PF437 PLATINUM TALEN‐MEDIATED NON‐VIRAL T CELL RECEPTOR GENE KNOCK‐IN FACILITATES UNIVERSAL T CELL GENOME EDITING FOR MANUFACTURING OF THERAPEUTIC IMMUNE CELLS

Abstract

Background:Adoptive transfer of T cells genetically modified to express T‐cell receptors (TCRs) directed against tumor‐specific antigens is an emerging therapeutic tool for refractory hematologic and non‐hematologic neoplasms. Although accumulating experiences in their clinical use have shown great promise, challenges still remain because mispairing of transduced TCR subunits with those of endogenous TCRs reduces expression of desired TCRs and could be responsible for graft‐versus‐host disease‐like complications by harmful mixed TCR dimers in a preclinical model. Furthermore, stable production of recombinant viral vectors widely used for TCR transduction is a time‐consuming and labor‐intensive process.Aims:To establish a universal protocol for manufacturing therapeutic TCR‐transduced T cells by avoiding such “misparing phenomena”, we aimed to develop a novel strategy for precise integration of TCR gene segments into the endogenous TCR locus by microhomology‐mediated end‐joining (MMEJ) with the use of highly efficient “Platinum Transcription Activator‐like Effector Nucleases (pTALENs)” and non‐viral DNA vectors, designated as the TAL‐TCR‐PITCh (TALEN‐mediated precise integration of TCR into chromosome) system.Methods:Two pairs of pTALENs were designed for targeted disruption of gene segments encoding T cell receptor alpha (TRA) and beta (TRB) constant regions and constructed using the Platinum Gate TALEN Kit (Addgene, USA). These pTALENs are structurally different from conventional TALENs and endowed with higher activities by non‐repeat‐variable di‐residues variations in the TALE repeats. As a vehicle for non‐viral transduction of desired TCR gene segments into genome‐edited T cells lacking endogenous TCRs, we constructed a TAL‐TCR‐PITCh vector containing the TRA‐TRB gene cassette and the target sequences for pTALENs for TRA. If cleaved by TRA‐pTALEN, this vector generates microhomologous DNA ends complementary to the pTALEN‐targeted sites in the TRA locus. Therefore, TAL‐TCR‐PITCh vector is precisely incorporated between the cleaved endogenous TRA genomic sites by MMEJ, a unique DNA double‐strand breaks repair mechanism that uses 5–25 bp alignment of microhomologous sequences locating internal to the broken DNA ends.Results:We first examined activities of pTALENs for TRA and TRB in Jurkat cells electroporated with pTALEN mRNA and confirmed dose‐dependent cleavage efficiencies; proportions of Jurkat cells lacking CD3/TCR expression reached to approximately 80% for TRA and 60% for TRB. T7 endonulease I assays and amplicon sequencing of the targeted sites also revealed successful digestions in similar proportion of pTALEN‐transduced cells. By using these pTALENs for TCR genome editing, we then performed knockout of TRA and TRB loci in primary human T cells obtained from healthy donors and could retrieve viable T cells lacking endogenous TCR expression with 40–50% efficiencies. TCR‐deficient T cells sorted by CD3‐negative selection were easily expandable by use of anti‐CD3/CD28 antibodies‐coated beads. As a proof‐of‐principle experiment to confirm the feasibility of our TCR knock‐in protocol, we could successfully produce gene edited primary T cells reprogrammed to express TCRs reactive against an immunogenic NY‐ESO‐1‐derived peptide in an HLA‐A∗02‐restricted manner by two‐step transduction of TRB‐pTALEN mRNA (step 1) and TAL‐TCR‐PITCh vector with TRA‐pTALEN mRNA (step 2).Summary/Conclusion:These results collectively suggest that pTALEN‐mediated TCR genome editing is feasible and provides a universal platform the production of TCR‐transduced therapeutic T cells with safer profiles.