CROI 2020 Abstract eBook
transcription at the integration site. Third, we examined cellular transcriptional landscape of cells treated with candidate HIV-1-suppressin agents to understand how these agents affect host cell environment. Fourth, to understand whether candidate HIV-1-suppressing agents can disrupt the proliferation dynamics of HIV-1-infected cells, we examined the frequency of HIV-1-infected cells from HIV-1-infected individuals upon ex vivo T cell activation with and without ex vivo treatment of candidate HIV-1-suppressing agents. Results: We identified four FDA-approved drugs – JAK1 inhibitor filgotinib, JAK1/2 inhibitor ruxolitinib, spironolactone and guanine synthesis inhibitor mycophenolic acid – which reduce HIV-1-GFP reporter expression in cell line models and HIV-1 RNA transcription in CD4+ T cells from HIV-1-infected individuals. Among them, filgotinib, spironolactone and mycophenolic acid suppress HIV-1-driven aberrant host gene transcription and aberrant oncogenic protein production in a HIV-1-reporter cell line model. Filgotinib alters host transcriptional landscape by changing host RNA processing involving intron retention and RNA splicing. During CD3/CD28 induced T cell activation and proliferation, filgotinib reduces the frequency of cells harboring inducible HIV-1 ex vivo. Conclusion: Filgotinib preferentially reduce the proliferation of HIV-1-infected cells upon T cell activation. HIV-1 suppressing agents serve as a new therapeutic approach to target the clonally expanding HIV-1-infected cells. 1 University of Nebraska Medical Center, Omaha, NE, USA, 2 Cornell University, Ithaca, NY, USA, 3 Temple University, Philadelphia, PA, USA, 4 Stanford University, Stanford, CA, USA Background: A key challenge in developing successful HIV-1 cure strategies rest in eliminating the integrated provirus from the genomes of infected CD4+ T lymphocytes and monocyte-macrophages. In a first step towards this end, we recently demonstrated success by the sequential use of long acting slow effective release (LASER) ART and CRISPR-Cas9 in achieving viral sterilization from a subset of infected humanized mice. We sought to improve upon the transduction and known immunogenicity of the adeno-associated virus 9 (AAV9; 1012 genome copies/mouse) by generating an HIV-1 pseudovirus enabling both CD4 and CCR5 receptor targeting. We hypothesize that virus-like particles, bearing antigenic resemblance to HIV but lacking infectivity, will utilize viral glycoprotein-120 (gp120) to specifically deliver curative agents to CD4+ cells. Methods: Viral matrix (HIV-1p17) and capsid (HIV-1p24) were genetically fused to biotinylated peptide (AviTag) and monomeric streptavidin (maxavidin) encoding sequences, respectively, to facilitate encapsulation of bioconjugated payloads. We generated VLPs (figure 1) by pseudotyping modified lentiviral structural proteins with dual-tropic HIV-189.6 envelope by co-transfection of plasmids in HEK293FT cells. A duplex LTR and gag splicing CRISPR-Cas9 systemwas inserted via plasmid. Non-gene payloads including streptavidin quantum dots, biotinylated fluorophore and a cabotegravir (CAB) prodrug were independently loaded in the VLPs. Results: VLPs retained the same 150nm size, spherical morphology, and targeting epitope (gp120) expression as native infectious HIV-1 but were replication incompetent. Using our bioconjugation system, streptavidin quantum dots and biotinylated fluorophore were detected in the VLPs at 1.4 and 3.6-fold above baseline measurements. In human PBMC, 57% of monocytes and 9.5% of CD4+ T cells co-localized with fluorescently labeled VLPs. VLPs bearing CRISPR-Cas9 showed gp120-mediated entry and robust excision of proviral DNA from HIV-1 infected CD4+ T cells. Conclusion: HIV-1 VLPs, engineered for loading with bioconjugated theranostic agents, direct payloads to CD4+ cell targets. VLPs specifically delivered proviral DNA excision therapy to HIV-infected T cells supporting the need for their development in HIV-1 cure strategies.
354 ROMIDEPSIN COMBINED WITH PRO-APOPTOTIC DRUGS REDUCE INTEGRATED HIV DNA
Youry Kim 1 , Ajantha Solomon 2 , Jennifer M. Zerbato 2 , Paul Cameron 2 , James McMahon 3 , Jenny L. Anderson 2 , Sharon R. Lewin 2 , for the Lewin Laboratory 1 University of Melbourne, Melbourne, VIC, Australia, 2 Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia, 3 The Alfred Hospital, Melbourne, VIC, Australia Background: Effective elimination of latently infected cells in people living with HIV (PLWH) on antiretroviral therapy (ART) through activation of HIV transcription will also require the induction of death of the infected cell. Given that HIV proteins, such as envelope and vpr, expressed late in the productive replication cycle, can induce apoptosis of CD4+ T-cells, we hypothesised that using latency reversing agents (LRAs) to induce expression of pro-apoptotic viral proteins combined with pro-apoptotic drugs will enhance the reduction of latently infected cells. Methods: Total CD4+ T-cells were isolated from peripheral blood collected by leukapheresis from PLWH on ART. CD4+ T-cells were treated with pro-apoptotic drugs (the phoshoinositide-3 kinase (PI3K) inhibitors, IPI-443, IPI-3063 and wortmannin or an inhibitor of B-cell lymphoma (Bcl)-2, venetoclax) for 24 hours, followed by treatment with five different latency reversing agents (LRAs; panobinostat, romidepsin, bryostatin, JQ1 or PMA/PHA) for 4 or 24 hours and then the pro-apoptotic drugs alone for a further 48 hours. We measured integrated HIV DNA and cell-associated unspliced (CA-US) HIV RNA by RT-qPCR. Results The combined treatment of romidepsin with each of the four pro-apoptotic drugs led to a greater decline in integrated HIV DNA versus either romidepsin or pro-apoptotic drug alone. Romidepsin together with 5nM venetoclax showed the greatest decline in integrated HIV DNA. Romidepsin or venetoclax alone resulted in a mean fold change (MFC) in HIV integrated DNA of 0.72 and 0.18, respectively while the combined treatment resulted in an MFC of 0.54. Panobinostat and JQ1 combined with 1μM venetoclax also led to a reduction in HIV integrated DNA (PNB+1μM VNX MFC=0.47; JQ1+1μM VNX MFC=0.60), compared to the decline resulting from each drug alone (PNB MFC=0.71; JQ1 MFC=0.88; 1μM VNX MFC=0.76). We observed increases in CA-US HIV RNA and the ratio of CA-US HIV RNA to integrated DNA following treatment of CD4+ T-cells with all four LRAs as well as each of the pro-apoptotic drugs alone or combined. Conclusion: Using CD4+ T-cells from PLWH on ART ex vivo, reduction of integrated HIV DNA could be significantly enhanced using the combination of romidepsin with either a PI3K or Bcl-2 inhibitor. The addition of a pro-apoptotic drug could potentially provide the ‘’kill’’ needed for effective ‘’shock and kill’’.
353 DEVELOPMENT OF A PSEUDOVIRUS DELIVERY SYSTEM FOR HIV-1 ELIMINATION Jonathan Herskovitz 1 , Mahmudul Hasan 1 , Wilson Blomberg 1 , Insiya Mukadam 1 , Jatin Machhi 1 , Maxim Oleynikov 2 , Kamel Khalili 3 , Bhavesh D. Kevadiya 4 , Benson Edagwa 1 , Channabasavaiah Gurumurthy 1 , Howard E. Gendelman 1
CROI 2020 122
Made with FlippingBook - professional solution for displaying marketing and sales documents online