In its simplest form, PTSelect™ calls for adding an miRNA-containing intron upstream of the gene of interest (GOI) that leads to transcription of both the GOI and eventual activation of an siRNA. The actual selection process is then performed by transfecting mRNA that containts an RNAi site that interacts with the siRNA from the original plasmid. The active siRNA cleaves the mRNA in cells that are stably transfected and producing the GOI thereby reducing translation of the mRNA, while uninhibited translation in cells without the siRNA.
Benefits – We foresee several major benefits to this overall approach:
Flexibility – The mRNA can encode (1) a death gene to kill unwanted cells similar to the current process; (2) a fluorescent marker for subsequent cell sorting using flow cytometry with no antibody required for the GOI; or (3) a cell surface marker for subsequent sorting using magnetic activated cell sorting.
Higher titers – By eliminating a resistance protein that would otherwise need to be expressed, we anticipate significant increases in titers of final product. The expression of the siRNA has no effect on the growth rate of the cells in comparison to slower growth experienced by cells expressing stably transfected genes. Transcription and translation are rate limiting steps in GOI production, and adding an intronic siRNA does not affect either process.
More accurate selection – Our system ties the levels of the siRNA marker directly to the GOI number by lying within the 3’ UTR of the GOI, in between the promoter and the ORF. Physically coupling the production of our trigger siRNA directly to the real-time production of the GOI mRNA from the same promoter will have two benefits. First, it will increase the efficiency of any selection methods because levels of the selection marker, the miRNA, and the GOI are in sync. Secondly, the selection can be optimized to produce results based on high productivity of transcription of the GOI mRNA, effectively killing all cells that have either no or low production of the GOI. It is also possible to use different siRNA for the heavy and light chains for antibodies for additional specificity in selection.
Complementary to existing processes – Our approach does not require new equipment to be purchased, a change to a proprietary cell line, or other vector/plasmid sequence modification. We anticipate this protocol to integrate well into any production environment.