SNAP-X: Development of a Mutagenesis Strategy and High Density Protein Array to Comprehensively Display Protein Variants

本申请是作为对特殊利益通知（NOSI）的行政补充提交的，该通知标识为NOT-CA-20-042。对高度传染性疾病，特别是由病毒引起的疾病的研究，对科学研究界提出了独特的生物安全挑战。与其失去这项研究可以提供的见解，还不如通过在没有完全感染性颗粒的情况下检查病毒编码的蛋白质来降低研究感染性病毒的风险。这种方法已被用于许多其他病毒，并已被证明在获取有价值的数据方面是有效的。然而，像SARS-CoV-2这样的病毒会发生突变，导致许多不同的毒株和序列变异，很难预测哪些突变会改变病毒的传播、感染症状或疫苗/治疗效果。鉴于癌症患者是Covid-19的高危人群，在癌细胞如何影响病毒感染或病毒如何影响癌症进展的背景下，情况就更加复杂了。以高通量、系统和全面（即每个突变）的方式检测病毒蛋白突变的方法完全缺乏。为了帮助解决这一挑战，我们建议利用我们创新的高通量诱变策略，全面生成sars - cov - 2s“刺突”蛋白每一个可能的点突变的质粒，作为人类进入受体的关键病毒识别蛋白，以加速功能研究和疫苗开发。由此产生的质粒集对于识别关键S蛋白变异的功能分析特别有用，这是当前Covid-19研究的一个重要领域。S蛋白突变质粒将通过在人细胞中表达变体质粒并确定哪些变体与不同的市售SARS-CoV-2 S蛋白抗体结合来验证。这些结果将通过免疫荧光染色证实。虽然我们设想这些变体被癌症研究界在他们的模型系统中使用，但它们也可以被一般的SARS-CoV-2研究界同样有效地使用。该方法避免了易出错PCR的大量突变偏差，并确保每个质粒只有一个突变，从而大大提高了随机诱变的效率。这一策略将被下一代测序（NGS）证实，与容易出错的PCR进行比较。在成功验证S蛋白变异库后，我们将为SARS-CoV-2基因组编码的另外三个关键蛋白生成突变质粒集。该行政补充属于我们的母公司IMAT奖的范围，这是一种技术开发R21机制，用于生成三种关键癌蛋白的所有突变体，并将表达的蛋白质变体连接回微阵列上的特定特征位置。在成功完成本行政补充后，我们打算准备SBIR I期，将每个SARS-CoV-2突变质粒分选到多孔板的不同孔中，以便为Covid-19研究界提供更多可用性。

This application is being submitted as an Administrative Supplement in response to the Notice of Special Interest (NOSI) identified as NOT-CA-20-042. Study of highly infectious diseases, especially those caused by viruses, poses unique biosafety challenges to the scientific research community. Rather than lose the insights this research can provide, common workarounds exist to reduce the risk of studying infectious virus by examining the proteins encoded by the virus in the absence of a fully infectious particle. This approach has been employed for many other viruses and has been proven effective in obtaining valuable data. However, viruses like SARS-CoV-2 mutate leading to many different strains and sequence variations, and it can be difficult to predict which mutations alter virus transmission, infection symptoms, or vaccine / treatment efficacy. Given that cancer patients constitute an at-risk population for Covid-19, this is even more complicated in the context of how cancer cells affect virus infection or how the virus affects cancer progression. Methods to examine virus protein mutations in a high throughput, systematic, and comprehensive (i.e. every mutation) manner are completely lacking. To help address this challenge, we propose to harness our innovative high throughput mutagenesis strategy to comprehensively generate plasmids of every possible point mutation of the SARS-CoV-2 S “spike” protein as the key viral recognition protein of the human entry receptor, towards accelerating functional studies and vaccine development. The resulting plasmid set is especially useful for functional assays to identify critical S protein variants, which is a vital area of current Covid-19 research. The S protein mutant plasmid set will be validated by expression of the variant plasmids in human cells and determination of which variants bind to different commercially available SARS-CoV-2 S protein antibodies. These results will be confirmed by immunofluorescence staining. While we envision these variants being employed by the cancer research community in their model systems, they can be used equally effectively by the SARS-CoV-2 research community in general. The proposed method significantly improves on random mutagenesis by error-prone PCR by avoiding its substantial mutational bias and ensuring exactly one mutation per plasmid for streamlined analysis. This strategy will be confirmed by next generation sequencing (NGS), comparing against error-prone PCR. Following successful validation assays for the S protein variant pool, we will generate mutant plasmid sets for three additional key proteins encoded by the SARS-CoV-2 genome. This Administrative Supplement is within the scope of our parent IMAT award, which is a technology development R21 mechanism, to generate all mutants of three key oncoproteins and link the expressed protein variants back to specific feature locations on a microarray. Upon successful completion of this Administrative Supplement, we intend to prepare a SBIR Phase I to sort each individual SARS-CoV-2 mutant plasmid into distinct wells of multiwell plates for additional availability to the Covid-19 research community.