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: 10239237
• 负责人: Alan B Moy
• 金额: $ 34.6万
• 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/10239237
• 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; 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; differentiation protocol; 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; regeneration potential; 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.

Cellular Engineering Technologies（CET）已提交此提案以响应RFA-GM- 19-001. CET提出了一个直接的第二阶段SBIR应用程序，以创建更多的可重复性 人诱导多能干细胞（iPSC）和创造用于生长、维持 和鉴定iPSC。iPSC制造和后续生产中的一个主要挑战是 分化是由于非随机的染色体变异导致的遗传不稳定性的出现。 突变。遗传不稳定性导致遗传变异的克隆扩张， iPSC异质性。促进遗传不稳定性的实验变量并不好 明白然而，癌基因依赖性重编程和延长细胞培养显然是 与遗传不稳定性有关。此外，现有的iPSC重编程方法适用于 临床前研究尚未优化以减轻感染性，炎性， 细胞治疗的肿瘤和遗传风险。因此，iPSC重编程应该是 标准化，包括非整合，无病毒和无癌基因的方法，这将 在贴壁和悬浮细胞中提供可再现的iPSC。这一里程碑将缓解 致癌和病毒效应，可以减少iPSC制造中的遗传不稳定性， 分化此外，如果生长因子能够促进iPSC的再生和分化， 显示完全人翻译后修饰（PTM）。虽然细菌制造 生长因子和非人糖基化肽和蛋白质在茎中普遍存在 在细胞领域，它们表现出比它们的天然人类对应物不同的生物活性。因此，使用 缺乏完全人PTM的生长因子可能会放大遗传不稳定性并扭曲细胞生长， iPSC和分化细胞的表型，特别是对于多个分化步骤， 需要多种生长因子。CET是一家生物技术公司， 人体干细胞和一流的非整合，无饲养，无病毒， 无致癌基因的iPSC重编程方法，该方法已被验证并发表于 贴壁细胞和悬浮细胞。此外，CET是选择的唯一来源制造商 出生后的干细胞这些能力使CET能够获得永生化的人类产后 设计用于完全人PTM的生物学生物处理的干细胞。因此，CET准备 通过制造过程开发iPSC和分化细胞， 不稳定该提案的重点是开发一个制造平台，以创建GLP 和GMP级iPSC，即使在随后的 神经祖细胞分化