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<p>One challenge in utilizing the Measles virus (MV) for cancer therapy is the number of virus particles required, nearly a million times greater than the amount reported for vaccination. This study aims to design and develop a cell line with increased production capacity to supply the required amounts of MV in oncolytic virotherapy. The sphingosine kinase 1 (SphK1) gene was inserted into a pIRES2-EGFP plasmid and transiently transfected into four cell lines: MRC-5, HEK293, Vero, and A549. Fluorescent light intensity was measured using flow cytometry, and the MV production titer was determined using the TCID50 method. Transient transfection of pIRES2-EGFP-SphK1 was associated with increases in MV yield of approximately 3 logs in HEK293, 2 logs in Vero and A549, and 1 log in MRC-5 cells compared to controls and has impacted the morphology of MRC-5 cells. The top 100 genes co-expressed with SphK1 were identified with the ARCHS4 RNA-seq data resource, and functional enrichment with EnrichR suggested involvement in interleukin and cytokine signaling, extracellular matrix organization, and stress responses. The observed results indicate that augmenting the expression of the SphK1 gene may enhance MV production and influence cellular behavior, although effects appear to be cell line-dependent. A better understanding of cell-specific S1P signaling and cytoskeletal regulation could assist in optimizing cell lines for scalable virotherapy production.</p> Graphical Abstract <p>This study presents a novel strategy to boost therapeutic-grade measles virus (MV) production for oncolytic virotherapy by transiently overexpressing the Sphingosine Kinase 1 (SphK1) gene in mammalian cells using a recombinant pIRES2-EGFP-SphK1 construct. SphK1 overexpression significantly increased MV titers (1–3 logs), especially in HEK293 and Vero cells. These results were supported by flow cytometric analyses and titration assays (TCID50). Furthermore, bioinformatic analyses revealed that enhanced replication may involve cytokine signaling, extracellular matrix (ECM) organization, and cytoskeletal regulation.</p> <p></p>

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Cell line engineering for enhanced measles virus production with sphingosine kinase 1 gene overexpression

  • 期刊论文

  • 发表日期：

    2025年12月6日

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  • Malihe Rastegarpanah,
  • Babak Negahdari,
  • Yazdan Asgari,
  • Mohammadali Mazloomi,
  • Kayhan Azadmanesh

摘要

One challenge in utilizing the Measles virus (MV) for cancer therapy is the number of virus particles required, nearly a million times greater than the amount reported for vaccination. This study aims to design and develop a cell line with increased production capacity to supply the required amounts of MV in oncolytic virotherapy. The sphingosine kinase 1 (SphK1) gene was inserted into a pIRES2-EGFP plasmid and transiently transfected into four cell lines: MRC-5, HEK293, Vero, and A549. Fluorescent light intensity was measured using flow cytometry, and the MV production titer was determined using the TCID50 method. Transient transfection of pIRES2-EGFP-SphK1 was associated with increases in MV yield of approximately 3 logs in HEK293, 2 logs in Vero and A549, and 1 log in MRC-5 cells compared to controls and has impacted the morphology of MRC-5 cells. The top 100 genes co-expressed with SphK1 were identified with the ARCHS4 RNA-seq data resource, and functional enrichment with EnrichR suggested involvement in interleukin and cytokine signaling, extracellular matrix organization, and stress responses. The observed results indicate that augmenting the expression of the SphK1 gene may enhance MV production and influence cellular behavior, although effects appear to be cell line-dependent. A better understanding of cell-specific S1P signaling and cytoskeletal regulation could assist in optimizing cell lines for scalable virotherapy production.

Graphical Abstract

This study presents a novel strategy to boost therapeutic-grade measles virus (MV) production for oncolytic virotherapy by transiently overexpressing the Sphingosine Kinase 1 (SphK1) gene in mammalian cells using a recombinant pIRES2-EGFP-SphK1 construct. SphK1 overexpression significantly increased MV titers (1–3 logs), especially in HEK293 and Vero cells. These results were supported by flow cytometric analyses and titration assays (TCID50). Furthermore, bioinformatic analyses revealed that enhanced replication may involve cytokine signaling, extracellular matrix (ECM) organization, and cytoskeletal regulation.