STTR Phase I: Improving Biomanufacturing in Chinese Hampster Ovary Cells Using Structurally Interacting RNA (sxRNA)

STTR 第一阶段：利用结构相互作用 RNA (sxRNA) 改善中国仓鼠卵巢细胞的生物制造

• 批准号: 1520807
• 负责人: Edward Eveleth
• 金额: $ 22.5万
• 依托单位: HocusLocus, LLC
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1520807&HistoricalAwards=false
• 关键词: STTR; Phase; Improving; Biomanufacturing; Chinese

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project is to develop the technology to power a more efficient platform for the most common method of production of biological therapeutics, the Chinese Hamster Ovary (CHO) cell line. Each batch production cycle of a given therapeutic is large and represents a significant investment and significant profit, and any methods to increase, even incrementally, the yield of such a batch would yield great profits. Currently, these cells split their metabolic energy into producing both the therapeutic, and a protein used to select for highest producers. The proposed system utilizes the transient expression of a different kind of selection marker - one that frees significant metabolic resources to focus solely on the production of the therapeutic once the selection phase is done. Increasing yields will drive down costs, and make these treatments available to a wider demographic. The biomanufacturing companies are searching for new methods to increase product yield, and are ready to spend significant time and resources to maintain their cutting edge status and profitability.This STTR Phase I project proposes to develop a selection method to significantly increase CHO cell biotherapeutic production. Current methods sacrifice yield because the cells must produce the selectable marker as well as the product, at a heavy burden to the cells. The proposed technology will use the transient expression of the selectable marker, glutamine synthase, tightly controlled by and active only in the presence of the gene of interest (GOI) through our structural interacting RNA (sxRNA) technology. Once the selection phase is complete, metabolic costs of selectable marker will cease and metabolic resources will go to GOI production. sxRNA links the expression of the selectable marker on an ectopic mRNA to a miRNA in the splicing product during GOI mRNA maturation. Stringency of selection is, therefore, directly proportional to GOI copy number and production. The plan is to optimize sxRNA technology through computational and empirical screens, and use the most suitable candidates in a side-by-side comparison with the traditional CHO cell glutamine synthetase system. Benchmarks for success will include assays for gene copy numbers by real-time PCR, and active product yield versus costs. Long-term stability of the GOI also will be monitored over many passages following the termination of the selection phase.

这个小型企业技术转移（STTR）项目的更广泛的影响/商业潜力是开发该技术，为最常见的生物疗法生产方法，中国仓鼠卵巢（CHO）细胞系提供更有效的平台。给定治疗性的每个批处理生产周期都很大，代表了巨大的投资和可观的利润，任何增加的方法，即使从逐步增加，这种批次的收益率也会产生巨大的利润。目前，这些细胞将其代谢能量分解为产生治疗性，并且是用于选择最高生产者的蛋白质。所提出的系统利用了不同类型的选择标记的瞬态表达 - 一种方法可以释放大量的代谢资源，仅一旦完成选择阶段就专注于治疗阶段的生产。增加产量将降低成本，并使这些治疗可用于更广泛的人群。生物制造公司正在寻找提高产品产量的新方法，并准备花费大量时间和资源来维持其最先进的地位和盈利能力。该STTR I阶段项目提议开发一种选择方法，以显着增加Cho Cell生物治疗生产。当前的方法牺牲产量，因为细胞必须产生可选的标记和产物，并承担沉重的细胞负担。所提出的技术将使用可选标记，谷氨酰胺合酶的瞬态表达，仅在感兴趣基因（GOI）的存在下，通过我们的结构相互作用RNA（SXRNA）技术，受到紧密控制和活跃。选择阶段完成后，可选标记的代谢成本将停止，代谢资源将用于GOI生产。 SXRNA将异位mRNA上选择标记的表达与GOI mRNA成熟期间的剪接产物中的miRNA联系起来。因此，选择的严格性与GOI复制号和生产成正比。该计划是通过计算和经验屏幕优化SXRNA技术，并在与传统的CHO细胞谷氨酰胺合成酶系统并排比较中使用最合适的候选者。成功的基准将包括实时PCR的基因拷贝数测定，以及主动产品收益率与成本。在选择阶段终止之后，还将在许多段落中监视GOI的长期稳定性。