Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Area of Science:

• Gene Therapy
• Molecular Biology
• Cell Biology

Background:

• Non-viral vectors are crucial for gene therapy but struggle with DNA delivery to post-mitotic cells due to inefficient nuclear entry.
• Post-mitotic cells, like dorsal root ganglion neurons (DRGN), present a significant challenge for conventional gene transfer methods.
• Overcoming the nuclear barrier is essential for advancing non-viral gene therapy efficacy.

Purpose of the Study:

• To develop and evaluate a hybrid mRNA/DNA system to improve gene expression in post-mitotic cells.
• To bypass the nuclear entry barrier for enhanced cytoplasmic gene expression.
• To assess the efficiency of this system in primary neuronal cultures.

Main Methods:

• Co-delivery of messenger RNA (mRNA) encoding T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid.
• In vitro transfection of primary dorsal root ganglion neuronal (DRGN) cultures.
• Cell-free assays to evaluate T7 RNAP activity and mRNA requirements.
• Characterization of mRNA structure, intracellular stability, and nucleic acid persistence.

Main Results:

• The hybrid mRNA/DNA system achieved 10- to 2200-fold higher gene expression in DRGN cultures compared to a cytomegalovirus (CMV)-driven plasmid.
• Expression levels were 30-fold greater than with a T7-driven plasmid alone.
• Minimal quantities of T7 RNAP mRNA significantly enhanced gene expression compared to T7-driven plasmids with or without T7 RNAP protein.

Conclusions:

• Co-delivery of mRNA with plasmid DNA is a potent strategy to enhance gene expression, overcoming nuclear barriers.
• This hybrid system represents a significant advancement for non-viral vector-mediated gene transfer in challenging cell types like DRGN.
• The findings pave the way for more effective gene therapy applications in neuroscience and other fields.