Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

This homology-mediated end-joining, or HMEJ-based protocol, offers a GLP-compatible approach for targeted non-viral engineering of highly functional CAR T or TCR-transgenic T-cells for immunotherapy.

The current GMP manufacturing of CAR T and TCR transgenic T-cells for immunotherapies remains expensive and requires a long production time. This non-viral method significantly reduces both the cost and the manufacturing time.

This protocol enables non-viral targeted integration of large DNA templates into primary human T-cells. In contrast, many other techniques exhibit reduced efficacy with larger DNA templates or require the use of randomly-integrating viral vectors.

This non-viral method achieves efficient targeted integration of transgenes into primary T-cells with low toxicity and preserved function in vitro and in vivo, supporting the clinical development of engineered T-cell therapies.

Efficient non-viral targeted engineering in primary human T-cells creates an opportunity to develop novel immune cell therapies more safely and effectively with future research focused on improving therapeutic efficacy across multiple disease indications and immune cell subsets.

[Narrator] To begin, prepare a recovery plate by adding 300 microliters of recovery media to a well of a 24-well tissue culture plate. Warm the plate in a 37 degrees Celsius and 5% carbon dioxide incubator. To prepare T-cells, harvest the stimulated T-cell and T-cell activation bead mixture by mixing them in the flask, and then moving them to an appropriate volume tube. Place the tube in the magnet to isolate the beads from the cells in suspension. Allow it to incubate for at least one minute. Without removing the tube from the magnet, transfer the media and T-cells to a new tube. Count the T-cells and transfer them to either 14-milliliter or 50-milliliter conical tubes. Top off the tube with PBS, and keep the cells in a 37 degrees Celsius and 5% carbon dioxide incubator until centrifugation. Next, prepare the T-cell mix by combining the primary cell solution and the supplement to create the master mix buffer. Centrifuge the T-cells in PBS at 200 g for 10 minutes at room temperature. Decant or aspirate the supernatant. Resuspend the T-cells in the master mix buffer at a concentration of 1 million cells per 18 microliters of master mix buffer. On ice, add reagents to a well of a 96-well plate for each condition. Now, prepare the electroporation mix by adding 18 microliters of T-cell mix to each well of the 96-well plate as required. Add additional master mix buffer to ensure a final total volume of 22 microliters for all electroporation conditions. Mark one end of the electroporation cuvette and its cap to perform the electroporation. Remove the cap from the cuvette. Gently pipette the electroporation mix from the wells of the 96-well plate up and down one to two times and load it into cuvettes. Recap the cuvette using the previously made marking to avoid putting the cap on backward. Tap the cuvette three to five times to remove potential air bubbles. Place the cuvettes in the electroporation equipment and perform electroporation. Gently remove the cuvette from the equipment and place it in the hood. Allow the cells to rest at room temperature for 10 to 15 minutes. After the rest period, withdraw 80 microliters of warmed recovery media from the recovery plate. Add the media to the cuvette gently, avoiding direct contact with cells. Gently pipette up and down once, and transfer the total reaction volume back to the recovery plate. Incubate the cells for 30 minutes at 37 degrees Celsius and 5% carbon dioxide. Add TCM to dilute the cells to a concentration of 1 million cells per milliliter. After dilution, restimulate the cells by adding T-cell activation beads at a ratio of 0.5 beads per cell to each well. Ensure that the beads have been washed to remove their storage buffer, and then resuspend them in TCM before adding to each well. Finally, on day three, remove the T-cell activation beads and analyze the cells. A high-efficiency knock-in of the giant minicircle construct into the TRAC locus of primary human T-cells was achieved using CRISPR-Cas9, resulting in 23.35% GFP expression. The negative control showed no GFP expression. There were no significant differences in fold expansion between the experimental and control conditions. Similarly, no significant differences were observed in cell viability between the experimental and control conditions.