The idea underlying the project was to prove the relevance of the development of a novel class of immunosuppressive drugs with a better safety that will selectively inhibit the proliferation of T lymphocytes by targeting a medically-relevant target named CTPS1 recently identified by the group of A. Fischer and S. Latour. Several points needed to be addressed to gain the validation of CTPS1 as a target. The excepted outcomes of the project were to amass the weight of data on the biology of CTPS1 needed for achieving a successful technology transfer to a start-up company that has been founded in partnerships with private partners in order to perform a drug discovery program including high throughput drug screenings. This background work included the development of cellular and animal models, the technical feasibility studies validating the plan for drug discover and pre-clinical studies.
To better evaluate the role of CTPS1 in vivo and for pre-clinical studies, several mice models have been developed and established during the project, including different mouse lines deficient for CTPS1 (CTPS1-knock out mice or CTPS1-KO), in which the inactivation of the ctps1 gene was be achieved in all tissues, hematopoietic tissues, T cells or in an inducible manner upon tamoxifen treatment. Only CTPS1-KO mice in all tissues and in hematopoietic tissues have been obtained before the end of the project and analyzed with standard methods including examination of the immune system. Results of these analyses indicate that 1) inactivation of the ctps1 gene in all tissues leads to embryonic lethality 2) inactivation of the ctps1 gene in hematopoietic tissues results in severe defects in erythroid, myeloid and lymphoid development. Because of the severity of these phenotypes, preclinical studies could not be performed in these mice. These results demonstrated the crucial role of CTPS1 in the embryonic development, which was not noticed in humans with a CTPS1 deficiency. To circumvent this difficulty and maintain the objectives of the project, mice models mimicking the human CTPS1 mutation (that was identified in a cohort of immune-deficient patients) have been engineered by standard recombination strategy and by CRISPR-Cas9 technology, but they were not yet available at the end of the project. Study of the different mice models will be pursued when available. During the project, several studies were also undertaken to identify and understand the mechanisms, signals and post-translational modifications underlying the expression and the function of CTPS1 in activated T lymphocytes. These studies led to the identification of transcription factors and signaling cascades required for CTPS1 expression in activated T cells. Investigations will be pursued to better characterize the role of these post-translational modifications, transcription factors and signaling cascades. In parallel of these studies, various tools and tests have been produced to validate the drug screening cascades and improve the feasibility of the drug discovery program, that were transferred to the start-up/industrial partner. These materials and methods included CTPS1-deficient cell lines (obtained by the CRIPR-Cas9 technology), assays for CTPS1 detection in tissues and blood samples, validation assays for the measurement of CTPS enzymatic activity from cell extracts.
Contacts with potential industrial and financial partners by the start-up have been established in order to move forward with the whole discovery program and clinical indications and developments. The data obtained during the project have helped for these contacts and discussions. Competitive analyses have been initiated from a market and scientific/clinical and will be continued. From these analyses, autoimmune diseases and Graft Versus Host Disease (GVHD) remain the first medical for the use of these novel immunosuppressive drugs. Several specific autoimmune diseases appear to be highly relevant (from a market and scientific/clinical perspectives) for the first potential clinical assays (as well as GVHD). Cancer is now considered as a possible new indication for CTPS1 inhibitors in particular T-cell acute lymphoblastic leukemia (T-ALL), for which there is a high unmet medical need. Importantly, these analyses did not reveal restrictions or contraindications to the drug discovery program.
In conclusion, the project allowed to develop and generate tools and knowledge supporting the relevance of the development of immunosuppressive drugs targeting CTPS1. Unfortunately, preclinical studies in mice could not be performed because of the embryonic lethality of CTPS1-deficient mice. However, the data, tools and methods engendered by the project helped start-up/industrial partner to find and convince new financial partners to secure and move forward with the drug discovery and clinical development program for the next years.
