Periodic Reporting for period 2 - EpiTune (Epigenetic fine-tuning of T cells for improved adoptive cell therapy)
Reporting period: 2020-07-01 to 2021-12-31
In this project, we want to tackle these obstacles from a new molecular angle, utilizing the profound impact of epigenetic mechanisms on the senescence process as well as on the functional imprinting of T lymphocytes. We propose to equip T lymphocytes with the required properties for their successful and safe therapeutic application, including their functional fine-tuning according to the clinical need by directed modifications of the epigenome ('Epi-tuning').
To reach these goals we want to: 1) clarify the epigenetic mechanisms in the acquisition of proliferation-induced cellular senescence to reveal strategies for their directed prevention and 2) identify epigenetic regions involved in stable functional imprinting of T cells in order to address them as molecular switches for the functional optimization of T cell products using state-of-the-art CRISPR/dCas9-mediated epigenetic editing approaches.
These innovative epigenetic "one-shot" manipulations during the in vitro expansion phase of T cell products should advance T cell therapy towards improved efficiency, stability as well as safety.
In order to switch DNA methylation states of promoters and enhancer at will and with that regulate gene expression for the improvement of T cell products, we established a technique of 'epigenetic editing' in our lab. This method is based on the CRISPR-Cas9 system: It harnesses the targeting mechanism of the system to allow the directed relocation of the editing complex selectively to the target region. However, instead of inducing changes in the genomic sequencing in the target region, as the regular Cas9 enzyme does, our editors delivers an epigenetic modifier to the target location, which will then induce DNA demethylation at the target site. This approach proved to work very efficiently in T cells, which were successfully transfected with plasmids coding for the complete editor complex. As a proof-of-concept study, demethylation of the regulatory element lead to the activation of the associated gene, proving that indeed DNA methylation switching can be achieved in primary human T cells and allows directed changes of the gene expression program in these cells. The results of this study we could successfully publish in the open access journal Frontiers in Immunology (Kressler et al., 2021). This method lays the basis for our aim of fine-tuning T cell products by modifying the epigenetic profile ('epi-tune'). While the approach works very well, its efficiency is currently limited since transfection of plasmids is rather toxic to primary human T cells. We are currently working on improving this limitation and apply it on the identified epigenetic elements undergoing DNA methylation changes during the Treg product generation culture.
Other breakthrough findings we expect to occur in the second half of the project, which hopefully will be less (strongly) impacted by the Covid19 pandemic as the first half of the project.