Robust delivery while preserving viability across cell populations

Data from the Xenon Electroporation System demonstrates superior non-viral gene editing and transfection performance. Additional data will be added to this page as it becomes available.


Knock-out and knock-in in CAR T cells

In these data graphs, chimeric antigen receptor (CAR) T cells were generated from three different donors by using Cas9/gDNA to knock out the endogenous T-cell receptor (TCR) and knock in an FMC CAR. As a benchmark, lentiviral transduction would be expected to achieve 20–40% success for this process.

 

Knock-out and knock-in performance with the Xenon system was strong. Across all three donors, successful transfection percentages (cells both knocked out and knocked in) ranged from about 22% to more than 45%, depending on the electroporation chamber used. Transfection efficiency on the Xenon instrument surpassed even the Neon Transfection System on which the process was developed. Cell viability—which must be balanced against transfection efficiency—exceeded 70% in all but one case, and was usually within 10% of the untransfected controls. Percentages of CD4 and CD8 T cells remained relatively consistent before and after electroporation.

Transfection efficiency

Knock-out and knock-in performance in CAR T cells. T cells from three donors were transfected with an FMC CAR construct using Cas9/gRNA on the Neon Transfection System (100 µL) or the Xenon Electroporation System with the SingleShot (1 mL) or MultiShot (9 mL) electroporation chamber, or were left untransfected (0). Cells were characterized after 72 hours as untransfected (TCRαβ+, gray), knocked out but not knocked in (TCRαβCAR, light blue), or successfully knocked out and knocked in (TCRαβCAR+, dark blue). Across all donors, successful knock-in percentages on the Xenon system ranged from 21.9% to 45.6%, exceeding even the Neon system.

Three bar graphs, representing three donors. Each bar graph includes a negative control, a sample run on the Neon system, and two samples run on the Xenon system.

Viability

Viability in CAR T cells. In the same experiment, cells were assessed for viability after 72 hours using trypan blue exclusion. For the three donors, cell viability on the Xenon system ranged from 64.6% to 83.6%.

Three bar graphs, representing three donors. Each bar graph includes a negative control, a sample run on the Neon system, and two samples run on the Xenon system.]

CD4 to CD8 T cell ratio

Preservation of CD4 vs CD8 T cell ratio. In the same experiment, CD4 and CD8 T cells were identified by flow cytometry. Non-electroporated cells (condition 0) were gated on live, single, and untransfected (TCRαβ+), while electroporated cells were gated on live, single, and knocked out (TCRαβ). Proportions of CD4 (light blue) and CD8 (dark blue) T cells remained largely consistent from non-electroporated cells to cells electroporated with the Neon system (100 µL) and with the Xenon system (1 and 9 mL).

A set of three complex bar graphs representing three donors, with data showing T cell surface markers CD4 and CD8 following electroporation at increasing volumes, compared to pre-electroporation cell surface marker values.

Flow cytometry analysis of electroporated cells

CAR T cell analysis by flow cytometry. In the same experiment, electroporated cells were analyzed by flow cytometry. Plots and analysis are representative of all three donors. (A) Gating on live single cells, 89.5% lost TCR function (TCRαβ) and thus were successfully knocked out, while 9.59% retained TCR expression (TCRαβ+) and thus were not successfully transfected. (B) Analysis of untransfected cells shows the percentages of CD4 (43.1%) and CD8 (51.7%) single-positive (SP) cells. Double-positive (DP), double-negative (DN), and V5+ (successfully knocked-in) populations are small and reflect noise. (C) Analysis of knocked-out cells similarly shows the percentages of CD4 (38.0%) and CD8 (49.5%) SP cells. 31.2% of the knocked-out cells were successfully knocked in (V5+), including 36.2% of CD4 and 30.2% of CD8.

A group of 9 flow cytometry dot plots showing the changes in cell populations after they have been transfected by electroporation.

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