CRISPR/Cpf1 and suicide gene as personalized approach for patients with TP53-mutated Chronic Lymphocytic Leukemia (CLL)

Chronic Lymphocytic Leukemia (CLL) is a very heterogenous disease caused by alterations in both chromosomes and genes, such as deletion of the 13q14, 11q22-23, 17p12, and trisomy of chromosome 12 or genetic mutations in the TP53, ATM, BRIC3, NOTCH. Within genetic abnormalities, TP53 mutations are detected in a small percentage of leukemia patients (about 10%). Currently adopted therapeutic choices (chemoimmunotherapy, targeted therapy, hematopoietic stem cell transplantation) are not effective due to the acquired abilities of TP53-mutated clones to escape the control systems. For this reason CLL patients harboring a mutated TP53 gene (point mutation, insertion or deletions) are grouped in the highest risk category according to the international prognostic index for chronic lymphocytic leukemia (CLL-IPI). Hence, one possible solution for these patients would be the development of a personalized therapy. In my study I designed and developed a CRISPR-Cpf1 system which has proved to be an efficacious technology to get rid of only mutated cancer cells. The system called CRISPR_LV_TK+, recently patented (WO2020/079574), is based on the locus specific delivery of the Herpes Simplex Virus – Thymidine Kinase (HSV-TK) suicide gene in cells bearing the target TP53 mutation detected in a CLL patient (p.Ser183*) in follow-up in our Unit. The approach was positively tested in HEK293T cell lines engineered for the target mutation. Following administration of ganciclovir, I was able to detect a high percentage of cell death only in the samples that have properly integrated the HSV-TK. In conclusion, the results show the high efficiency and specificity of the CRISPR_LV_TK+ system, and opens avenues to be applied as personalized therapy not only for CLL but for different kinds of cancers caused by specific mutations in distinct genes which are the driver of therapy resistance.