Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR

Hamada M1, Nishio N2, Okuno Y3, Suzuki S3, Kawashima N1, Muramatsu H1, Tsubota S4, Wilson MH5, Morita D6, Kataoka S1, Ichikawa D1, Murakami N1, Taniguchi R1, Suzuki K1, Kojima D1, Sekiya Y1, Nishikawa E1, Narita A1, Hama A1, Kojima S1, Nakazawa Y6, Takahashi Y7.
Source: EBioMedicine
Publication Date: (2018)
Issue: 2352:
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
PBMC, human
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

To produce piggyBac-CD19 CAR-T cells, we used 4D-Nucleofector device and P3 Primary Cell 4D-Nucleofector X Kit (Lonza, Basel,
Switzerland) for gene transfer. Briefly, we used program EO-115 to electroporate 1 × 107 PBMC with 5 µg each of pIRII-CAR.CD19 transposon plasmid and pCMV-piggyBac transposase plasmid. Transfection efficiency figure 2a, 30-55%, comparison with retriviruses and lentivirus with similar results.


Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.