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.

本申请作为行政补充材料提交，以响应标识为 NOT-CA-20-042 的特别利益通知 (NOSI)。高传染性疾病的研究，特别是由病毒引起的疾病，给科学研究界带来了独特的生物安全挑战。存在常见的解决方法，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，而不是失去这项研究可以提供的见解。这种方法已用于许多其他病毒，并已被证明可以有效获取有价值的数据。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，并且很难预测哪些突变会改变病毒传播、感染症状或疫苗/治疗效果。鉴于癌症患者构成了 Covid-19 的高危人群，考虑到癌细胞如何影响病毒感染或病毒如何影响癌症进展，情况变得更加复杂。完全缺乏高通量、系统、全面（即每一个突变）方式检测病毒蛋白突变的方法。为了帮助应对这一挑战，我们建议利用我们创新的高通量诱变策略，全面生成 SARS-CoV-2 S“刺突”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，以加速功能研究和疫苗开发。由此产生的质粒集对于识别关键 S 蛋白变体的功能测定特别有用，这是当前 Covid-19 研究的一个重要领域。 S 蛋白突变质粒组将通过在人类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得到证实。虽然我们设想癌症研究界在其模型系统中使用这些变体，但一般 SARS-CoV-2 研究界也可以同样有效地使用它们。该方法通过避免其显着的突变偏差并确保每个质粒恰好有一个突变以进行简化分析，显着改进了易错 PCR 的随机诱变。该策略将通过下一代测序 (NGS) 与容易出错的 PCR 进行比较来证实。在成功验证 S 蛋白变体库后，我们将为 SARS-CoV-2 基因组编码的另外三种关键蛋白生成突变质粒集。本行政补充文件属于我们的母公司 IMAT 奖项的范围，该奖项是一种技术开发 R21 机制，用于生成三种关键癌蛋白的所有突变体，并将表达的蛋白变体连接回微阵列上的特定特征位置。成功完成本行政补充后，我们打算准备 SBIR 第一阶段，将每个单独的 SARS-CoV-2 突变体质粒分选到多孔板的不同孔中，以供 Covid-19 研究界使用。