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

SNAP-X：开发诱变策略和高密度蛋白质阵列以全面展示蛋白质变体

• 批准号: 10203604
• 负责人: Mary Szatkowski Ozers
• 金额: $ 12.91万
• 依托单位: PROTEOVISTA, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203604
• 关键词: 2019-nCoV; Address; Administrative Supplement; Affect; Amino Acid Substitution; Antibodies; Area; Award; Back; Binding; Biological Assay; Biological Models; COVID-19; Cancer Patient; Cells; Collection; Communicable Diseases; Communities; Data; Development; Ensure; Exploratory/Developmental Grant; Genome; Human; Immunofluorescence Immunologic; Individual; Infection; Link; Location; Methods; Mutagenesis; Mutate; Mutation; Oncoproteins; Parents; Pathogenicity; Pharmaceutical Preparations; Phase; Plasmids; Point Mutation; Populations at Risk; Protein Array; Proteins; Receptor Cell; Research; Risk; S-nitro-N-acetylpenicillamine; Small Business Innovation Research Grant; Stains; Symptoms; Treatment Efficacy; Vaccines; Validation; Variant; Viral; Viral Proteins; Viral Structural Proteins; Virus; Virus Diseases; anticancer research; cancer cell; cell determination; density; improved; innovation; insight; interest; mutant; mutation screening; next generation sequencing; novel; particle; receptor; response; technology development; tumor progression; vaccine development; viral transmission

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.

该申请被作为行政补充提交，以响应被确定为非CA-20-042的特殊利益通知（NOSI）。对高度传染病的研究，尤其是由病毒引起的疾病，对科学研究界面临着独特的生物安全挑战。与其失去这项研究所能提供的见解，不如存在常见的解决方法，以减少研究感染性病毒的风险，通过检查在没有完全传染性粒子的情况下由病毒编码的蛋白质。这种方法已用于许多其他病毒，并已被证明有效地获得了有价值的数据。然而，诸如SARS-COV-2突变的病毒导致许多不同的菌株和序列变化，并且很难预测哪些突变会改变病毒传播，感染症状或疫苗 /治疗效率。鉴于癌症患者构成了Covid-19的高危人群，因此在癌细胞如何影响病毒感染或病毒影响癌症进展的背景下，这更加复杂。完全缺乏以高通量，系统和全面（即每种突变）方式检查病毒蛋白突变的方法。为了帮助解决这一挑战，我们建议利用创新的高吞吐量诱变策略，以全面地产生SARS-COV-2 S“ SPIKE”蛋白的每种可能点突变的质粒，作为人类进入受体的关键病毒识别蛋白，以加速功能研究加速和疫苗开发。所得的质粒集对于鉴定关键S蛋白变异的功能测定特别有用，这是当前Covid-19研究的重要领域。 S蛋白突变质粒集将通过人类细胞中变异质粒的表达来验证，并确定哪些变体与不同的市售SARS-COV-2 S蛋白抗体结合。这些结果将通过免疫荧光染色确认。尽管我们设想这些变体在其模型系统中被癌症研究界雇用，但总体而言，SARS-COV-2研究社区可以同样有效地使用它们。提出的方法通过避免其实质突变偏置并确保每个质粒的一个突变来显着改善随机诱变，从而通过流线型分析来确保一个突变。与容易出错的PCR相比，下一代测序（NGS）将确认此策略。在成功验证S蛋白变体池之后，我们将为SARS-COV-2基因组编码的另外三个密钥蛋白生成突变质粒集。这种行政补充是在我们的父级IMAT奖的范围内，即技术开发R21机制，旨在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与微阵列上的特定特征位置联系起来。成功完成此管理补充剂后，我们打算准备SBIR I期，将每个单独的SARS-COV-2突变体质粒分类为多层板的不同井，以便为Covid-19的研究社区提供更多可用性。