The CDC estimates that in the U.S., 1,144,500 people aged 13 years and older are living with HIV infection, with approximately 180,900 (15.8%) others infected but undiagnosed (CDC, 2013). Strict adherence to highly active/combination anti-retroviral therapy (HAART/cART), prevents full-blown AIDS. However, HAART/cART fails to cure HIV infection as it has little effect on CD4+ cells infected with latent forms of the virus. If a patient no longer adheres to their prescribed regimen of HAART/cART, the latent pool quickly rebounds into full-blown HIV infection. Thus, HIV-AIDS is still far from eradicated. Issues with Current Solutions & How Product Meets Unmet Needs Current methods of quantifying the latent reservoirs include the quantitative viral outgrowth assay (Q-VOA), PCR and RT-PCR. Q-VOA is accepted as the most accurate method, but is a time and resource intensive procedure. PCR grossly overestimates the latent pool through detection of unintegrated as well as nonfunctional virus DNA. RT-PCR can be used to detect viral RNA to 20-50 virus particles per mL and thus reduces the time to result of QVOA and is generally applicable for measuring viral load, but does not directly detect replication-competent latent HIV-infected cells. Q-VOA, the accepted quantitation standard, is currently available only at relativel few AIDS research facilities, due to its intensive resource and labor requirements. This product will introduce a real-time molecular assay to detect transcriptionally-competent HIV mRNA directly from latently infected cells isolated from HAART/cART patients. With validation against the Q-VOA standard, this assay has the potential to provide high-throughput, real-time, and lower-cost quantitation of the latent HIV- infected reservoirs in the body and significantly accelerate testing and discovery of a cure for HIV infection. Summary of Approach The product proposed is a real-time quantitative autoligation detection reaction (qLDR), which uses fluorogenic probes for chemical ligation in a thermocycling amplification reaction. qLDR allows for real-time and accurate quantification of the level of HIV mRNA present in CD4+ latent HIV-infected cells. The proposed assay will employ modified fluorogenic nucleic acid probes for superior stability and highly specific HIV RNA detection for quantifying latent HIV-infected reservoirs. Collaborators and Unique Resources Jan Biotech, Inc., with expertise in molecular diagnostic development, will collaborate with Dr. David Putnam, a chemist in the Department of Chemical and Biomolecular Engineering of Cornell University. Dr. Harris Gelbard, investigating the phenomenon of latent reservoir-induced neuroAIDS at the University of Rochester Center for AIDS Research (CFAR), and CFAR will provide consultation and HAART/cART CD4+ samples. Cell lines will be provided by the NIH AIDS Reagent Program; Q-VOA validation testing will be performed by CARE. Phase I Specific Aims Specific Aim 1: Develop spliced-RNA detection assay for quantitation of latent HIV-1 infected cells Specific Aim 2: Test qLDR with HAART patient CD4+ cells and validate against Q-VOA How Anticipated Results will Justify Phase II and Further Product Development Superior performance of qLDR is expected compared to Q-VOA and Q-VOA with RT-PCR, with real-time, sensitive and specific detection of spliced HIV mRNA directly from latent HIV-infected CD4+ cells from HAART/ cART patients. Successful Phase I validation against the Q-VOA standard will justify Phase II full validation and product development to produce a high-throughput commercial ready laboratory research assay platform. Additional Time and Funding Necessary to Bring Product to Market after Phase I Completion It is anticipated that a high-throughput laboratory research product can be brought to market as a laboratory assay kit for research purposes at the completion of the Phase II, two years after the Phase I work has been completed. It is anticipated that an additional 2-3 years and funding through a Phase II bridge award will be needed to perform the clinical trials required for FDA approval as a clinical diagnostic.
The CDC estimates that in the U.S. 1,144,500 people aged 13 years and older are living with HIV infection, with approximately 180,900 (15.8%) others who are infected but undiagnosed (CDC, 2013). Highly active anti-retroviral therapy (HAART)/combination anti-retroviral therapy (cART), while keeping HIV infection in remission, fails to eradicate reservoirs of latent HIV- infected cells in patients. Strategies targeting this latent reservoir are now being tested in clinical trials, and well-validated high-throughput assays that quantify this reservoir are urgently needed to facilitate the complete eradication of HIV-AIDS.
