HocusLocus Inc. — Department of Health and Human Services STTR Phase II: 400

Description

PROJECT SUMMARY Over $billion dollarsworth of biological productsincluding antibodiesvaccines and proteinsare produced each yearThis continues to grow worldwide at aboutyearmaking biopharmaceuticals a fairly recession proofgrowing and pro table industryThis shift towards biopharmaceuticals re ects a fundamental shift within the pharmaceutical industryBiomanufacturers are under constant pressure to reduce costs by increasing productivity of their cell culturing activitiesWe believeby taking advantage of post transcriptional regulationthat we can achieve aimprovement in productive yielddefined as titeras well as significant reductions in workload and timeFor perspectiveaimproved titer from each cell in production could save $billion in biomanufacturing costsCurrent biomanufacturing practice is to express a drug resistance gene as a selectable proxy for successfully transformed cellswhich are then sub cloned and screened for production of the GOIThese basic techniques have not changed in any substantial way in overyearsbut they have two major drawbacksthey require the production of the antibiotic resistant gene which competes with the GOI for scarce translational resources placing an additional metabolic burden on the cellsandthe selection marker or antibiotic resistance gene production is not directly coupled to the GOI giving the potential for false positivesOur protocolPTSelectuses post transcriptional regulation as an alternative to using a drug resistance gene by using an siRNA coupled as an intron to the GOIRather than force the cells to transcribe and translate an additional drug resistance gene that serves as a proxy for GOI expressionwe introduce a custom siRNA into an intron upstream of the GOIExpression of the tethered siRNA and GOI is thus directly coupled making future selection potentially more accurateWe then use mRNA to perform selection with an mRNA has sequences that are perfectly complementary to the siRNA to induce RNA interferenceRNAiultimately down regulating the death gene mRNAThusthe more the GOI is producedthe more the siRNA is also produced which results in more degradation of the mRNAInstead of adding a chemical to select for resistancewe transfect a death gene encoding mRNA into the cellsor a fluorescent marker than identifies desired cells using fluorescence activated cell sortingFACSor a cell surface marker gene mRNA coupled with magnetic bead Ab to perform magnetically activated cell sortingMACSThis project will allow us to finished development of these techniques and performa a comparison study between current resistance gene products and our new PTSelect product