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Epigenetic fine-tuning of T cells for improved adoptive cell therapy

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

Adoptive T cell therapy is a promising approach in various clinical settings, from target-specific immune reconstitution fighting cancer and chronic infections to combating undesired immune reactivity during auto-immunity and after organ transplantation. While first promising therapies are now available in the oncological field, several limitations have still to be overcome in order to unfold the full potential of this precision medicine approach. Among them is 1) the acquisition of senescence of T cells during the required in vitro expansion phase which limits their survival and fitness after infusion into the patient, and 2) the functional plasticity of T cells, which is sensitive to the inflammatory environment they encounter after transfusion and which might result in a functional switch from the desired effect (e.g. immunosuppressive) to the opposite one (pro-inflammatory).

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.
As the first step to reach our goals, we analyzed epigenetic changes occurring during the generation of T cell products. For this, we profiled genome-wide DNA methylation, one of the most relevant epigenetic marks, in regulatory T lymphocytes (Treg) at different time-points during expansion culture. Samples for this were acquired from the Berlin Center for advanced therapies (BeCAT), which hosts a lab generating therapeutic cell products under good manufacturing practice (GMP) conditions for the use in clinical trials and in compassionate use applications. These detailed analyses revealed two key findings relevant to our project aims: 1. There was a progressive genome-wide DNA methylation loss occurring in the heterochromatic parts of the genome which correlated to the expansion rate of the culture and hence correlated to the degree of proliferation-induced senescence. In functional studies, we found first indications on the underlying molecular causes for this proliferation-induced heterochromatic demethylation. In the next steps, we will further analyze the consequences of this effect for T cell function and senescence acquisition and search for approaches to prevent or even reverse these culture-induced alterations. 2. We identified a number of regulatory regions (i.e. promoters and enhancers) in the genome, which undergo DNA methylation changes during generation of Treg products. Several of them might negatively influence the function and identity of the final products. Therefore, such elements qualify as promising target regions for epigenetic editing approaches aiming at improving therapeutic Treg products. The results of this deep DNA methylation profiling study of Treg products is currently in revision for publication in a relevant scientific journal.

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.
Although epigenetic profiling reveals many important features of a cellular population, detailed epigenetic analyses of therapeutic T cell products as one part of the quality control assessment after manufacturing, is currently lacking. Therefore, our in depth DNA methylation profiling of Treg products during manufacturing is unprecedented and hopefully will be the start to include epigenetic profiling to the portfolio of standard techniques for the quality control measures of T cell products.
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.
Visual project aims