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.

这个小企业技术转让（STTR）项目更广泛的影响/商业潜力是开发技术，为最常见的生物疗法生产方法--中国仓鼠卵巢（CHO）细胞系--提供更高效的平台。给定治疗剂的每个批量生产周期都很大，并且代表着显著的投资和显著的利润，并且任何增加（甚至递增地）这种批量的产率的方法都将产生巨大的利润。目前，这些细胞将其代谢能量分解为产生治疗药物和用于选择最高生产者的蛋白质。所提出的系统利用不同种类的选择标记的瞬时表达-一旦选择阶段完成，释放显著的代谢资源以仅专注于治疗剂的生产的选择标记。增加产量将降低成本，并使这些治疗方法适用于更广泛的人群。生物制造公司正在寻找新的方法来提高产品产量，并准备花费大量的时间和资源来保持其领先地位和盈利能力。该STTR I期项目提出开发一种选择方法，以显着提高CHO细胞生物素产量。目前的方法牺牲了产率，因为细胞必须产生选择性标记以及产物，这对细胞来说是沉重的负担。所提出的技术将通过我们的结构相互作用RNA（sxRNA）技术使用选择性标记谷氨酰胺合酶的瞬时表达，该选择性标记谷氨酰胺合酶由感兴趣的基因（GOI）严格控制并且仅在感兴趣的基因（GOI）存在下才有活性。一旦选择阶段完成，选择标记的代谢成本将停止，代谢资源将用于GOI生产。sxRNA在GOI mRNA成熟期间将异位mRNA上的选择性标记的表达与剪接产物中的miRNA联系起来。因此，选择的严格性与GOI拷贝数和产量成正比。该计划是通过计算和经验筛选来优化sxRNA技术，并在与传统CHO细胞谷氨酰胺合成酶系统的并排比较中使用最合适的候选物。成功的基准将包括通过实时PCR检测基因拷贝数，以及活性产品产量与成本。在选择阶段结束后，还将在多次传代中监测GOI的长期稳定性。