PEN photoporation for the genetic engineering of therapeutic mesenchymal stromal cells and T cells

Project Information

Penphomet

Grant agreement ID: 101158879

DOI

10.3030/101158879

EC signature date 26 February 2024

Start date 1 March 2024

End date 28 February 2027

Funded under

• The European Innovation Council (EIC)

Total cost

€ 2 497 711,25

EU contribution

€ 2 497 711,25

Coordinated by

TRINCE
Belgium

Periodic Reporting for period 1 - Penphomet (PEN photoporation for the genetic engineering of therapeutic mesenchymal stromal cells and T cells)

Reporting period: 2024-03-01 to 2025-02-28

Summary of the context and overall objectives of the project

Adoptive cell therapy has emerged as a promising strategy to treat cancer. It relies on patient-derived cells, such as T cells and mesenchymal stromal cells (MSCs), which are genetically engineered to become better equipped to fight cancer cells. While ex vivo genetic modification of T cells and MSCs has traditionally been performed with viral vectors, they come with concerns about safety, sustainable production and high development costs. Electroporation is a non-viral alternative transfection technology, but can lead to significant gene expression changes, phenotypic alterations, and decreased therapeutic potency. Recently, photoporation with electrospun photothermal nanofibers (PEN photoporation) was demonstrated to provide a safer alternative with minimal impact on the cell’s functionality and phenotype. The technology makes use of photothermal nanofibers which, upon stimulation with laser light, can transiently permeabilize cells to allow gene-modifying effector molecules to enter the cells. Having been thoroughly demonstrated and validated in a research setting (TRL4), this project aims to bring the PEN photoporation technology to TLR6 by developing hard- and software for automated high-throughput transfections of T cells (>1B cells/h) and MSCs (>10M cells/h). The technology will be extensively tested and validated in the cGMP compliant laboratories of the project partners for the genetic engineering of T cells and MSCs. At the same time, steps will be taken to prepare for commercialization and market deployment. By the end of the project a fully automated and validated high-throughput prototype system will be available for installation at centralized cell production facilities or ready for integration in point-of-care cell manufacturing equipment. This project aligns with the Micro-Nano-Bio challenge as it combines nanotechnology with microfluidics to enhance genetically engineered cell therapy products.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the first project year, we have successfully developed a cell culture plate containing photothermal nanofibers for the photoporation of cells. Experiments demonstrated, however, that photoporation with biodegradable polymeric particles was more efficient than with photothermal nanofibers. Therefore, it was decided to continue with those sensitizers instead, for which we could demonstrate efficient transfection of T cells and MSCs with relevant biological molecules, such as mRNA.

In-house built photoporation equipment was installed at the research facilities of the project partners, with which they could demonstrate that results on the transfection of T cells and MSCs are reproducible.

Hard- and software is being developed for high-throughput photoporation of cells.

It was demonstrated that photoporation of T cells and MSCs happens equally efficiently in microfluidic chambers as in traditional well plates.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The possibility of transfecting T cells and MSCs in microfluidic devices opens up the possibility of developing closed and automated cGMP compliant photoporation equipment, ready for integration in cell therapy manufacturing facilities. Further work will focus on upscaling and demonstrating efficient transfection of these cells with relevant biomolecules other than mRNA, such as pDNA and gene editing enzymes.