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

这一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。精选的疫苗和基因疗法是通过病毒载体提供的，但精确的剂量鲜为人知，这导致了质量控制方面的担忧。目前的测量技术是间接的、不精确的、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精确的诊断基准和适当的治疗剂量至关重要。该项目开发了一种测量病毒浓度和传染性的新技术。拟议的项目涉及病毒量化技术，因为它们受到几个限制。典型的传染性分析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，自然病毒并不是为了便于识别而设计成含有荧光转基因的，而且一些颗粒不会含有病毒基因组--这导致了那些含有基因“有效载荷”的病毒与空载体之间的竞争。一般来说，病毒颗粒在给定样本中的密度、传染性和复制效率鲜为人知。该项目提出了一项具有三项功能的技术：量化粒子总数，测量基因组包含单位，以及评估给定病毒样本的传染性。这种新方法结合了利用生物物理特性识别病毒颗粒的纳米孔技术和单分子RNA荧光原位杂交，以区分受感染细胞中的病毒RNA单分子。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.