Nanoneedle Array-Electroporation Facilitates Intranuclear Ribonucleoprotein Delivery and High Throughput Gene Editing

Area of Science:

• Cell Biology
• Immunology
• Biotechnology
• Gene Editing

Background:

• Dendritic cells (DCs) are key regulators of T cell immunity with significant therapeutic potential for cancer and autoimmune diseases.
• Efficient gene editing in DCs is essential for understanding their function and enhancing therapeutic efficacy.
• Conventional transfection methods face significant challenges in delivering genetic material into DCs due to their inherent resistance.

Purpose of the Study:

• To develop an efficient and scalable method for intranuclear gene delivery and editing in dendritic cells.
• To overcome the limitations of existing gene editing techniques in hard-to-transfect cells like DCs.
• To demonstrate the efficacy of a novel delivery system for precise genetic modification of DCs.

Main Methods:

• Development of a hollow nanoneedle array (HNA) system for intranuclear delivery.
• Integration of nano-electroporation with the HNA for simultaneous membrane perforation.
• Direct delivery of Cas9/sgRNA ribonucleoprotein (RNP) complexes into the nucleus of primary DCs.

Main Results:

• The HNA system successfully reached the cell nucleus, positioning membranes for efficient delivery.
• Simultaneous perforations of nuclear and plasma membranes enabled direct RNP entry.
• Achieved efficient knockout of the PD-L1 gene in primary DCs, validating the system's effectiveness.

Conclusions:

• The developed HNA-based system provides a powerful new tool for direct intranuclear gene editing in dendritic cells.
• This method offers a scalable solution for overcoming transfection barriers in DCs and other challenging cell types.
• The technology holds promise for advancing DC-based immunotherapies and fundamental research.