Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 600.00K | Year: 2012
DESCRIPTION (provided by applicant): 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 moietiesthat 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 endof 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 theZNA 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: 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.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 2.83M | Year: 2009
DESCRIPTION (provided by applicant): We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use a lateral flow device to detect amplification products. Such devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several moderate complexity and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 200 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). Preliminary studies suggest a limit of detection of 100 copies/mL plasma when nucleic acids extracted from plasma samples with a sequence-specific capture method are used as templates for RT-HDA. We propose to further test the specificity and sensitivity of the HIV test with clinical specimens obtained from 5 high-risk patient recruitment sites. Assays will be performed at BioHelix, and at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. In addition, we propose to perform a validation study at the National Institute for Communicable Diseases (NICD) of South Africa to gain clearance to sell kits in South Africa. Finally, we may also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. By the end of Phase II, we will have preliminary data for submission of an IDE to the FDA to kick-off a clinical study to seek pre market approval (PMA) for sale of the assay system for human diagnostics in the US. PUBLIC HEALTH RELEVANCE: More than 90 % of the 40 million human immunodeficiency virus (HIV)-infected persons live in Third World countries (see www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm). Unfortunately, performing HIV molecular tests in resource poor settings is difficult. BioHelix's proprietary helicase dependent amplification (HDA) platform is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. HDA is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). Indeed, HDA can be performed in a simple 100 water bath. To illustrate the potential of this technology for the third world, we currently sell HDA reagents as part of a teaching kit for use in high schools (sold through Carolina Biological Supply). BioHelix is combining this HDA technology with a low cost (~ 2- 3.75) disposable device specifically designed to perform molecular tests without contaminating the laboratory with amplicons. The device uses a lateral flow system to detect amplification products. Lateral flow devices are common in many moderately complex, and CLIA-waived tests listed on the Food and Drug Administration (FDA) device database. We believe this combination of characteristics makes our proposed product ideal for the HIV vaccine trial sites. A low cost nucleic acid screening test will help identify those people and reduce the spread of the HIV epidemic as well as benefit participants in HIV vaccination trials who become infected with the virus. An estimated 16 to 22 million persons aged 18-64 years are tested annually for HIV in the United States (see http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5347a4.htm). Most of the near patient testing relies on rapid tests like OraQuick while most hospital screening programs use enzyme immuno assay (EIA). Walensky et al. (2008) recently reported that using western blot alone as a confirmation test for oral rapid tests provides conclusive HIV status in only 50% of patients at first follow-up. Such patients are typically asked to return for testing in ~ 1 month. In contrast, performing an HIV-1 RNA test to confirm the rapid test, when a western blot fails to do so, improves this rate to 96.2% in the first round of testing. Considering that our assay system relies of minimal instrumentation we feel our test can offer an additional layer of confirmation for OraQuick positives at the near patient setting and in the developing world and thus relieve the need for asking patients to return for testing in 1 month.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 546.87K | Year: 2010
DESCRIPTION (provided by applicant): This SBIR-AT-NIAID Phase I application addresses the problem of malaria diagnostics in resource- limited settings. BioHelix, has developed an isothermal nucleic acid amplification chemistry called helicase dependent amplification (HDA) that is tolerant of the DNA amplification inhibitors present in whole blood. This proposal will focus on the application of this technology to the detection of malarial asexual parasites without nucleic acid extraction. Based on our preliminary results, the expected sensitivity of the species-specific molecular test (~100 nucleic acid copies/5L blood) better than that possible with current rapid tests for asexual stages (200 copies of parasite/5L blood). Our specific objectives for this 2-year-long project are: 1. Develop an HDA assay to detect P. falciparum, P. vivax, P. ovale, and P. malariae. 2. Develop competitive internal controls (CIC) for the species-specific assay, as well as a 2-plex assay to detect the CIC with the targets in whole blood. 3. Develop manufacturing processes for the proposed assay kit that will enable field-use of the technology. 4. Evaluate the performance of the assay relative to the gold-standard tests. 5. Obtain CE marking for the assay. 6. Submit a pre-IDE to FDA as a preliminary step to seeking FDA clearance for the test. PUBLIC HEALTH RELEVANCE: As malaria is the leading cause death in endemic regions, the current treatment approach is not based on laboratory, or microscopy testing because establishing quality-assured microscopy in rural and resource- poor settings is difficult. The liberal use of anti-malarial drugs that results from such practices presents a problem in that resistance to first line drug treatments is wide-spread. This SBIR-AT-NIAID proposal focuses on the development of a species-specific for the detection of Plasmodium in blood. More sensitive, species- specific, and stage-specific diagnostic tests will help focus drug treatment, and may slow the spread of anti- malarial drug resistance in the pathogen population. Based on our preliminary results, the expected sensitivity of the species-specific DNA test (~100 nucleic acid copies/5L blood) will be better than that possible with current rapid tests for asexual stages (200 copies of parasite/5L blood). A more sensitive test that can be performed in the field near patients would greatly enhance malaria fighting activities. We anticipate tests in the range of 7- 10 for the HDA tests should be possible, if total test volumes reach 200,000.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 482.81K | Year: 2011
DESCRIPTION (provided by applicant): This STTR-AT-NIAID seeks to develop an integrated nucleic acid system based on research done by Catherine Klapperich's laboratory at Boston University. The BU lab-on-a-chip includes a micro solid phase extraction (lt SPE) column, flap valves and hydrophobic vents to gate fluid movements through micro channels, and multiple reaction chambers for experimental replicates and control reactions. Dr. Klapperich's laboratory has successfully performed nucleic acid amplifications in the chip using BioHelix's isothermal amplification process. Biohelix's proprietary technology is called helicase-dependent amplification (HDA). It uses DNA helicases to separate DNA strands during exponential amplification at a constant temperature of65:C. Like the polymerase chain reaction (PCR), HDA assays use a competitive internal control (i.e., a template DNA of known concentration spiked into the raw sample) that can be amplified by the same primers as the analyte, but detected separately; henceallowing us to detect amplification inhibitors in direct clinical samples. The objectives of Phase I are to: 1) modify the BU microfluidic device design to incorporate a lateral flow strip as a means of detecting amplification products using the naked eye; and 2) demonstrate the feasibility of performing integrated tests using the device from aim 1 using CT and NG spiked at 104 in 1 mL urine to establish proof of concept for integrated assays performed in a modified BU chip that includes a lateral flow strip. Our specific aims for Phase II will be to develop a lateral flow reader sub-system, integrate it with the BU instrument to build a pre-commercial looks-like / works-like prototypes, simplify chip design and fabrication to allow for pilot scale manufacturing, and implement a pilot scale manufacturing for the disposable at BioHelix / Quidel. At this stage Qiagen is the most likely manufacturer for the reader. At the conclusion of Phase II, we should be ready for clinical validation of the new assay system. PUBLIC HEALTH RELEVANCE: This Phase I STTR-AT-NIAID project seeks proof-of-concept project will focus on the most abundant sexually transmitted disease (STD) pathogens: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). The scientific literature clearly shows that molecular testing is the most sensitive means of detecting CT and NG and the molecular CT/NG high throughput screening market is currently valued at over 300M/year. Moreover, CDC urges STD clinics to test patients with POC tests if health care workers suspect these patients are unlikely to return to the STD clinic to learn the results of the test. Unfortunately, there are no point-of-care (POC) CT NG molecular tests, and existing POC molecular testing systems like the GeneXpertare too costly for use in STD clinics. This proposed project would seek to remedy to this short coming. We propose to develop a low-cost POC molecular diagnostic system using a design developed by Dr. Catherine Klapperich's laboratory at Boston University(BU). Although the current BU disposable can perform our proprietary isothermal amplification reactions, it does not allow for low cost, instrument-free detection of amplification products; i.e., a fluorescence microscope can be used to detect product formation but this is not a commercially viable option. The device we envisage for Phase I will incorporate a lateral flow strip as a means of detecting the presence or absence of nucleic acid amplification products by simple visual inspection. BioHelix has experience in developing molecular tests using lateral flow based detection.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.89M | Year: 2008
DESCRIPTION (provided by applicant): The objective of our NIAID Biodefense Phase I SBIR program was to develop, and commercialize field deployable nucleic acid analysis devices for use in biodefense applications. We have begun to sell IsoAmp II kits that will allow our clients to develop home brew assays (we list customers in the commercialization plan). For example, the LANL has obtained NIH funding (R01) to develop an assay system for detecting influenza virus using our IsoAmp II assay formulation (http: //www.lanl.gov/science/1663/flu.php). These kits contain an enhancer protein, discovered in our Phase I, that accelerates helicase dependent amplification (HDA); i.e., a thermostable single strand DNA binding protein. HDA chemistry now allows for the speci fic amplification of both ribonucleic acids (RNA), and deoxyribonucleic acids (DNA). Moreover, we have optimized our primer design process to facilitate assay development. We also collaborated with the Keck Graduate Institute (KGI) to develop a small, port able nucleic acid analysis device (a.k.a. Rabbit). Finally, we have developed a number of DNA probe based assays for biodefense targets; e.g. Bacillus anthracis and Ebola virus. In this Phase II, we propose to put into place a functioning QMS at BioHelix, manufacture cGMP reagents and devices that perform the our assays, and to validate our assay system for typing HSV-1 and HSV-2 with a large number (~1,000) clinical samples. The data generated from this clinical study will be used to apply for regulatory c learance from FDA for the sale of this diagnostic system. We have selected HSV as a target for this first clinical assay because this virus is the lead cause of sexually transmitted disease (STD) in the United States. In addition, molecular testing for HSV DNA has greater sensitivity than the traditional viral culture (sensitivity of more than 95 percent, compared with 75 percent for culture). Despite this fact, molecular tests are not as widely used as culture for the diagnosis of genital herpes because of the higher cost of molecular tests. Moreover, molecular testing is viewed as a complex process requiring the isolation of DNA from clinical samples. Finally, as there are no FDA cleared commercial molecular assays for HSV, implementing such tests in clini cal laboratories is burdensome. We believe we can offer a solution to this problem. Genital and oral swab specimens collected in Cellmatics Transport Kit buffer (Becton Dickinson, Sparks, MD) can be diluted 1/2000 for direct analysis by the IsoAmp HSV assa y. This dilution based sample processing will greatly reduce the degree of complexity of our assay system. In addition, we have begun to collaborate with GE Healthcare to develop a Ready-To-Go(tm) IsoAmp formulation for our assays that will eliminate the n eed for multiple, precise volumetric measurements. BioHelix and GE Healthcare plan to develop a formulation under design control in order to manufacture it under cGMP. The instrument developed by KGI will also be transferred to a cGMP manufacturer (Source Scientific), such that by mid-2009 we expect to have a cGMP assay system ready for clinical validation studies.