Scalable continuous-flow electroporation platform enabling T cell transfection for cellular therapy manufacturing.

Area of Science:

• Biotechnology
• Cellular Therapy Manufacturing
• Gene Editing Technologies

Background:

• Viral vectors are a manufacturing bottleneck for cellular therapies.
• Current electroporation methods lack throughput and scalability for large-scale cell manufacturing.
• Efficient non-viral transfection methods are needed for primary cell engineering.

Purpose of the Study:

• To present a novel electroporation platform for rapid, reproducible, and scalable cell transfection.
• To demonstrate the platform's efficiency in delivering genetic material (DNA and mRNA) to primary human T cells.
• To showcase therapeutic applications, including CRISPR/Cas9 mediated gene editing for T cell receptor (TCR) knockdown.

Main Methods:

• Development of a novel electroporation platform designed for both small-volume research and large-scale manufacturing.
• Optimization of transfection protocols for primary human T cells using plasmid DNA and mRNA.
• Assessment of cell viability and transfection efficiency post-electroporation.
• Demonstration of CRISPR/Cas9 gene editing for TCR knockdown in T cells.
• Evaluation of the system's scalability and throughput.

Main Results:

• High transfection efficiency achieved for mRNA delivery (>95%) with minimal cell viability loss (<2%).
• Demonstrated scalability with an experimental throughput of 256 million cells/min.
• Successful CRISPR/Cas9 mediated knockdown of T cell receptor (TCR) expression in primary T cells.
• Reproducible transfection of primary human T cells with both DNA and mRNA.

Conclusions:

• The novel electroporation platform overcomes limitations of traditional methods for cellular therapy manufacturing.
• The system enables efficient and scalable non-viral engineering of primary T cells.
• This technology addresses unmet needs in the development of advanced cell-based therapies.