In Vitro Synthesis, Delivery, and Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac...

Area of Science:

• Molecular Biology
• Gene Therapy

Background:

• Nonviral gene transfer is crucial for therapeutic and bioproduction applications.
• The piggyBac transposon system offers large cargo capacity but faces safety concerns due to persistent transposase expression.
• Genotoxic effects like multiple integrations can arise from traditional DNA-based transposase delivery.

Purpose of the Study:

• To develop a safer, transient gene transfer method using piggyBac transposase mRNA.
• To overcome limitations of DNA-based transposase delivery, reducing genotoxic risks.
• To optimize mRNA production and delivery for efficient transposition in mammalian cells.

Main Methods:

• In vitro synthesis and quality verification of piggyBac transposase mRNA.
• Evaluation of various transfection reagents for efficient mRNA delivery.
• Assessment of mRNA bioavailability and transposition efficiency in HeLa and mesenchymal stromal cells.

Main Results:

• Successful production and quality control of synthetic mRNA.
• Identification of effective transfection reagents for mRNA delivery.
• Demonstrated efficient piggyBac transposition in HeLa cells and mesenchymal stromal cells, supporting hematopoiesis.

Conclusions:

• Delivering piggyBac transposase as mRNA provides a transient expression window, enhancing safety.
• The described method facilitates efficient and safe gene transfer for potential therapeutic applications.
• This approach supports gene insertion in relevant cell types for bioproduction and regenerative medicine.