The method is relatively simple and allows efficient gene editing of rhesus macaque B cells. The method allows the testing of gene-edited B cells for therapeutic purposes in rhesus macaques. It is also suitable for preclinical B cell therapy research.
The general method may be adapted for other cell types. If the procedure is followed correctly, one should not expect struggles. However, as gRNA cutting efficiency and a good quality batch of recombinant AAV6-providing template are critical.
To begin, prewarm the B cell culture media in a 37-degree-Celsius water bath. Thaw the B cell stimulants on ice. Prepare an appropriately-sized tube containing a pre-warmed thawing media.
Thaw one to two vials of primary rhesus macaque splenocytes or PBMCs in a 37-degree-Celsius water bath. Decant the splenocytes into the prepared tube containing a pre-warmed thawing medium. Rinse the cryotubes to collect all the cells.
Centrifuge the cells at 200 G for 10 minutes at room temperature. Discard the supernatant and resuspend the cells in 10 milliliters of thawing medium for washing. Repeat the centrifugations three times to remove the freezing medium.
After the last centrifugation, resuspend the cells at an estimated five times 10 to the six cells per milliliter in a B cell culture medium. Combine 10 microliters of cell suspension with 10 microliters of 0.4%trypan blue and count the cells on a hemocytometer. Adjust the cell concentration to three times 10 to the six cells per milliliter with B cell culture medium according to the cell count.
Then, add the B cell stimulants to their final concentrations and mix. Transfer the cells to a cell culture dish and incubate them at 37 degrees Celsius with 5%carbon dioxide for 48 hours. After settling, cells should form a dense monolayer and small clusters of cells may be already visible.
After about two days, cells should be healthy, irregular, and have formed visibly bigger clusters. After activating the B cells, prepare the reagents for electroporation and transduction. Prewarm DMSO nuclease-free duplex buffer, buffer T, and buffer E or E2 from the electroporation kit to room temperature.
Thaw the rAAV6 homology-directed repair template, or HDRT, and B cell stimulants on ice. Resuspend the CRISPR Cas9 single-guide RNA, or sgRNA, at 100-micromolar in duplex buffer. Reconstitute for 10 minutes at room temperature and mix by vortexing and flicking.
Place the reconstituted sgRNA on ice until use. For 10-microliter electroporation, prepare 550 microliters of B cell culture medium with stimulants and 1%DMSO. Add 50 microliters of this medium with or without antibiotics into a 48-well cell culture plate.
Then, add rAAV6 HDRT to the medium in the wells, up to 20%of the volume of the well. Prewarm the prepared dish and remaining medium in an incubator at 37 degrees Celsius with 5%carbon dioxide. Next, for 10-microliter electroporation, prepare 1.15 microliters of ribonucleoprotein, or RNP, by mixing 0.4 microliters of 61-micromolar Cas9 with 0.75 microliters of 100-micromolar sgRNA in duplex buffer.
Incubate the RNP for 15 minutes at room temperature before mixing it with the cells. After incubation, multiple RNPs can be combined if more than one locus is to be targeted simultaneously. Next, prepare the cells for electroporation.
Leave the cells at room temperature to avoid temperature shocks. After incubation, harvest the cells into an appropriate vessel. Rinse the dish with DPBS to collect the maximum number of cells.
Centrifuge the cells at 200 G for 10 minutes. Discard the supernatant and resuspend the cells in DPBS at approximately two times 10 to the six cells per milliliter, based on the number of cells put into the culture. Combine equal volumes of cell suspension and 0.4%trypan blue and count using a hemocytometer.
After centrifuging and discarding the supernatant, resuspend the cells in pre-warmed buffer T at 5.55 times 10 to the seven cells per milliliter, based on the cell count. Set up the transfection system by turning the machine on and setting it to 1, 350 volts, 15 milliseconds, and one pulse. Place the pipette station inside the laminar flow hood.
For each set of 10 electroporations, prepare a transfection tube with three milliliters of buffer E.Insert the tube into the pipette station. Combine 1.15 microliters of RNP with nine microliters of cells per electroporation. Prepare at least 20%more to avoid bubbles and incubate at room temperature for one minute before electroporation.
Aspirate 10 microliters of RNP and cell mixture into the electroporation tip on an electroporation pipette. Insert the loaded pipette into the pipette station and start the electroporation. Ensure the tips are completely free of air bubbles to prevent arcing.
Immediately eject the electroporated cells into the prepared pre-warmed small volume of medium, with or without rAAV6, inside the 48-well. Add control samples without transfection to the culture wells. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for four to six hours.
At the end of the incubation, add 450 microliters of pre-warmed B cell culture medium containing stimulants, DMSO, and with or without antibiotics into the plate. Perform genomic DNA analysis after 24 hours. Quantify the editing efficiency with digital droplet polymerase chain reaction, or PCR, using a primer outside the homology arm and a primer inside the insert.
Perform PCR to amplify the insertion site and Sanger sequencing to verify correct editing. To analyze the protein levels, culture the cells for 40 to 48 hours after electroporation to allow protein expression changes and perform an analysis by flow cytometry. The production of rAAV6 using tetracycline-enabled, self-silencing adenoviral helper resulted in the production of four times 10 to the 10 GC per milliliter of cell culture medium, outperforming the production using a helper-free triple transfection by 30-to 40-fold.
The optional purification resulted in the elimination of the majority of CD3+T cells and CD14+and CD16+myeloid cells, with purities of 80 to 95%CD20+B cells being routinely obtained. Gene editing of primary rhesus macaque B cells is shown here. This study maintained high cell viability, while simultaneously deleting the light chain expression in 80%B cells.
The majority of the B cells still expressed isotype IgM. The addition of rAAV6 encoding the antibody 1485 HDRT resulted in gene editing and antibody 1485 surface expression in 16 to 21%of the B cells, albeit at a lower fluorescence intensity for antibody chains than on unedited B cells. The addition of 1%DMSO and extended, concentrated incubations with the rAAV6 HDRT generally increased the editing efficiency.
Using this method, typically five to 20%and up to 40%editing efficiency was achieved, depending on the individual rhesus macaque and the quality of the rAAV6 HDRT batch. While performing the electroporation, ensure to have enough mix for transfection and there are no bubbles in the tip. The cells can be analyzed by any desired method or an autologous transfer into the donor rhesus macaque can be performed.
