STTR Phase I: Harnessing CRISPR-mediated silencing for the one-step optimization of protein production strains

这项小型企业技术转移（STTR）项目的更广泛/商业影响将是开发一种设计大肠杆菌的技术，该技术将简化作为蛋白质纯化一部分的污染细胞蛋白质的去除。尽管生物制药生产取得了重大进展，但蛋白质纯化仍然是生产中成本最高、限制最多的环节之一，对初创企业和财富500强企业都产生了负面影响。这个STTR项目旨在通过开发一个平台来识别对下游加工施加最大负担的细胞蛋白，然后在生产菌株中关闭这些基因，从而直接解决这一挑战。该平台可以很容易地在大部分生物制造行业中实施，有可能降低消费者在不断发展的医疗保健市场中的成本，并减少将新疗法推向市场所需的时间和投资。这个STTR I期项目建议开发一种单一结构，可以协调和可调地沉默在通过离子交换色谱纯化蛋白质疗法时遇到的最繁重的污染蛋白的表达。由此产生的结构可以很容易地结合到几乎任何大肠杆菌菌株中，为商业化和专有生产菌株提供了一个简单的附加。拟议的I期工作的计划结果是一个最小可行产品，通过离子交换色谱纯化，大大提高了代表性生物治疗蛋白的捕获和纯度。相关构建体将协调并完全关闭污染非必需基因的表达，并精细调节污染必需基因的表达，以提高靶捕获和纯度，同时保持细胞生长。这种结构将是迈向商业化产品的重要一步，它将彻底改变在广阔的生物制药行业中如何进行下游加工。

The broader/commercial impacts of this Small Business Technology Transfer (STTR) project will be the development of a technology to engineer E. coli that will simplify the removal of contaminating cellular proteins as part of protein purification. Despite major advancements in biopharmaceutical manufacturing, protein purification remains one of the most costly and restrictive aspects of production that impacts negatively impacting start-ups and Fortune 500 companies alike. This STTR project seeks to directly tackle this challenge by developing a platform to identify the cellular proteins that exert the greatest burden on downstream processing, and then shut off these genes in the production strain. This platform could be readily implemented across much of the biomanufacturing industry, with the potential to reduce costs to consumers in the ever-burgeoning healthcare market and ease the time and investment required to bring new therapeutics to market. This STTR Phase I project proposes to develop a single construct that can coordinately and tunably silence expression of the most burdensome contaminating proteins that are encountered when purifying protein therapeutics via ion exchange chromatography. The resulting construct could be readily incorporated into virtually any E. coli strain, offering a simple add-on to both commercialized and proprietary production strains. The planned outcome of the proposed Phase I work is a minimal viable product that substantially boosts the capture and purity of a representative biotherapeutic protein purified via ion-exchange chromatography. The associated construct will coordinately and completely turn off the expression of contaminating, non-essential genes, and finely tune the expression of contaminating, essential genes to improve target capture and purity while preserving cell growth. This construct will represent an important step toward a commercial product that could revolutionize how downstream processing is conducted in the expansive biopharmaceuticals industry.