Nucleofection of K562, CD34+ HSCs and H9 cells

2,E+5 K562 cells were transfected with 3µg PCR-eGFP or mini-eGFP or plasmid-eGFP by 4D-Nucleofector System (Lonza) using the SF Cell Line 4D-Nucleofector X Kit S (V4XC-2032) according to the manufacturer’s instructions. The FF-120 program was used.

2,E+4 CD34+ HSCs were transfected with 1?µg mini-eGFP or plasmid-eGFP by 4D-Nucleofector System (Lonza) using the P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza) according to the manufacturer’s instructions. The EO-100 program was used.

4,E+5 H9 embryonic stem cells (ESCs) were transfected with 1?µg mini-eGFP or plasmid-eGFP by 4D-Nucleofector System (Lonza) using the P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza) according to the manufacturer’s instructions. The CB-150 program was used.

After Nucleofection, cells were resuspended with prewarmed cell medium and transferred into plate and incubated at 37°C in 5% CO2. Cell viability and transfection efficiency were evaluated 2 days after Nucleofection by cell counting and fluorescence-activated cell sorting (FACS) analysis, respectively.

Culturing primary human fresh umbilical cord blood-derived T cells

T cells were stimulated with anti-CD3/CD28 Dynabeads at a bead to T cell ratio of 1:1 and cultured in X-Vivo 15 medium (Lonza), supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine and 1 mM sodium pyruvate in the presence of 300 IU/mL recombinant human interleukin (IL)-2.

Generation of mini-019-CAR-T cells

T cells were activated for 3 days and then 1,0E+6 cells were transfected with 3µg mini-019-CAR DNA by 4D-Nucleofector System (Lonza) using the P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza) according to the manufacturer’s instructions. The EO-115 program was used. After Nucleofection, cells were resuspended in 1?mL prewarmed T cell medium and transferred into 12-well cell plate and incubated at 37°C in 5% CO2. Cell viability and transfection efficiency were evaluated 2 days after Nucleofection by cell counting and FACS analysis, respectively.

Lonza Summary:

Minicircle vectors have been used to deliver Sleeping Beauty system to generate CAR-T cells.

In this study, the authors utilize the Nucleofector 4D technology to deliver episomal minicircle DNA vector into human primary T cells, hCD34+, hESC H9 and K-562 lines generating an integration-free modified cells. After cultivation and stimulation in the X-vivo15 media system, the mini-019-CAR-T engineered human T cells showed a remarkable efficacy in vitro and in vivo on a cancer mouse model, comparable to lentivirus-modified CAR-T cells.

The data suggest that transgene integration-free T cells constitute an attractive option for clinical applications. In fact, the usage of the Nucleofection delivery method shows a robust alternative to lentiviral and retroviral delivery method eliminating the risk of insertional mutagenesis. Shorter CAR-T persistence, due to the non-integrative nature of the transgene, helps reducing side effects such as cytokine storm or off-tumour toxicity. When needed, multiple rounds of infusion of the Nucleofector engineered CAR-T cells should help eliminate cancer cells efficiently. Finally, this would reduce significantly the cost of the immunotherapy, as the clinical-grade virus production is a major reason driving the high cost of viral-based clinical use.