Nat Nanotechnol. 2017 Aug;12(8):813-820 

In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers

Matthias T. Stephan


摘要:

An emerging approach for treating cancer involves programming patient-derived T cells with genes encoding disease-specific chimeric antigen receptors (CARs), so that they can combat tumour cells once they are reinfused. Although trials of this therapy have produced impressive results, the in vitro methods they require to generate large numbers of tumour-specific T cells are too elaborate for widespread application to treat cancer patients. Here, we describe a method to quickly program circulating T cells with tumour-recognizing capabilities, thus avoiding these complications. Specifically, we demonstrate that DNA-carrying nanoparticles can efficiently introduce leukaemia-targeting CAR genes into T-cell nuclei, thereby bringing about long-term disease remission. These polymer nanoparticles are easy to manufacture in a stable form, which simplifies storage and reduces cost. Our technology may therefore provide a practical, broadly applicable treatment that can generate anti-tumour immunity ‘on demand’ for oncologists in a variety of settings.All of the plasmids used in this project were custom-cloned by vectorbuilder.com.The following piggyBac transposon gene expression vectors were used:These two control plasmids contain the same component as plasmids (1) and (2), respectively, but encode the tumour-irrelevant P4-1BBz CAR (ref. 9) instead of the leukaemia-specific 19-41BBz CAR. P4-1BBz retargets T lymphocytes to prostate-specific membrane antigen (PSMA), a protein expressed in prostate cancer cells and the neovasculature of various solid tumours in humans, but absent in mice.The following regular plasmid gene expression vector was used:This plasmid encodes the hyperactive iPB7 piggyBac transposase8 under the control of the EF1alpha promoter. A WPRE sequence was inserted between the stop codon and the SV40 polyA.The following lentivirus gene expression vector was used:This construct’s gene expression cassette is identical to plasmid (1), only cloned into the vectorbuilder.com lentiviral backbone (third generation).We thank I. Stanishevskaya and D. Ehlert (cognitionstudio.com) for the design of the illustrations. We also thank M. Sadelain (Memorial Sloan-Kettering Cancer Center, New York, New York) for the Eμ-ALL01 cell line, and for the DNA construct that encodes an all-murine CD19-specific CAR. This work was supported in part by the Fred Hutchinson Cancer Research Center’s Immunotherapy Initiative with funds provided by the Bezos Family Foundation, a New Idea Award from the Leukemia & Lymphoma Society, the Phi Beta Psi Sorority, the National Science Foundation (CAREER, award no. 1452492 and EAGER award no. 1644363), and the National Cancer Institute of the National Institutes of Health under award no. R01CA207407. M.T.S. was also supported by a Research Scholar Grant (RSG-16-110-01 – LIB) from the American Cancer Society.Additional informationSupplementary information is available in the online version of the paper. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Competing financial interestsThe authors declare no competing financial interests.
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