A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells

Abstract

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.

Plasmonic nanobubbles for target cell-specific gene and drug delivery and multifunctional processing of heterogeneous cell systems

“…Cell injection and transfection methods are similarly limited. Three major approaches deliver plasmids with viral, nonviral using plasmid carriers and non-viral using external energy methods [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] . While viruses offer greater efficacy of gene transfer, non-viral methods provide better safety and are usually less immunogenic.…”

“…PNBs with their much higher cellular specificity overcome this limitation because they do not depend upon the non-specific uptake of PNB-generating gold nanoparticles [20][21][22][23][24] . External energy-based methods use sono-, electro-and opto-poration of cells, of which electroporation/nucleofection is most widely used, despite its poor selectivity and cellular toxicity [12][13][14] . Gene transfer also employs laser methods through heating, shockwave generation, optical breakdown and bubble generation [15][16][17][18] .…”

“…Ideally both elimination and transfection should be highly efficient, cell-specific and fast. Existing methods [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] , however, lack such characteristics, especially those of multi-functionality and cellular selectivity when applied to heterogeneous cell systems. As a result, current cell processing for cell and gene therapies is often slow, expensive and labor intensive and is compromised with high cell losses and poor selectivity, thus limiting the efficacy and availability of these cell therapies.…”

Cellular Therapies: Gene Editing and Next-Gen CAR T Cells

“…Transient CAR-T expression was achieved by ex vivo electroporating T cells with mRNA coding for CARs or TCR s (Yoon et al 2009 ;Rabinovich et al 2009 ). High-throughput mRNA electroporation can resulting in 80 % or more of the cells expressing the construct (Choi et al 2010 ). Multiple mRNA can be co-delivered and expressed, as can one or more gRNA and Cas9 mRNA.…”

Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor

“…The introduction of mRNA into activated T cells is efficient and as technologies improve to synchronously electroporate large numbers of cells, it may be possible to overcome the expected loss of CAR expression from transient transfection by repeatedly electroporating and administering CAR ϩ T cells and NK cells. 133,147 Regarding the risks of transposition, a review has been published discussing the risks and benefits of the SB system for clinical application which addresses the low potential for genotoxicity. 148 For additional reviews on insertional mutagenesis in genetically modified T cells, please refer to Table 2.…”