Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins

Area of Science:

• Stem Cell Biology
• Immunology
• Molecular Biology

Background:

• Generating autologous pluripotent stem cells requires efficient DNA-free reprogramming methods.
• Transfecting cells with RNA offers a direct way to express reprogramming proteins without genetic modification.
• However, long RNA transfection triggers a strong innate immune response, inhibiting cell growth and causing cell death.

Purpose of the Study:

• To develop DNA-free reprogramming techniques using RNA transfection.
• To overcome the innate immune response that limits repeated RNA transfection.
• To enable sustained expression of reprogramming proteins for therapeutic applications.

Main Methods:

• Screened siRNA cocktails to identify those that desensitize cells to exogenous RNA.
• Targeted specific viral pathways involved in innate immune signaling.
• Knocked down interferon-beta (Ifnb1), Eif2ak2, and Stat2 to suppress the immune response.

Main Results:

• Combined knockdown of Ifnb1, Eif2ak2, and Stat2 rescued cells from the immune response to frequent long RNA transfection.
• Primary human fibroblasts were successfully transfected every 24 hours with RNA encoding reprogramming proteins (Oct4, Sox2, Klf4, Utf1).
• Encoded proteins were active, and expression was maintained for many days through multiple cell divisions.

Conclusions:

• Suppressing innate immunity enables frequent, successful transfection with protein-encoding RNA.
• This technique provides precise control over protein expression levels and timing, valuable for research.
• The method advances the development of RNA-based reprogramming and directed-differentiation strategies.