A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

CAR T cell therapy has achieved unprecedented success in treating certain pathological cancers. In this study, we introduced a nonviral, long-integrating approach for generating transient CAR T cells and provide targeted procedures for assessing CAR T cell function. Our research aims to generate safe and efficacious CAR T cells in a more cost-effective approach.

CAR T cells are typically generated by viral transduction. Retroviruses and antiviruses are the most common viral vectors for CAR gene delivery. Manufacture viral vectors is complex and costly, and the viral transduction induces random and permanent CAR-encoding DNA integration into the T cell genome, which may cause safety concerns.

Our protocol offers a nonviral approach for generating CAR T cells that only transitively express CAR. This method uses mRNA for CAR expression and employs electroporation to deliver the mRNA into T cells. This has no gene integration to T cell genome, and the manufacturing procedures are simpler and more cost-effective.

To begin, linearize the CAR plasmid DNA with the restriction enzyme BGL2 and perform phenol chloroform isoamyl alcohol DNA extraction procedure. After purifying the linearized DNA, verify the size and integrity of the template with agarose gel electrophoresis. For in vitro transcription, mix the reaction components and incubate the reaction mixture at 37 degrees Celsius for at least two hours.

After the transcription, add two microliters of ribonuclease III, DNase I to the reaction. Mix gently and incubate for 15 minutes at 37 degrees Celsius to degrade the template DNA. Purify the transcribed CAR MRNA with an RNA cleanup kit and dilute it in nuclease-free water.

Measure the mRNA concentration using a spectrophotometer before storing it at minus 80 degrees Celsius. Thaw the in vitro transcribed and purified car mRNA sample on ice. Suspend one times 10 to the power of six cryopreserved human peripheral blood mononuclear cells in one milliliter of CAR T medium.

To activate the T cells, add an equal number of pre-washed human T activator CD3/CD28 beads. Culture and expand the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for several days. Add one milliliter of CAR T medium per well to two wells of a 12-well plate and incubate at 37 degrees Celsius for equilibration.

Now, transfer five times 10 to the power of six T cells to a sterile tube. To remove the CD3/CD28 beads, place the tube onto a magnetic stand and carefully pipette the cell suspension into a new tube. Centrifuge the tube at 300 G for five minutes at room temperature.

After discarding the supernatant, resuspend the cell pellet in one milliliter of sterile PBS. Centrifuge the tube again and resuspend the cell pellet in 200 microliters of Neon buffer T.Mix five micrograms of CAR MNA diluted in the Neon buffer T and 100 microliters of cells in a total volume of 125 microliters. Add 25 microliters of Neon buffer T to the remaining 100 microliters of cells, which serves as a negative control.

Set the Neon electroporation system to 1, 800 volts, 10 milliseconds, and a single pulse. Use the Neon pipette to aspirate the T cells CAR mRNA mixture into a Neon 100 microliter tip and electroporate the cells. Immediately transfer the electroporated cells into the equilibrated culture medium in the 12-well plate and return the plate to the incubator to expand the cells until they are ready for use.

Retrieve the T cells transfected with CAR mRNA for flow cytometric analysis as early as six hours after the electroporation. Mix the CAR T cells several times with a pipette and transfer at least one times 10 to the power of five cells to a five-milliliter fluorescence-activated cell sorting, or FACS, tube. Add three milliliters of cold wash buffer to the tube and centrifuge the tube at 500 G for five minutes at room temperature.

After discarding the supernatant, resuspend the cell pellet in 100 microliters of wash buffer. Add two microliters of fluorescence-labeled detection antibody and seven AAD viability dye to the suspended cells. Mix the sample and incubate it on ice for 30 minutes, avoiding light exposure, then wash the cells with three milliliters of cold wash buffer and centrifuge the tube again at 500 G for five minutes at room temperature.

After discarding the supernatant, resuspend the cells in 100 microliters of wash buffer and immediately analyze the cells using a flow cytometer. Remove the medium from the tumor cells expressing the CAR antigen on the cell surface. After washing the cells with PBS, incubate them with one milliliter of 0.05%trypsin EDTA solution for one minute at 37 degrees Celsius, then prepare a single cell suspension in the appropriate culture medium at a density of one to five times 10 to the power of five cells per milliliter.

Next, add 50 microliters of pre-warmed tumor cell culture medium to the E-plate and place it in a real-time cell analysis, or RTCA instrument, to measure background impedance, then add 100 microliters of the tumor cell suspension to each well and equilibrate the plate at room temperature for 30 minutes. Place the E-plate back into the RTCA instrument and monitor the cell index for 24 hours, with measurements taken every five to 15 minutes. The following day, prepare the CAR T cell suspension.

Pause the cell index recording and remove the E-plate from the RTCA instrument. Tilt the plate slightly and carefully remove 50 microliters of culture medium from each well without disturbing the tumor cells, then add 100 microliters of CAR T cells or control T cells to each well. Return the E-plate to the RTCA instrument and resume cell index monitoring for at least 24 hours, with sweeps taken every five to 15 minutes.

Stop the cell index monitoring and analyze the cytotoxicity result. For cytokine secretion analysis, transfer the supernatant from each well into a round bottom or V-shaped 96-well plate. Centrifuge the 96-well plate at 300 G for five minutes at room temperature and carefully transfer the supernatants into a new 96-well plate.

Finally, measure the cytokine levels in the supernatants using commercial ELISA kits. The CAR mRNA sequence was validated to be approximately two kilobases in size by gel electrophoresis. Over 50%of activated T cells expressed the CAR on day one post-electroporation, increasing to over 70%on day two before declining to approximately 10%by day five.

CAR T cells showed significant cytotoxicity against HER2 positive SK-OV-3 tumor cells, indicated by a sharp reduction in tumor impedance, whereas control T cells demonstrated minimal effect. High levels of interferon gamma secretion were detected in the CAR T cell culture medium, but not in the control group.