A method to enhance non-viral transfection of cells

Unmet Need

The process of introducing exogenous materials into cells, or transfection, is necessary for applications such as cellular engineering research, delivering proteins as therapeutics, cell therapy, and DNA vaccination. The commonly used viral-based transfection methods are becoming less favorable for many applications due to their immunogenicity, high manufacturing cost, limited delivery capacity, and inability to delivery therapeutically important proteins like Cas9. Non-viral delivery approaches, such as electrotransfection, avoid these limitations but are currently inefficient for clinical applications. There is an urgent need to improve the efficiency of non-viral transfection methods.

Technology

Duke inventors have developed a method for enhancing the molecular cargo delivery into cells intended for various biomedical applications. The enhancement of the electrotransfer is mediated by incorporation of non-degradable sugars into cell membranes, causing enlargement of lysosomes and formation of large (>500 nm) amphisome-like bodies (ALBs). The changes in subcellular structures redirect transport of cargo to ALBs rather than to lysosomes, reducing cargo degradation in cells. This technology has been demonstrated to significantly improve the efficiency of non-viral delivery of plasmid DNA, mRNA, Sleeping Beauty (SB) for inserting a gene of interest into mammalian cell genome, and the CRISPR/Cas9 system for genome editing.

Advantages

  • This electrotransfection method is safe, easy to use, and low cost
  • The electrotransfer of the SB system, which has been used to generate CAR-T cells, with this technology led to >3-fold higher eTE
  • Demonstrated an increased editing efficiency of up to 27% for with CRISPR/Cas9 system
Images showing sucrose-induced enlargement of lysosomes. HEK293 cells expressing LAMP1-RFP were pretreated with sucrose; and Cy5-pDNA was electrotransferred into the cells, followed by staining with LTG. Images were acquired 60 min after electrotransfer.

Duke File (IDF) Number

T-005341

Inventor(s)

  • Yuan, Fan
  • Cervia, Lisa
  • Chang, Chun-Chi "Chi"
  • Mao, Mao "Mao"

For more information please contact

College

Pratt School of Engineering