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

Motoharu Hamada, Nobuhiro Nishio, Yusuke Okuno, Satoshi Suzuki, Nozomu Kawashima, Hideki Muramatsu, Shoma Tsubota, Matthew H Wilson, Daisuke Morita, Shinsuke Kataoka, Daisuke Ichikawa, Norihiro Murakami, Rieko Taniguchi, Kyogo Suzuki, Daiei Kojima, Yuko Sekiya, Eri Nishikawa, Atsushi Narita, Asahito Hama, Seiji Kojima, Yozo Nakazawa, Yoshiyuki Takahashi, Motoharu Hamada, Nobuhiro Nishio, Yusuke Okuno, Satoshi Suzuki, Nozomu Kawashima, Hideki Muramatsu, Shoma Tsubota, Matthew H Wilson, Daisuke Morita, Shinsuke Kataoka, Daisuke Ichikawa, Norihiro Murakami, Rieko Taniguchi, Kyogo Suzuki, Daiei Kojima, Yuko Sekiya, Eri Nishikawa, Atsushi Narita, Asahito Hama, Seiji Kojima, Yozo Nakazawa, Yoshiyuki Takahashi

Abstract

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.

Keywords: CD19 CAR-T cell; Integration site mapping; Tag-PCR; piggyBac transposon.

Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1
Fig. 1
Schematic outline of the tag-PCR method of integrated site mapping compared with standard LAM-PCR and nrLAM-PCR methods. Linear amplification-mediated polymerase chain reaction (LAM-PCR) (A), and non-restriction LAM-PCR (nrLAM-PCR) (B) start with the amplification of vector–genome junctions using biotinylated primers, hybridizing close to the end of the known vector DNA sequence. Biotinylated single strand (ss)DNA is captured on magnetic particles. LAM-PCR then uses restriction enzyme digestion and linker ligation after double strand (ds)DNA synthesis. The product is amplified by nested PCR with linker- and vector-specific primers. In nrLAM-PCR, the ssDNA linker sequence is directly ligated to the unknown end of the pre-amplified ssDNA following amplification by nested PCR with linker- and vector-specific primers. Additional PCR is performed to add sequencing specific adapters to these (nr)LAM-PCR products. In tag-PCR (C), the tagmentation enzyme randomly fragments genomic DNA and tags the fragments with adapters at both ends. DNA fragments containing the vector–genome junction are amplified with a primer that is complementary to the adapter sequence and the other primer that is complementary to the vector sequence and tagged with the additional adapter sequence. A second PCR is performed to add the complete adapter sequences necessary for sequencing. Finally, the libraries are analyzed by a next-generation sequencer. Estimated durations are indicated alongside each step. IR, inverted repeat; LC, linker cassette; ssLC, single strand linker cassette.
Fig. 2
Fig. 2
Chimeric antigen receptor expression. The frequency of chimeric antigen receptor (CAR) expression was measured by flow cytometry using anti-idiotype antibodies after CAR-T cells had been cultured for 14 days. (A) CAR expression for the piggyBac, retrovirus, and lentivirus vectors obtained from three healthy donors. Each donor's CAR expression is shown by a single data point. (B) Representative results of CAR expression in primary T cells obtained from donor 3. The x- and y-axes indicate the fluorescence intensity and frequency of events, respectively.
Fig. 3
Fig. 3
Frequency of integration into or around genomic elements. Frequency of integration in genes (A), exons (B), 5 kb windows around transcriptional start sites (TSSs) (C), and 5 kb windows around CpG islands (D). Gray bars indicate the results from random simulation. * P < .001; † P < .01; ‡ P < .05; NS, not significant.
Fig. 4
Fig. 4
Integration frequency around transcriptional start sites and CpG islands. (A) The frequency of insertion within a distance of 500 bp from the nearest transcriptional start site (TSS). (B) The frequency of insertion within a distance of 500 bp from the nearest CpG island. The frequencies for the piggyBac, retrovirus, and lentivirus vectors are illustrated in blue, orange, and yellow, respectively. The gray dotted lines represent simulated random integration events.
Fig. 5
Fig. 5
Frequency of integration into or around transcriptional start site of proto-oncogenes and gene safe harbors. The frequency of integration within proto-oncogenes (A), within 50-kb windows around transcriptional start sites (TSSs) of proto-oncogenes (B), and gene safe harbors (C). Gray bars indicate results from random simulation. * P 

Fig. 6

Validation of tag-PCR system by…

Fig. 6

Validation of tag-PCR system by target capture sequencing. Comparison of integration site mapping…

Fig. 6
Validation of tag-PCR system by target capture sequencing. Comparison of integration site mapping of piggyBac CAR-T cell generated by tag-PCR and that by target capture sequencing. We calculated several parameters, including frequency of integration in genes (A), exons (B), 5-kb windows around transcriptional start sites (TSSs) (C), 5-kb windows from CpG islands (D), the patterns of insertions around TSSs (E), CpG islands (F), integration within proto-oncogenes (G), 50-kb windows around TSS of proto-oncogenes (H), and gene safe harbors (I) using data from both methods.
Fig. 6
Fig. 6
Validation of tag-PCR system by target capture sequencing. Comparison of integration site mapping of piggyBac CAR-T cell generated by tag-PCR and that by target capture sequencing. We calculated several parameters, including frequency of integration in genes (A), exons (B), 5-kb windows around transcriptional start sites (TSSs) (C), 5-kb windows from CpG islands (D), the patterns of insertions around TSSs (E), CpG islands (F), integration within proto-oncogenes (G), 50-kb windows around TSS of proto-oncogenes (H), and gene safe harbors (I) using data from both methods.

References

    1. Bishop D.C., Xu N., Tse B., O'Brien T.A., Gottlieb D.J., Dolnikov A., Micklethwaite K.P. Piggybac-engineered T cells expressing CD19-specific CARs that lack IgG1 Fc spacers have potent activity against B-ALL xenografts. Mol Ther. 2018;26(8)
    1. Brentjens R.J., Riviere I., Park J.H., Davila M.L., Wang X., Stefanski J., Taylor C., Yeh R., Bartido S., Borquez-Ojeda O., Olszewska M., Bernal Y., Pegram H., Przybylowski M., Hollyman D., Usachenko Y., Pirraglia D., Hosey J., Santos E., Halton E., Maslak P., Scheinberg D., Jurcic J., Heaney M., Heller G., Frattini M., Sadelain M. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood. 2011;118:4817–4828.
    1. Carbonaro D.A., Zhang L., Jin X., Montiel-Equihua C., Geiger S., Carmo M., Cooper A., Fairbanks L., Kaufman M.L., Sebire N.J., Hollis R.P., Blundell M.P., Senadheera S., Fu P.Y., Sahaghian A., Chan R.Y., Wang X., Cornetta K., Thrasher A.J., Kohn D.B., Gaspar H.B. Preclinical demonstration of lentiviral vector-mediated correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency. Mol Ther. 2014;22:607–622.
    1. Caruccio N. Preparation of next-generation sequencing libraries using Nextera technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition. Methods Mol Biol. 2011;733:241–255.
    1. Crooks G.E., Hon G., Chandonia J.M., Brenner S.E. WebLogo: a sequence logo generator. Genome Res. 2004;14:1188–1190.
    1. Galvan D.L., Nakazawa Y., Kaja A., Kettlun C., Cooper L.J., Rooney C.M., Wilson M.H. Genome-wide mapping of PiggyBac transposon integrations in primary human T cells. J Immunother. 2009;32:837–844.
    1. Geurts A.M., Yang Y., Clark K.J., Liu G., Cui Z., Dupuy A.J., Bell J.B., Largaespada D.A., Hackett P.B. Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther. 2003;8:108–117.
    1. Gogol-Doring A., Ammar I., Gupta S., Bunse M., Miskey C., Chen W., Uckert W., Schulz T.F., Izsvak Z., Ivics Z. Genome-wide profiling reveals remarkable parallels between insertion site selection properties of the MLV retrovirus and the piggyBac transposon in primary human CD4(+) T cells. Mol Ther. 2016;24:592–606.
    1. Grupp S.A., Kalos M., Barrett D., Aplenc R., Porter D.L., Rheingold S.R., Teachey D.T., Chew A., Hauck B., Wright J.F., Milone M.C., Levine B.L., June C.H. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368:1509–1518.
    1. Hacein-Bey-Abina S., Le Deist F., Carlier F., Bouneaud C., Hue C., De Villartay J.P., Thrasher A.J., Wulffraat N., Sorensen R., Dupuis-Girod S., Fischer A., Davies E.G., Kuis W., Leiva L., Cavazzana-Calvo M. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med. 2002;346:1185–1193.
    1. Hacein-Bey-Abina S., Von Kalle C., Schmidt M., McCormack M.P., Wulffraat N., Leboulch P., Lim A., Osborne C.S., Pawliuk R., Morillon E., Sorensen R., Forster A., Fraser P., Cohen J.I., de Saint Basile G., Alexander I., Wintergerst U., Frebourg T., Aurias A., Stoppa-Lyonnet D., Romana S., Radford-Weiss I., Gross F., Valensi F., Delabesse E., Macintyre E., Sigaux F., Soulier J., Leiva L.E., Wissler M., Prinz C., Rabbitts T.H., Le Deist F., Fischer A., Cavazzana-Calvo M. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–419.
    1. Harkey M.A., Kaul R., Jacobs M.A., Kurre P., Bovee D., Levy R., Blau C.A. Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis. Stem Cells Dev. 2007;16:381–392.
    1. Huang X., Guo H., Tammana S., Jung Y.C., Mellgren E., Bassi P., Cao Q., Tu Z.J., Kim Y.C., Ekker S.C., Wu X., Wang S.M., Zhou X. Gene transfer efficiency and genome-wide integration profiling of sleeping beauty, Tol2, and piggyBac transposons in human primary T cells. Mol Ther. 2010;18:1803–1813.
    1. Huye L.E., Nakazawa Y., Patel M.P., Yvon E., Sun J., Savoldo B., Wilson M.H., Dotti G., Rooney C.M. Combining mTor inhibitors with rapamycin-resistant T cells: a two-pronged approach to tumor elimination. Mol Ther. 2011;19:2239–2248.
    1. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant. 2013;48:452–458.
    1. Kebriaei P., Singh H., Huls M.H., Figliola M.J., Bassett R., Olivares S., Jena B., Dawson M.J., Kumaresan P.R., Su S., Maiti S., Dai J., Moriarity B., Forget M.A., Senyukov V., Orozco A., Liu T., McCarty J., Jackson R.N., Moyes J.S., Rondon G., Qazilbash M., Ciurea S., Alousi A., Nieto Y., Rezvani K., Marin D., Popat U., Hosing C., Shpall E.J., Kantarjian H., Keating M., Wierda W., Do K.A., Largaespada D.A., Lee D.A., Hackett P.B., Champlin R.E., Cooper L.J. Phase I trials using sleeping beauty to generate CD19-specific CAR T cells. J Clin Invest. 2016;126:3363–3376.
    1. Kochenderfer J.N., Feldman S.A., Zhao Y., Xu H., Black M.A., Morgan R.A., Wilson W.H., Rosenberg S.A. Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor. J Immunother. 2009;32:689–702.
    1. Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–2079.
    1. Manuri P.V., Wilson M.H., Maiti S.N., Mi T., Singh H., Olivares S., Dawson M.J., Huls H., Lee D.A., Rao P.H., Kaminski J.M., Nakazawa Y., Gottschalk S., Kebriaei P., Shpall E.J., Champlin R.E., Cooper L.J. piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum Gene Ther. 2010;21:427–437.
    1. Morita D., Nishio N., Saito S., Tanaka M., Kawashima N., Okuno Y., Suzuki S., Matsuda K., Maeda Y., Wilson M.H., Dotti G., Rooney C.M., Takahashi Y., Nakazawa Y. Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac transposon-engineered T cells. Mol Ther Methods Clin Dev. 2018;8:131–140.
    1. Mueller P.R., Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989;246:780–786.
    1. Nakazawa Y., Huye L.E., Dotti G., Foster A.E., Vera J.F., Manuri P.R., June C.H., Rooney C.M., Wilson M.H. Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes. J Immunother. 2009;32:826–836.
    1. Ochman H., Gerber A.S., Hartl D.L. Genetic applications of an inverse polymerase chain reaction. Genetics. 1988;120:621–623.
    1. Papapetrou E.P., Lee G., Malani N., Setty M., Riviere I., Tirunagari L.M., Kadota K., Roth S.L., Giardina P., Viale A., Leslie C., Bushman F.D., Studer L., Sadelain M. Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol. 2011;29:73–78.
    1. Paruzynski A., Arens A., Gabriel R., Bartholomae C.C., Scholz S., Wang W., Wolf S., Glimm H., Schmidt M., von Kalle C. Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing. Nat Protoc. 2010;5:1379–1395.
    1. Sadelain M., Papapetrou E.P., Bushman F.D. Safe harbours for the integration of new DNA in the human genome. Nat Rev Cancer. 2011;12:51–58.
    1. Schmidt M., Schwarzwaelder K., Bartholomae C., Zaoui K., Ball C., Pilz I., Braun S., Glimm H., von Kalle C. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR) Nat Methods. 2007;4:1051–1057.
    1. Thornhill S.I., Schambach A., Howe S.J., Ulaganathan M., Grassman E., Williams D., Schiedlmeier B., Sebire N.J., Gaspar H.B., Kinnon C., Baum C., Thrasher A.J. Self-inactivating gammaretroviral vectors for gene therapy of X-linked severe combined immunodeficiency. Mol Ther. 2008;16:590–598.
    1. Ustek D., Sirma S., Gumus E., Arikan M., Cakiris A., Abaci N., Mathew J., Emrence Z., Azakli H., Cosan F., Cakar A., Parlak M., Kursun O. A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology. Infect Genet Evol. 2012;12:1349–1354.
    1. Vera J., Savoldo B., Vigouroux S., Biagi E., Pule M., Rossig C., Wu J., Heslop H.E., Rooney C.M., Brenner M.K., Dotti G. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood. 2006;108:3890–3897.
    1. Wilson M.H., Coates C.J., George A.L., Jr. PiggyBac transposon-mediated gene transfer in human cells. Mol Ther. 2007;15:139–145.

Source: PubMed

Patrocinadores e Colaboradores

Condições médicas

Intervenções de drogas

3
Se inscrever