Evaluation of Self-delivering FANA Antisense Oligonucleotide Lead Compounds for HIV Therapy

Project Summary/Abstract: Human immunodeficiency virus 1 (HIV-1) is the primary cause of acquired immune deficiency syndrome (AIDS) that affects over a million people in the United States alone. Advances in treatment have significantly prolonged the lives of those infected with HIV-1, with combinatorial antiretroviral therapy (cART) being the current standard in therapeutic care. However, cART alone cannot achieve complete eradication of the virus. Besides, drawbacks such as drug resistance development and severe side effects (e.g. premature aging, cancer, and cardiovascular disease) remain critical issues in cART therapy. Therefore, there is a need for a treatment, with efficient delivery and a favorable safety profile, that can reduce the HIV-1 viral load to undetectable levels. A promising approach is to utilize RNA silencing to treat HIV-1 infection by targeting the dimerization initiation site (DIS), a replication signal in the 5’ untranslated region (5’-UTR). Dimerization is initiated when the 5andapos;-UTR undergoes a conformational change, allowing the DIS loops of two RNA genomes to base pair. This forms a kissing-loop (KL) dimer, which then leads to the subsequent packaging of the viral RNA. It is known that mutation or inhibition of the DIS severely restricts viral infectivity. Here we propose to test if 2’-deoxy-2’-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASOs), targeting DIS, will prevent viral replication. FANA ASOs will be designed to cleave (RNase H-dependent) or block viral RNA, thus inhibiting viral replication post-exposure. In a preliminary study using HIV-1 infected human peripheral blood mononuclear cells (PBMCs), DIS-targeting FANAs inhibited HIV-1 replication for as long as two weeks, after single doses of 400 nM and 3 µM doses (IC50 = 200 nM). FANA ASOs were also tested as prophylactics in vitro, at significantly lower concentrations, to prevent viral infection and amplification for up to 2 weeks. FANA ASOs were also designed to bind to DIS RNA without cleaving it, which would prevent the formation of the kissing loop structure that is necessary for replication. AUM- DIS-G9 emerged as the lead compound from our RNase H-dependent in vitro studies, while AUM-DIS-G0 will be the lead compound for our RNase H-independent trials. In this study, we will adopt a systematic approach to design and assess these two FANA lead ASOs targeting DIS. Our first aim is to evaluate the reduction in viral replication after treating with our two lead compounds, from our preliminary data, in a humanized NSG mouse model susceptible to HIV-1 infection. The second aim is to assess the potential of FANA ASOs as cART replacement in vivo, considering their prophylactic success in vitro, in a latently infected humanized mouse model. The third Aim is to perform standard pharmacokinetic and ADME studies on the lead compound from Aims 1 and 2 in vivo assessments. The goal of the proposed study is the development of a next generation antisense therapy for HIV.Project Narrative Human immunodeficiency virus (HIV-1) infection is a chronic, widespread disease. Combinatorial antiretroviral therapy (cART) is a standard treatment for HIV-1, that carries the risk of drug resistance and severe long-term side effects. We propose to develop 2’- deoxy-2’-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASOs) targeting HIV-1 dimerization initiation site (DIS), to block viral packaging, budding and subsequent infection to other host cells. Our two lead compounds, obtained from a published in vitro preliminary study, will be assessed for in vivo viral replication prevention and cART replacement using humanized NSG mice. Additionally, the lead compounds will be evaluated for standard pharmacokinetics, ADME, and genotoxicity studies as a first step to bringing the technology to the clinic.