213. Detection of Integration Sites of CD19 Chimeric Antigen Receptor Gene in a Non-Viral Gene Transfer System

Abstract

BACKGROUND: Chimeric antigen receptor (CAR)-modified T cells targeting CD19 have exhibited marked activity in hematological malignancies, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and B-cell lymphomas. Many of these results have been obtained using CD19-CAR T cells established by retro- or lentiviral vectors. We developed a non-viral gene-transfer method using the piggyBac transposon system wherein the CD19-CAR gene was integrated into the genome by cut paste mechanism of the transposase. In this system, the expression of CAR was permanent, and the in vitro and in vivo cytotoxic activity on several cancer cell lines was confirmed. In the process of such gene transfer, however, insertional mutagenesis and subsequent activation of proto-oncogenes are a concern. In this report, we aimed to detect the integration sites of CAR genes to evaluate the safety of our CD19-CAR T cells. METHOD: CD19-CAR T cells were produced by transfecting two plasmids containing piggyBac and a CD19-CAR gene by electroporation. We detected the integration sites of CAR genes by inverse polymerase chain reaction and subsequent next-generation sequencing using MiSeq. An analytic pipeline was developed to identify and to classify the integration sites. Proto-oncogenes were defined according to the Cancer Gene Census of the Catalogue of Somatic Mutations in Cancer database. RESULTS: We detected 181 independent integration sites of which five were in exons (CASP10, F2R, CYP51A1-AS1, ULBP2, and SLC24A2) and 113 in introns. The integration sites demonstrated no preference for specific sites. Five integration sites (2.8%) were in the introns of known proto-oncogenes (CDK6, MAML2, RAD51B, RUNX1, and EP300). This percentage of integrations into proto-oncogenes is comparable with that of random integration (2.4%) and lower than that of retro- and lentiviral vectors (6.3-10.4%) according to previous report using marker genes (Galvan et al. J Immunother 2009). CONCLUSION: We confirmed that our piggyBac-mediated transduction system randomly transduced the CAR gene into the genomic DNA. Therefore, this system is considered safer than viral vector system in terms of genotoxicity for CAR transduction into human T cells; however, the possibility of leukemic transformation caused by insertional mutagenesis should be carefully monitored by in vivo clonal analysis.