PFI-TT: Developing an integrated platform for high accuracy measurements of viral particle count and infectious titer

该创新-技术转化伙伴关系（PFI-TT）项目的更广泛影响/商业潜力是测量临床或研究环境中的病毒浓度。 选择疫苗和基因疗法是通过病毒载体传递的，但精确的剂量知之甚少，导致质量控制问题。目前的测量技术是间接的、不精确的和非标准的;它们需要时间和专门的设备。病毒颗粒的准确测量对于诊断的精确基准和治疗剂的适当剂量至关重要。该项目开发了一种新技术来测量病毒浓度和传染性。拟议的项目涉及病毒定量技术，因为它们受到一些限制。典型的感染性测定高度依赖于用于病毒感染的特定细胞系，导致实验室之间的高度变异性。此外，天然病毒没有被改造成含有荧光转基因以便于识别，并且一些颗粒将不含有病毒基因组-导致含有基因“有效载荷”和空载体之间的竞争。一般来说，对给定样品中病毒颗粒的密度、感染性和复制效率知之甚少。该项目提出了一种具有三种功能的技术：量化颗粒总数，测量含有基因组的单位，以及评估给定病毒样品的感染性。该新方法结合了纳米孔技术，利用生物物理特性识别病毒颗粒和单分子RNA荧光原位杂交，以区分感染细胞中的病毒RNA的单分子。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this Partnerships for Innovation - Technology Translation (PFI-TT) project is to measure viral concentrations in clinical or research settings. Select vaccines and gene therapies are delivered via viral vectors, but the precise dosing is poorly known, leading to quality control concerns. Current measurement techniques are indirect, imprecise, and nonstandard; and they require time and specialized equipment. Accurate measurement of viral particles is critical for precise benchmarking of diagnostics and appropriate dosing of therapeutics. This project develops a new technology to measure viral concentrations and infectivity. The proposed project addresses viral quantification techniques as they suffer from several limitations. Typical infectivity assays are highly dependent on the specific cell line used for viral infection, leading to high variability between labs. Furthermore, natural viruses are not engineered to contain a fluorescent transgene for easy identification, and some particles will not contain the viral genome - leading to competition between those containing the gene “payload” and empty vectors. In general, the density, infectivity, and replication efficiency of viral particles in a given sample is poorly known. This project advances a technology with three functioins: quantifying the total number of particles, measuring genome-containing units, and assessing infectivity of a given sample of the virus. The new method combines nano-pore technology using biophysical properties to identify viral particles with single-molecule RNA fluorescence in situ hybridization to discriminate down to single molecules of viral RNA in infected cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.