Our laboratory protocol enables the combination of human genome editing with CAR T cell therapy. The laboratory procedure is the approach with CRISPR-Cas9 at targeting up to three genes with high efficiency. And finally, our approach is able to be translated into human clinical trials.
The methods described can be applied to other CAR constructs and target genes. The basis of the techniques are robust enough to be translated to larger scale and to academic and industry collaborators for further development. When attempting this protocol for the first time, limit the number of groups in the guide RNA screen to enable accurate incubation times.
Adhering to the culture conditions and seeding density as described have proven to be critical in our experience. To begin, obtain autologous peripheral blood mononuclear cells, or PBMCs, from healthy volunteer donors and isolate CD4 and CD8 positive T cells using commercially available CD4 and CD8 selection kits. Combine CD4 positive and CD8 positive T cells in a one to one ratio and incubate 3 million cells per milliliter in R10, supplemented with 5 nanograms per milliliter of each human IL7 and human IL15 overnight at 37 degrees Celsius.
On the next day, count the T-cells and centrifuge 5 to 10 million cells at 300 times G for five minutes. Discard all supernatant and wash the cell pellet in reduced serum minimal essential media. Re-suspend the pellet in 100 microliters of nuclear affection solution according to the manufacturer's instructions.
While washing the cells, prepare a ribonucleoprotein, or RNP complex, by incubating 10 micrograms of Cas9 nuclease with 5 micrograms of single guide RNA for 10 minutes at room temperature. Include a mock control without single guide RNA. Combine the re-suspended cells with the RNP complex and add 4.2 microliters of four micromolar electroporation enhancer.
Mix well and transfer into electroporation cuvettes. Electroporate the cells using pulse code EH111, then incubate 5 million cells per milliliter in R10, supplemented with 5 nanograms per milliliter human IL7 and human IL15 at 30 degrees Celsius for 48 hours in 12 well plates. After the incubation, proceed with T-cell activation and expansion.
Design the sequencing primers by entering the sequence of the PCR amplicon into a standard primer design software. The design software will suggest multiple primer sequences that are suitable for Sanger sequencing. Choose forward and reverse primers that bind with the amplicon at least 150 base pairs upstream or downstream of the gRNA cut site to ensure good sequencing quality around the indels.
Use TIDE analysis to detect knockout efficiency at the genomic level. The algorithm accurately reconstructs the spectrum of indels from the sequence traces and calculates R square values. After activation, T-cells are expanded in culture and cryopreserved for future studies.
During the expansion, the population doubling and volume changes are tracked throughout the protocol for both mock and edited CAR T cells. No significant changes were detected in the proliferation and activation during the expansion. Once the cells were cryopreserved, levels of CAR expression were determined for further functional studies for both the mock edited and knockout CAR T cells.
No significant changes were observed. Knockout efficiency can be determined using multiple techniques. In these representative flow cytometry plots, the PDCD1 and TRAC loci were targeted using single guide RNA, showing an efficiency of 90%for the PDCD1 single guide RNA and 98%for the TRAC single guide RNA across multiple healthy donors.
It is important to make sure that the molar ratios of Cas9 and guide RNAs are accurate. During the procedure, the cells should always be kept at four degrees Celsius and not be left in the electroporation solution for extended periods of time. After CAR preservation, the CRISPR engineered CAR T cells can be used for in-vitro and in-vivo functional assays, such as cytotoxicity assays, cytokine production and syngenetic or xenograph tumor mouse models.
Our approach using CRISPR Cas9 technology is now being applied to clinical trials. The approach can be used both for cancer, as well as a non-malignant genetic disorders, such as hemoglobinophaties, which affect millions of children and adults worldwide.
