Rapid Non-Viral Platform for Generation of Genetically Modified T Cells for Therapy

Abstract Cancer immunotherapyparticularly genetically engineered adoptive T cell transferhas shown great potential for the treatment of cancer patientsThe use of T cells engineered to express a specific T cell receptorTCRsor chimeric antigen receptorCARsto treat cancer has generated durable cures for many cancer patients and has resulted in the first FDA approved CAR T therapy to treat childhood acute lymphoblastic leukemia inMost gene therapies rely on viral methods to genetically modify human primary cellsHoweverviral delivery method is expensivepoorly reproducible and associated with several safety concerns including insertion in or near genes that may cause malignancy and generation of replication competent virusThusnon viral DNA delivery methodssuch as Sleeping Beauty and piggyBachave been employed to generate CAR T cellsAlthough these non viral delivery methods have the advantage of lower costimmunogenicityand regulatory considerationsthey have been limited by their low transposition efficiency in primary human hematopoietic cellsIn this applicationwe propose to rationally optimize a recently discovered transposonTcBuster to deliver CARs to T cellsTo this endwe further enhance our already very active hyperactive mutants of the TcBuster transposase and optimize the delivery of the transposon into cellsFollowing optimization of the TcBuster transposon systemwe will combine these improvements with our proprietary methods to transfect T cells efficiently and safelyand test the immunotherapeutic effectiveness of TcBuster delivered CAR into T cellsThe successful completion of this project will result in the comprehensive methods to produce CAR T cells delivered by TcBuster which we will license to pharmaceutical companies to produce highly efficient CAR T immunotherapyMore broadlythese methods could be expanded beyond immunotherapeutic cancer applications to various infectious diseases in which gene delivery by TcBuster in T cells could be advantageous