Presenter of 1 Presentation
GENE EDITING OF APDS1 T CELLS
Abstract
Background and Aims
Activated PI3kinase Delta Syndrome type 1 (APDS1) is caused by gain of function mutations affecting the PIK3CD gene. The project aims to develop a gene editing approach to correct ex vivo patient T cells and reinfuse them. This treatment may be used to treat severe viral infections, or serve as a pre-requisite for a milder conditioning before hematopoietic stem cell transplantation.
Methods
T cells were modified using a nuclease targeting the PIK3CD gene and an AAV. Nuclease cleavage frequency was assessed by digital droplet PCR. Successful editing was analyzed by flow cytometry based on the expression of a reporter gene. Phospho-AKT level and cytotoxic activity were analyzed to evaluate the functional activity of corrected T cells.
Results
A highly specific nuclease achieved a reproducible cleavage efficiency of 80% and in conjunction with the AAV a gene editing efficiency of 20% at the PIK3CD locus. High basal level of phosphorylated AKT in APDS1 patient T cells was normalized in corrected patient cells. After T cell receptor activation phosphorylation of AKT could still be induced in corrected cells. Gene editing improved the impaired cytotoxic activity of repeatedly rechallenged APDS1 patient T cells towards lymphoblastoid B cell lines in the presence of blinatumomab.
Conclusions
We developed a protocol allowing efficient gene editing of APDS1 patient T cells. Corrected APDS1 T cells displayed normalized level of AKT phosphorylation and increased cytotoxic activity indicating the potential of our approach as a new treatment for ADPS1.