Improving The Reproducibility and Genetic Stability of IPSC and Differentiated Cells Through Oncogene-Free Reprogramming and Fully Human Growth Factors

通过无癌基因重编程和全人类生长因子提高 IPSC 和分化细胞的再现性和遗传稳定性

• 批准号: 10080387
• 负责人: Alan B Moy
• 金额: $ 37.5万
• 依托单位: CELLULAR ENGINEERING TECHNOLOGIES, INC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2022-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10080387
• 关键词: 3-Dimensional; Biological; Biotechnology; Cell Culture Techniques; Cell Differentiation process; Cell Therapy; Cells; Clonal Expansion; Computer Analysis; Coupled; DNA; Drug Screening; Exhibits; Genetic; Genetic Risk; Genomics; Goals; Growth Factor; Heterogeneity; Human; Inflammatory; Link; Manufacturer Name; Measurement; Mediating; Methods; Mutation; Oncogenes; Oncogenic; Peptides; Phase; Phenotype; Post-Translational Protein Processing; Proteins; Protocols documentation; Publishing; RNA; Reference Standards; Regenerative Medicine; Reproducibility; Risk; Safety; Small Business Innovation Research Grant; Source; Standardization; Suspensions; System; Technology; TimeLine; Viral; Virus; base; bioprocess; c-myc Genes; cellular engineering; design; drug discovery; genetic analysis; genetic variant; improved; induced pluripotent stem cell; innovation; manufacturing process; neoplastic; nerve stem cell; postnatal; postnatal human; pre-clinical; pre-clinical research; relating to nervous system; relational database; response; scale up; stem cell differentiation; stem cells; tool

Cellular Engineering Technologies (CET) has submitted this proposal in response to RFA-GM- 19-001. CET has proposed a direct Phase II SBIR application to create more reproducible human induced pluripotent stem cells (iPSCs) and create methods for growing, maintaining, and authenticating iPSCs. A major challenge in iPSC manufacturing and subsequent differentiation is the emergence of genetic instability that result from non-random chromosomal mutations. Genetic instability results in clonal expansion of genetic variants that increases iPSC heterogeneity. The experimental variables that promote genetic instability are not well understood. Yet, oncogene-dependent reprogramming and prolong cell culturing are clearly linked to genetic instability. Moreover, prior iPSC reprogramming methods adapted for preclinical research have not been optimized to mitigate against the infectious, inflammatory, neoplastic and genetic risks for cell therapy. Thus, iPSC reprogramming should be standardized to include non-integrating, virus-free and oncogene-free methods, which would offer reproducible iPSC in adherent and suspension cells. This milestone would mitigate oncogenic and viral effects that could reduce genetic instability in iPSC manufacturing and differentiation. Further, iPSC reproducibility and differentiation would improve if growth factors displayed fully human posttranslational modification (PTM). While bacterial-manufactured growth factors and non-human glycosylated peptides and proteins are ubiquitous in the stem cell field, they exhibit differential bioactivity than their native human counterparts. Thus, using growth factors that lack a fully human PTM may amplify the genetic instability and distort cell phenotype of iPSC and differentiated cells, particularly for multiple differentiation steps that require multiple growth factors. CET is a biotechnology company with a diverse pipeline of human somatic stem cells and a first-in-class non-integrating, feeder-free, virus-free and oncogene-free iPSC reprogramming approach that has been validated and published for adherent cells and suspension cells. Moreover, CET is the sole source manufacturer of select postnatal stem cells. These capabilities allowed CET to obtain immortalized human postnatal stem cells designed for biologic bioprocessing of fully human PTM. Thus, CET is poised to develop iPSC and differentiated cells through manufacturing processes that mitigate genetic instability. The focus of this proposal will be to develop a manufacturing platform to create GLP and GMP-grade iPSC with the least amount of genetic instability even after subsequent neuroprogenitor cell differentiation.

细胞工程技术公司(CET)已提交此提案，以回应RFA-GM- 19-001。CET提出了直接第二阶段SBIR应用程序，以创建更具重复性的应用程序 人类诱导多能干细胞(IPSCs)，并创造出生长、维持、 以及对IPSC进行认证。IPSC制造和后续的主要挑战 分化是由非随机染色体引起的遗传不稳定性的出现。 突变。遗传不稳定性导致基因变异的克隆性扩张，增加了 IPSC异质性。促进遗传不稳定的实验变量并不是很好 明白了。然而，癌基因依赖的重新编程和延长细胞培养显然是 与遗传不稳定有关。此外，现有的IPSC重编程方法适用于 临床前研究尚未优化以缓解传染性、炎症性、 细胞治疗的肿瘤和遗传风险。因此，IPSC重新编程应该是 标准化以包括非整合、无病毒和无癌基因的方法，这将 在贴壁细胞和悬浮细胞中提供可复制的IPSC。这一里程碑将缓解 致癌和病毒效应，可减少IPSC制造中的遗传不稳定性 差异化。此外，如果生长因素，IPSC的重复性和差异性将得到改善 显示了完全的人类翻译后修饰(PTM)。而细菌制造的 生长因子和非人糖基化多肽和蛋白质在茎中普遍存在。 在细胞场，它们表现出与原生人类对应的不同的生物活性。因此，使用 缺乏完全人类PTM的生长因子可能会放大遗传不稳定性并扭曲细胞 IPSC和分化细胞的表型，特别是在多个分化步骤中 需要多种增长因素。CET是一家生物技术公司，拥有多种流水线 人体干细胞和一流的非整合、无饲养层、无病毒和 已验证并发布的无癌基因IPSC重新编程方法 贴壁细胞和悬浮细胞。此外，CET是SELECT的唯一来源制造商 出生后干细胞。这些能力使CET获得了不朽的人类出生后 为完全人类PTM的生物生物处理而设计的干细胞。因此，CET准备好了 通过制造工艺开发IPSC和分化细胞，以减轻基因 不稳定。这项提案的重点将是开发一个制造平台，以创建GLP GMP级IPSC的遗传不稳定性最小，即使在 神经前体细胞分化。