A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells.
A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells.
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DOI：
10.1007/s10544-010-9440-3
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发表时间：
2010-10
影响因子：
2.8
通讯作者：
Cooper LJ
中科院分区：
文献类型：
--
作者：
Choi Y;Yuen C;Maiti SN;Olivares S;Gibbons H;Huls H;Raphael R;Killian TC;Stark DJ;Lee DA;Torikai H;Monticello D;Kelly SS;Kebriaei P;Champlin RE;Biswal SL;Cooper LJ
已经证明嵌合抗原受体（CAR）可以直接识别在B细胞恶性肿瘤的细胞表面上表达的CD19分子，而不依赖于主要组织相容性复合体（MHC）。尽管使用CD19特异性CAR的肿瘤T细胞疗法是有希望的，但这种方法依赖于使用将CAR稳定整合到T细胞染色体中的表达载体。为了避免表达整合转基因可能发生的潜在遗传毒性，我们使用高通量微电穿孔装置从mRNA表达了CD19特异性CAR转基因。本研究使用微电穿孔仪完成，以实现体外转录的CAR mRNA到已离体数值扩增的人T细胞中的有效和高通量非病毒基因转移。mRNA的电转移避免了与载体和转基因整合相关的潜在遗传毒性，并且高通量能力克服了预期的瞬时CAR表达，因为重复的电穿孔轮次可以替换已经失去转基因表达的T细胞。我们制造并测试了一种高通量微电穿孔仪，该仪器可以在10分钟内电穿孔2×108个原代T细胞。电穿孔后，高达80%的传代T细胞表达CD19特异性CAR。视频延时显微镜（VTLM）证明了基因操作的T细胞特异性裂解CD19+肿瘤细胞的重定向效应功能。我们的生物医学微型装置，其中T细胞被瞬时和安全地修饰为肿瘤特异性，然后可以重新输注，提供了一种重定向T细胞特异性的方法，这对过继免疫疗法的发展具有影响。
It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2×108 primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical micro-device, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.
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病毒特异性的T细胞设计为共表达肿瘤特异性受体：神经母细胞瘤个体中的持久性和抗肿瘤活性。
影响因子：
82.9
作者：
通讯作者：
--
影响因子：
12.6
作者：
Wang, Hsiang-Yu;Bhunia, Arun K.;Lu, Chang
通讯作者：
Lu, Chang
影响因子：
11.2
作者：
Singh, Harjeet;Manuri, Pallavi R.;Cooper, Laurence J. N.
通讯作者：
Cooper, Laurence J. N.
DOI：
10.1073/pnas.85.3.856
发表时间：
1988-02-01
影响因子：
11.1
作者：
MILLER, JF;DOWER, WJ;TOMPKINS, LS
通讯作者：
TOMPKINS, LS
影响因子：
4.2
作者：
Mitchell, Duane A.;Karikari, Isaac;Sampson, John H.
通讯作者：
Sampson, John H.