SBIR-STTR Award

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.

Public Health Relevance Statement:


Public Health Relevance:
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.

Project Terms:
13 year old; Acquired Immunodeficiency Syndrome; Adherence (attribute); AIDS neuropathy; Anti-Retroviral Agents; assay development; Biological Assay; Blood specimen; Calibration; CD4 Positive T Lymphocytes; Cell Line; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chemicals; Chronic; Clinical; clinical application; Clinical Trials; Collaborations; collaboratory; Consultations; cost; design; Detection; Development; Diagnostic; Disease remission; Engineering; Funding; high throughput screening; Highly Active Antiretroviral Therapy; HIV; HIV Infections; HIV-1; Howard Temin Award; Individual; internal control; Investigation; Laboratories; Laboratory Research; Length; Life; Ligation; Marketing; Measures; meetings; Messenger RNA; Methods; Molecular; novel; Nucleic Acid Probes; Oligonucleotide Probes; Patient Monitoring; Patients; Performance; Phase; prevent; Procedures; product development; Production; programs; public health relevance; Reaction; Reagent; Regimen; Research; research facility; Research Personnel; Resources; Rest; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA Sequences; RNA Splicing; Sampling; Site; Small Business Technology Transfer Research; Solutions; Technology; Testing; Time; transcriptome sequencing; Translations; United States National Institutes of Health; Universities; Validation; Viral; viral DNA; Viral Genome; Viral Load result; viral RNA; Virion; Virus; Work

Public Health Problem 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. For most HIV-positive individuals, available treatments can only control HIV infection and delay progression to AIDS. HAART has substantial side effects and fails to prevent at least 50% of patients from developing HIV-1 associated neurocognitive disorders. Pre-exposure prophylaxis (PrEP) has been an exciting development for reduction of HIV acquisition risk and transmission, but it remains unclear when it is safe to discontinue PrEP treatment. The ultimate value of the SBIR Phase II project will demonstrate the ability of our technology to predict time to viral load rebound after treatment interruption, which remains a critical unmet need in the HIV cure field. Issues with Current Solutions & How Product Meets Unmet Needs Current methods of quantifying the latent reserve include the quantitative viral outgrowth assay (Q-VOA), PCR and RT-PCR. Q-VOA is a time and resource intensive procedure while 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. Jan Biotech’s HIV qLDR offers clear advantages including: 1) detection of latent reservoir cells in the blood without activation; 2) direct detection of RNA to improve quantitative ability that is lost in other assays; 3) nonenzymatic amplification, which relieves the need for RNA purification and its attendant loss of RNA; 4) detection of short segments of RNA, providing effective detection even in the presence of RNase degradative enzymes; 5) Jan Biotech’s qLDR PNA probes enable the use of short sequences complementary to highly conserved regions of the HIV-1 genome, which allows for breadth of detection across the highly genetically diverse HIV-1 subtypes. Summary of Approach The Phase I qLDR probe sets differentiated between latent and activated HIV-infected cells in peripheral blood, which has great promise for accurate measurement of the level of the replication-competent latent reservoir. The Phase II Specific Aims will demonstrate the ability of the assay to predict time to viral load rebound after treatment interruption using ex vivo and in vivo samples. Testing will evaluate the utility of the assay as a clinical endpoint to guide treatment interruption decisions and facilitate HIV remission and cure discovery. Collaborators and Unique Resources Jan Biotech, Inc., with expertise in molecular diagnostic development, will consult with Jonathan Li, MD, MMSc Assistant Professor of Medicine, Harvard Medical School, in a AIDS Clinical Trials Group (ACTG) study. Judith Currier, MD, MSc, Vice Chair of the ACTG Network will oversee sample collection from the treatment interruption study, A5345; Q-VOA cross-validation testing will be performed by Southern Research Institute. Phase II Specific Aims Specific Aim 1: HIV qLDR prediction of replication-competent latent reservoir Task 1: Optimization of qLDR probe sets Task 2: Ex vivo rebound after treatment interruption (TI) Specific Aim 2: HIV qLDR prediction of HIV rebound during Analytical Treatment Interruption Task 1: HIV qLDR prediction of Viral Rebound after Analytical Treatment Interruption (ATI) Task 2: Comparison of HIV qLDR to other assays used in ATI study Specific Aim 3: Preclinical Validation of HIV qLDR Task 1: Validation of qLDR performance characteristics Task 2: QVOA validation Market after Phase II Completion Both Gilead and Bionor are commercially interested in the technology and look forward to results generated from the Phase II and the ACTG study. Regulatory approval will be sought for commercialization.

Public Health Relevance Statement:
Relevance to Public Health The CDC estimates that 1.2 million people aged 13 years and older are living with HIV infection in the U.S., with approximately 156,000 (13%) undiagnosed. 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. The proposed latent HIV diagnostic has the potential to predict whether an individual can safely end treatment and remain virus-free.

Project Terms:
13 year old; Acquired Immunodeficiency Syndrome; Adverse effects; Africa; Aftercare; AIDS clinical trial group; Anti-Retroviral Agents; assay development; base; Biological Assay; Biotechnology; Blood; Blood specimen; Cause of Death; CD4 Positive T Lymphocytes; Cell Count; Cell Line; Cells; Centers for Disease Control and Prevention (U.S.); Characteristics; Clinical; Clinical Trials; Collaborations; Combined Modality Therapy; commercialization; Consult; Detection; Development; Diagnostic; Disease remission; DNA; Enzymes; Evaluation; Genome; high throughput screening; Highly Active Antiretroviral Therapy; HIV; HIV Infections; HIV Seropositivity; HIV-1; HIV-associated neurocognitive disorder; Human; improved; in vivo; Individual; interest; Interruption; Letters; Measurement; Measures; medical schools; Medicine; Methods; molecular diagnostics; Monitor; Participant; Patients; Performance; peripheral blood; Pharmacotherapy; Phase; pre-clinical; pre-exposure prophylaxis; preclinical evaluation; Preclinical Testing; Predictive Analytics; Preparation; prevent; Procedures; Process; professor; Public Health; Reaction; Research; Research Institute; Resources; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Risk; RNA; RNA purification; RNA Splicing; sample collection; Sampling; Small Business Innovation Research Grant; Technology; Testing; Time; transmission process; treatment adherence; Universities; Validation; Viral; Viral Load result; viral rebound; viral RNA; Virion; Virus; Vulnerable Populations