Some patients suffer from life-threatening complications caused by normally harmless chronic viruses such as cytomegalovirus (CMV) as adverse effect of immunosuppressive therapy, e.g. required after transplantation. Mostly, potent anti-viral drugs can manage these complications, however many of these drugs are toxic, some patients are irresponsive or develop resistances. Thus, specific regeneration of the endogenous anti-viral immune response by adoptive anti-viral T-cell therapy (AVTT) is an attractive alternative treatment. In some cases, our conventional approach of adoptive AVTT only controls the virus temporally, probably due to malfunctions of transferred T-cells caused by immunosuppressants.
We applied electroporation to transfer nucleoprotein complexes of the nuclease CRISPR-associated protein 9 with a site-specific single guide RNA (sgRNA) to generate immunosuppressant-resistant T-cells by vector-free knockout (k.o.) of the cell-intrinsic target protein, which is required for the drug's immunosuppressive function. We proofed the concept by assessment of the function of CMV-specific T-cell products in the presence of distinct immunosuppressive drugs, upon CMV-specific stimulation.
We successfully developed a GMP-compliant protocol with a sgRNA, which efficiently knocks out the target in CMV-specific T-cell products (Tk.o.CMV-TCPs). We confirmed functionality of T-/-CMV-TCPs in the presence of the immunosuppressant. Specificity was demonstrated by loss of function of Tk.o.CMV-TCPs in the presence of an immunosuppressant from the same class requiring a different adaptor protein. Tk.o.CMV-TCPs showed improved anti-viral cytokine production even in the presence of classical triple immunosuppression applied in organ transplantation. Currently, we are investigating off-target effects to exclude safety concerns as prerequisite for ultimate translation of Tk.o.CMV-TCP to first in-human application.