Stabilization of the RNA Complement of Enveloped Viruses: Beta Testing and Manufacturing Scale-up of a Novel Filter Paper Product for the Field Collection of Dried Blood Spots

有包膜病毒 RNA 补体的稳定性：用于现场采集干血斑的新型滤纸产品的 Beta 测试和生产放大

• 批准号: 10153685
• 负责人: Nan Su
• 金额: $ 100万
• 依托单位: GENTEGRA, LLC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-08 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10153685
• 关键词: Air; Bacteria; Biological Assay; Biological Markers; Blood; Blood Preservation; Businesses; Caliber; Cellulose; Chemicals; Clinical; Collaborations; Collection; Complement; Congenital Abnormality; Cryopreservation; DNA; DNA Viruses; Data; Dengue; Development; Fingers; Flavivirus; Formulation; Free Radicals; Funding; Genome; Goals; HIV; HIV-1; Human; International; Laboratories; Length; Link; Liquid substance; Marketing; Medical; Methods; Microcephaly; Modeling; Molecular Epidemiology; Mutation; Neurologic; Paper; Pathology; Phase; Planets; Production; Public Health; Publications; Publishing; Quantitative Reverse Transcriptase PCR; RNA; RNA Stability; RNA Virus Infections; RNA Viruses; RNA analysis; Ramp; Reagent; Recovery; Refrigeration; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Review Literature; Sales; Sampling; Screening procedure; Ships; Site; Small Business Innovation Research Grant; Specimen; Speed; Spottings; Structure; Technology; Temperature; Testing; Time; Travel; Tropical Climate; Universities; Validation; Viral; Viral Epidemiology; Virus; Whole Blood; Work; ZIKA; base; detection limit; healing; inhibitor/antagonist; manufacturing scale-up; new technology; news; next generation sequencing; novel; nuclease; pandemic disease; preservation; pressure; prototype; research and development; sample collection; screening; small molecule; success; tool; vector; viral RNA; viral detection; virology; virus envelope

Abstract. 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.

抽象的。 1960年，基于滤纸的样本收集与古特里卡革命性的血液为基础的 通过采集伤口采血筛选出生缺陷小分子生物标志物 直接在滤纸上，以形成干血斑（DBS）。在2000年，古特里卡 （Whatman 903）DBS样本收集（由该团队和许多其他团队）扩展到DNA 生物标志物以及血液传播细菌和DNA病毒的分析。 DNA的巨大成功，已经将研究转移到了“更高”的目标，即收集和 保留DBS的高度不稳定的RNA补体：重点是使 无冷冻采血。这支持RNA病毒感染的筛查。许多 随后出现了出版物，以确定Whatman 903 DBS用于HIV， 寨卡病毒和其他新出现的RNA病毒。 好消息是，这几项已发表的研究已经证实，病毒RNA可以 通过定量RT-PCR（qRT-PCR）从手指穿刺获得的DBS中回收，如果 收集和储存“适当”。 坏消息是，已发表的研究表明，可以获得足够的RNA稳定性 仅在不现实的条件下（4 ℃，-20 ℃储存）。在热带环境条件下 这些研究得出结论，DBS的收集和运输 不起作用：即RNA在不到一周的时间内降解超过qRT-PCR检测限。 该阶段IIB SBIR专注于一对全新滤纸的beta测试， 产品（GT-A和GT-B）由申请人团队创建。这将是第一次， 在真实的热带条件下收集/运输病毒血;连续40 ℃储存2周， 产生用于标准qRT-PCR分析的RNA。第二B阶段开发的核心技术， 最初由GenTegra在DARPA的资助下开发，使用两种不同的方法来病毒 RNA保存：GT-A，稳定DBS中病毒的结构，从而利用 由完整结构提供的天然保护;和GT-B，立即裂解病毒， 在DBS中同时引入一组新的核酸酶和自由基抑制剂。 IIB期β测试将由两个顶级实验室进行验证：ATCC（针对寨卡病毒）和斯坦福大学（针对HIV-1）， 对于这两种技术。GenTegra将与其他病毒进行平行测试。在完成此 IIB阶段，一种或两种产品将准备好进行国际销售和营销， GenTegra和Ahlstrom-Munksjö之间的现有合作。