Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell...

Area of Science:

• Stem Cell Biology
• Virology
• Gene Therapy

Background:

• Reprogramming differentiated cells into induced pluripotent stem cells (iPSCs) typically requires simultaneous delivery and sustained expression of multiple transcription factors.
• Existing gene delivery systems face challenges in efficiency, control over gene expression, and complete removal of exogenous reprogramming factors, posing risks like malignant transformation.
• Achieving autoregulated pluripotency necessitates the replacement of exogenous factors with endogenous counterparts and the complete removal of delivery vectors.

Purpose of the Study:

• To develop a novel gene delivery and expression system capable of efficient and controlled cell reprogramming.
• To create a system that allows for the stable expression of reprogramming factors and their subsequent complete removal from target cells.
• To establish a tool for advanced cell reprogramming and stem cell research with minimized risks of malignant transformation.

Main Methods:

• Development of a replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic viral variant.
• Utilizing the SeVdp vector for efficient delivery and stable cytoplasmic expression of up to four exogenous reprogramming genes (Oct4/Sox2/Klf4/c-Myc) in various mammalian cells.
• Employing siRNA to interfere with viral transcription/replication, leading to the rapid and thorough elimination of SeVdp genomic RNA from target cells.

Main Results:

• The SeVdp vector efficiently delivered and stably expressed multiple reprogramming factors in diverse cell types, including human hematopoietic stem cells.
• Interference with viral transcription/replication via siRNA effectively removed the SeVdp vector from reprogrammed cells without chromosomal integration.
• SeVdp-mediated delivery of Oct4/Sox2/Klf4/c-Myc reprogrammed mouse primary fibroblasts into Nanog-positive induced pluripotent stem cells with approximately 1% efficiency.

Conclusions:

• The developed SeVdp vector is a versatile and efficient tool for cell reprogramming, overcoming limitations of existing systems.
• This system facilitates the controlled expression and subsequent removal of exogenous reprogramming factors, enhancing safety and enabling autoregulated pluripotency.
• The SeVdp vector holds significant potential for advancing induced pluripotent stem cell generation and applications in stem cell research.