SBIR-STTR Award

The Flaviviruses, especially Dengue, Zika and Yellow Fever have been a major public health concern for centuries in semi and tropical climates, including parts of the southern US. Presently, these viruses are the cause of more than 300 million infections per year. Global travel, alteration of the underlying mosquito vector’s geographical range, the possibility of RNA genome mutation resulting from vector changes and new environmental selective pressures all appear to have influenced the spread of Flavivirus infection to new human populations. More recently, Zika, with the proposed link to microcephaly and other neurological birth defects, has thrust into the spotlight an urgent need for an economical, international-scale screening of susceptible populations. We believe there is a critical need to develop new technology to collect, transport and recover blood or blood products containing this pervasive class of unstable enveloped RNA virus, in the complete absence of any sort of protection via refrigeration. The goal of this new technology is the recovery of intact viral genomes to support viral load and nucleic acid testing: to improve the diagnoses of Flavivirus infection and, coupled with new, now- inexpensive methods of Next Gen sequencing, to provide the real possibility of discovering how the viral genome may be mutating under selective pressure. The overarching requirement for such new sample collection and preservation technology is that it must be robust enough to preserve even the most unstable of the Flavivirus RNAs, yet inexpensive enough to support both population scale public health screening and sophisticated molecular epidemiology to detect mutational drift (via Next-Gen sequencing) while being deployable in a way that facilitates implementation in isolated, tropical, poor, susceptible populations in the complete absence of cryogenic preservation. GenTegra, in collaboration with Ahlstrom (the world-leader in medical grade filter paper for newborn screening) have recently co-developed a medical grade cellulose paper that supports long term stabilization of the DNA in blood at ambient temperature. In this Phase II SBIR, GenTegra will leverage that ongoing collaboration with Ahlstrom, coupled with our expertise in the development and implementation of the new stabilization chemistries, to develop a completely new filter paper based product for Flavivirus RNA field collection and preservation. To achieve those highly Innovative and Significant goals, we propose 3 Specific Aims, to be executed over 2 years. Upon successful completion, this program will yield a product prototype that will be ready for manufacturing scale-up and international commercial deployment. The Investigators and Research Environment among the collaborating institutions (GenTegra, ATCC, and Ahlstrom) are of international caliber with respect to delivering this (21st century) approach to paper-based biosample collection. It should be noted that, although this SBIR is purely focused on Flavivirus, the technology may be generally applicable to ambient temperature collection and transport of all RNA viruses in animals and man.

Public Health Relevance Statement:
Project Narrative We describe a two-year Phase II SBIR program, comprising a collaboration between GenTegra LLC (a small California biotech) The ATCC (a large Virginia not-for-profit) and Ahlstrom (a multinational paper company, with its US Medical Products division in Pennsylvania) that is focused on development of a completely new approach to the collection and preservation of RNA viruses, especially the Dengue and Zika Flaviviruses. This new technology is based on ambient temperature preservation of the virus, as collected in the field from finger prick blood applied directly to filter paper. It is proposed that this SBIR-funded technology, which is based on a new class of inexpensive, chemically treated filter paper that has been invented with DARPA support, will revolutionize the use of sophisticated RNA testing in epidemiology and public health screening, where the samples of interest must be collected in low resource environments.

Project Terms:
Animals; Award; Biological; Biological Assay; Biological Preservation; Biotechnology; Blood; Blood Preservation; blood product; Caliber; California; Cellulose; Chemicals; Chemistry; Collaborations; Collection; commercialization; Congenital Abnormality; Coupled; cryogenics; Dengue; Dengue Virus; Development; Diagnosis; DNA; elastomeric; Eligibility Determination; Environment; Epidemiology; Fingers; Flaviviridae; Flaviviridae Infections; Flavivirus; Flavivirus Infections; Formulation; Funding; Geography; Goals; Heat Stress Disorders; Human; improved; Infection; innovation; Institution; interest; International; Lead; Legal patent; Link; Logistics; man; manufacturing scale-up; Medical; Methods; Microcephaly; Microspheres; Modeling; Molecular Epidemiology; Mutate; Mutation; Neonatal Screening; Neurologic; new technology; next generation sequencing; novel; novel strategies; Nucleic Acid Amplification Tests; Paper; Pennsylvania; Phase; Plasma; Plasticizers; Population; Porifera; Preparation; pressure; Printing; Production; programs; Protocols documentation; prototype; Public Health; Recovery; Refrigeration; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; RNA; RNA Viruses; S-Adenosylmethionine; sample collection; Sampling; screening; Shipping; Small Business Innovation Research Grant; Solid; Technology; Temperature; Testing; Travel; Tropical Climate; vector; vector mosquito; Viral Genome; Viral Load result; viral RNA; Virginia; Virus; Work; Yellow Fever; Yellow fever virus; Zika Virus

In 1960, filter paper based sample collection with Guthrie Cards revolutionized blood-based screening of small molecule biomarkers of birth defect: via the collection of heal stick blood directly onto filter paper, to form a dried blood spot (DBS). In the 2000's, Guthrie Card (Whatman 903) DBS sample collection was extended (by this Team and many others) to DNA biomarkers and to analysis of blood-borne bacteria and DNA viruses. That substantial success with DNA, has shifted research to the “loftier” goal of collecting and preserving the highly unstable RNA complement of DBS: with emphasis on tools to enable the refrigeration-free collection of blood. This supports screening of RNA virus infections. Numerous publications have subsequently emerged, to establish the use of Whatman 903 DBS for HIV, Zika and other emerging RNA viruses. The Good News is, those several published studies have confirmed viral RNA can be recovered by quantitative RT-PCR (qRT-PCR) from DBS obtained from a finger prick, if collected and stored “appropriately”. The Bad News is, published studies have shown that adequate RNA stability can be obtained only under unrealistic conditions (4C, -20C storage). Under conditions of tropical ambient temperatures (30C-40C) those studies have concluded that DBS collection and transport does not work: i.e. RNA degrades beyond the qRT-PCR detection limit within less than a week. This Phase IIB SBIR is focused on beta testing of a pair of fundamentally new filter paper based products (GT-A & GT-B) created by the Applicant Team. For the first time, it will be possible to collect/ship viremic blood under realistic tropical conditions; 2 weeks of continuous 40C storage, yielding RNA for standard qRT-PCR analysis. The core technology exploited in this Phase IIB, originally developed by GenTegra with DARPA funding, uses two different approaches to viral RNA preservation: GT-A, to stabilize the structure of the virus in a DBS, thus exploiting the natural protection afforded by that intact structure; and GT-B, to instantly lyse the virus, simultaneously introducing into the DBS, a novel panel of nuclease and free radical inhibitors. Phase IIB beta test is to be validated by two top labs: ATCC (for Zika) and Stanford (for HIV-1), for both technologies. GenTegra will in parallel test with other viruses. Upon completion of this Phase IIB, one or both products will be ready for international sales & marketing, supported by the Existing collaboration between GenTegra and Ahlstrom-Munksjö.

Public Health Relevance Statement:
Project Narrative A Phase IIB SBIR is described, which is focused on testing and validation of two filter paper based products, GT-A and GT-B, invented by GenTegra, to preserve the blood borne RNA viruses that may be collected in the field as a dried blood spot, delivered from a finger prick. HIV-1 and Zika will be the first test cases to be validated, at two top international laboratories: the ATCC (Zika) and Stanford (HIV-1). It is proposed that both products will be unique in molecular epidemiology as they will be the first to stabilize RNA viruses, in the face of extremely primitive field conditions (no cold storage capability), to support advanced methods of RNA based viral detection and sequencing.

Project Terms:
Air; Bacteria; base; Biological Assay; Biological Markers; Blood; Blood Preservation; Businesses; Caliber; Cellulose; Chemicals; Clinical; Collaborations; Collection; Complement; Congenital Abnormality; Cryopreservation; Data; Dengue; Detection; Development; DNA; DNA Viruses; Fingers; Flavivirus; Formulation; Free Radicals; Funding; Genome; Goals; healing; HIV; HIV-1; Human; inhibitor/antagonist; International; Laboratories; Length; Link; Liquid substance; manufacturing scale-up; Marketing; Medical; Methods; Microcephaly; Modeling; Molecular Epidemiology; Mutation; Neurologic; new technology; news; next generation sequencing; novel; nuclease; pandemic disease; Paper; Pathology; Phase; Planets; preservation; pressure; Production; prototype; Public Health; Publications; Publishing; Quantitative Reverse Transcriptase PCR; Ramp; Reagent; Recovery; Refrigeration; Research; research and development; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Review Literature; RNA; RNA analysis; RNA Stability; RNA Virus Infections; RNA Viruses; Sales; sample collection; Sampling; screening; Screening procedure; Ships; Site; Small Business Innovation Research Grant; small molecule; Specimen; Speed; Spottings; Structure; success; Technology; Temperature; Testing; Time; tool; Travel; Tropical Climate; Universities; Validation; vector; Viral; viral detection; Viral Epidemiology; viral RNA; virology; Virus; virus envelope; Whole Blood; Work; Zika Virus