In this Phase I project, we propose to investigate a novel primer chemistry and probe detection system called the Zip nucleic acids (ZNA"). ZNAs are oligonucleotides conjugated to a number of cationic spermine moieties that enhance the effective concentration of primers and probes near nucleic acid targets. This property has been reported to enhance the speed and sensitivity of RT-PCR (Moreau et al. 2009). ZNAs are compatible with Taqman detection formats (Paris et al. 2010). We expect this detection technology will further accelerate our RNA detection assays as well as increase our accuracy. We will compare the performance of ZNA to LNA DNA dual labeled (Tong et al. 2008, Li et al. 2010) and CPT probes. By the end of the study we will have completed the analytical study stage required for the commercial release of an IsoAmp HIV-1 quantitative assay. Our phase I research plan includes 4 aims: 1. Design and test ZNA primers targeting all subtypes of HIV-1, 2. Design and test with both ZNA taqman and ZNA cycling probes for the HIV assay, 3. Develop a simple work flow for extraction of RNA from dry blood spots (DBS) and dry plasma spots (DPS), and 4. Test the sensitivity and specificity of assays using the ZNA technology in combination with IsoAmp in a panel of HIV-1 isolates. At the conclusion of Phase I, we will be ready to identify the best probe technology to develop a commercial IsoAmp HIV-1 quantitative assay for commercial distribution in the US and abroad. We will also have a clear indication of the type of sample extraction method that best suits HDA viral load testing. In Phase II, we would develop a pre-IDE for a multi-site clinical study plan to seek FDA approval for sale in the US. We would also explore commercial release in the rest of the World.
Public Health Relevance Statement: Human immune-deficiency virus (HIV) viral load testing is the standard of care for monitoring anti-retroviral therapy in the United States and Europe. HIV quantitative tests rely of high throughput systems that use the polymerase chain reaction (PCR) and all suffer one major limitation: a need for expensive instrumentation, and skilled personnel to operate the equipment (Fiscus et al. 2006). In the developing World, low-cost CD4+ monitoring tests (Rodriguez et al. 2005) are used because of economic drivers, despite the fact that CD4+ monitoring lags a rise in viral RNA load in cases of therapeutic failure (Vaidya et al. 2010). BioHelix has developed an isothermal nucleic acid amplification chemistry called helicase dependent amplification (HDA) that can solve this problem. This technology has 4 advantages over PCR methods of viral load testing: 1) it relies on a low-cost instrument (1/10 the cost of PCR machines), 2) it can amplify RNA faster than DNA and can match the fastest PCR assays (Goldmeyer et al. 2007), 3) it is more tolerant of base variations in primers and probes than PCR, and 4) it is more tolerant to amplification inhibitors found in clinical samples than PCR. In this Phase I project, we will explore the potential application of Zip nucleic acids (ZNA) to enhance the performance of our HIV assays (Tang et al. 2010). By the end of the study we will have completed the analytical study stage required for the commercial release of an IsoAmp HIV-1 quantitative assay. In Phase II, we would develop a pre-IDE for a multi-site clinical study plan to seek FDA approval for sale in the US.
Project Terms: Adherence (attribute); Affinity; Anti-Retroviral Agents; Back; base; Binding (Molecular Function); Biological Assay; Blood; Chemistry; Clinical; cost; cost effective; Country; design; Detection; DNA; DNA-Directed DNA Polymerase; Dose; Economically Deprived Population; Economics; Enzymes; Equipment; Europe; Failure (biologic function); Fluorescence; Goals; health care delivery; helicase; Human; Human Resources; Immune; inhibitor/antagonist; instrument; instrumentation; internal control; Label; Laboratory Personnel; Licensing; locked nucleic acid; Logistics; Measurement; melting; Methods; Minor; Molecular; Molecular Conformation; Monitor; Mortality Vital Statistics; Multi-Institutional Clinical Trial; National Institute of Allergy and Infectious Disease; novel; Nucleic Acid Probes; Nucleic Acids; Oligonucleotides; Patients; Performance; Pharmaceutical Preparations; Phase; phase 2 study; Plasma; Polymerase Chain Reaction; Problem Solving; Property; Reporting; Research; Resources; response; Rest; Reverse Transcription; Risk; RNA; Sales; Sampling; Sensitivity and Specificity; Signal Transduction; Small Business Innovation Research Grant; Sodium Chloride; Specimen; Speed (motion); Spermine; Spottings; Staging; standard of care; System; Taq Polymerase; Technology; Temperature; Testing; Therapeutic; Time; Training; United States; Variant; Viral load measurement; Viral Load result; viral RNA; Virus; Work
