sxRNA-Select: Using RNAs for Positive Selection of Cell Lines, Enabling High-Yield Biotherapeutic Production

Sxrna Technologies, Inc., Albany NY

Investigators

Abstract

Abstract - sxRNA Technologies, Inc (sxRNA Tech) is developing a rapid cloning technology, sxRNA- Select, that will decrease the time it takes to clone mammalian cells and increase the protein yields thereof. Biologics have emerged as a powerful and impactful class of therapeutics but are time-consuming and expensive to develop and produce, with a major bottleneck created by the cloning selection process. Cell-line development requires screening hundreds to thousands of clones. Current methodologies for selecting cells that have stably incorporated genes of interest (GOIs) use antibiotic and/or metabolic selection approaches, which are multi-phased and time-consuming. The very expression of these proxy proteins expends significant cellular resources which can slow proliferation and limit total protein yield. Additionally, toxic selection agents must be maintained throughout the selection process, further slowing growth, and causing the need for a processing step to remove them which risks cell line stability and productivity. The aim of this STTR program is to develop a plug- and-play structurally interacting RNA (sxRNA) cloning kit that reduces time to cell line selection while increasing protein yield. Selection is controlled using sxRNA, an RNA-based technology that enables specific selection based on the unique expression of an RNA expressed as part of the GOI being cloned. sxRNA- Select uses the production of a microRNA (the trigger) imbedded into a GOI intron. The expressed trigger then interacts with a co- and/or subsequently-transfected transient mRNA (the sxRNA-bait), modulating its translation. The trans-interaction of the sxRNA-trigger with the mRNA/sxRNA-bait is used to analogically select cells expressing the GOI the most and can be used as a proxy for GOI expression level. Critically, because the mRNA/sxRNA-bait degrades within days, high producing stable clones do not waste valuable cellular resources on maintaining the production of a selectable marker. The company previously developed the sxRNA technology as a tool for use in cell culture applications. Main areas of technical risk center around the efficacy of simultaneous and/or subsequent transfection of plasmids and sxRNA-bait, effective ratiometric protein production, and directly demonstrating the time and yield advantage of the approach compared to state-of-the- art methods available to therapeutic developers today. For this Phase I project, sxRNA, in collaboration with the University at Albany-SUNY, will undertake three specific aims: 1) optimizing the protocol for delivery of plasmids and sxRNA-bait, 2) demonstrating selection for ratiometric protein expression, and 3) conducting a head-to-head yield comparison with industry-standard approaches including metabolic and antibiotic selection. At the conclusion of this work, sxRNA Tech will have a minimum viable product for rapid cell cloning based on positive selection with sxRNAs. Phase II will then focus on the combination of positive selection and negative selection, the relatively simpler process. Further development will include the expression of even more complex trispecifics and a scale-up of the manufacturing process to ensure that demand can be met once commercialized.