottrell 2 , Jason Brenchley 3 , Jeffrey Lifson 4 , Brandon Keele 4 , Andrew Badley 5 , Guido Silvestri 1 , Deanna A. Kulpa 1 , Mirko Paiardini 1 1 Emory University, Atlanta, GA, USA, 2 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 3 National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA, 4 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 5 Mayo Clinic, Rochester, MN, USA Background: In spite of the success of antiretroviral therapy ART in suppressing HIV replication, effective strategies to limit the establishment and maintenance of the HIV reservoir are needed. Recent data highlighted BCL-2 as a key regulator for the establishment, persistence, and expansion of the CD4+ T cell reservoir, protecting infected cells from virus or CD8 T cell-induced cell death. Thus, there is a strong rationale behind promoting apoptosis of latently HIV-infected cells via BCL-2 inhibition. Herein, we evaluate in SIV-infected, ART-treated rhesus macaques (RMs) the effect of the BCL-2 inhibitor venetoclax, alone or combined with a latency-reversing strategy (CD8a cell depletion), on cellular dynamics and the size of the SIV reservoir. Methods: 12 RMs were intravenously infected with barcoded SIVmac239 and initiated ART 2 weeks post-infection. RMs were distributed into 3 arms: 4 RMs received only ART; 4 RMs received 10 daily doses of venetoclax as a subcutaneous injection starting at ART initiation; and 4 RMs received venetoclax in conjunction with a single dose of the anti-CD8a depleting antibody MT807R1 at ART initiation. Animals were followed until day 70 post-infection. Results: Venetoclax and venetoclax plus MT807R1 treated RMs had a slower viral plasma load decay rate than control RMs, with prolonged on-ART viremia. In blood, venetoclax depleted CD4+ T cells, including their central and effector memory subsets. This was accompanied by decreased total SIV-DNA in peripheral blood mononuclear cells and intact SIV-DNA in sorted CD4+ T cells, consistent with a preferential loss of infected cells following venetoclax administration. Furthermore, and despite the slower plasma viral load decay, venetoclax-treated animals showed a faster intact SIV-DNA decay rate, consistent with killing of SIV-DNA harboring cells. This effect was specific to the treatment and not sustained after the interruption of Venetoclax. Conclusion: This study shows that BCL-2 inhibition at ART initiation can delay the establishment of latency and transiently deplete infected cells by promoting the elimination of SIV-infected circulating CD4+ T cells. The results support the concept that BCL-2 expression favors the survival of infected cells and represents a potential therapeutic target. Further studies are needed to explore the effect of BCL-2 inhibition during long-term ART and prolonged exposure to BCL-2 inhibitors. CD4-Targeted mRNA Delivery of Tat Reverses HIV-1 Latency Edward Kreider 1 , Vincent H. Wu 1 , Jayme M. Nordin 1 , Francesco Pennino 2 , Amie Albertus 1 , Tyler Papp 1 , Obinna Uzosike 1 , Khumoekae Richard 2 , Carol Cheney 3 , Karam Mounzer 1 , Hamideh Parhiz 1 , Michael R. Betts 1 , Daniel T. Claiborne 2 , Drew Weissman 1 , Luis J. Montaner 2 1 University of Pennsylvania, Philadelphia, PA, USA, 2 Wistar Institute, Philadelphia, PA, USA, 3 Merck & Co, Inc, Rahway, NJ, USA Background: Interventions that reverse viral latency are needed for an HIV cure. The viral protein Tat drives HIV expression but is toxic and difficult to deliver in a targeted manner. We tested whether CD4-targeted delivery of Tat encoding mRNA could safely reverse latency. Methods: mRNA encoding wildtype and nontoxic Subtype B and D Tat variants were made and transfected into TZM-bls to assess transactivation. Five J-Lat clones were treated with Tat mRNA-LNPs or small molecule agents (romidepsin, prostratin, pabinostat, or AZD5582) and evaluated using flow cytometry, live cell imaging, or single cell multiomic (RNA+ATAC) profiling. Ibalizumab (anti CD4) was conjugated to mRNA-LNPs to generate CD4-targeted mRNA-LNPs. Primary CD4 T cells were treated with CD4-targeted Tat or GFP mRNA-LNPs to

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CROI 2024 139