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Efficient episomal expression vector for human transitional carcinoma cells

M J Cooper1, S Miron

  • 1Department of Medicine, University Hospitals of Cleveland, Case Western Reserve University School of Medicine, OH 44106.

Human Gene Therapy
|October 1, 1993
PubMed
Summary
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BK virus (BKV) episomes efficiently replicate in bladder cancer cells, enabling high gene expression and stable inheritance. This offers a promising tool for gene therapy in transitional carcinoma treatment.

Area of Science:

  • Molecular Biology
  • Virology
  • Cancer Research

Background:

  • Developing efficient expression vectors for human transitional carcinoma cells is crucial for gene therapy.
  • Episomal vectors offer potential advantages over integrated vectors for stable gene expression.

Purpose of the Study:

  • To evaluate the replication activity of BK virus (BKV) and Epstein-Barr virus (EBV) derived episomal vectors in HT-1376 bladder carcinoma cells.
  • To assess the stability and gene expression levels of BKV-derived episomes in HT-1376 cells.

Main Methods:

  • Southern blot analysis to detect episomal replication and integration.
  • Stable transfection of HT-1376 cells with BKV-derived episomes containing a neomycin resistance gene.
  • Quantitative analysis of gene expression and episomal copy number.

Related Experiment Videos

  • Soft agar cloning and withdrawal of selection pressure to assess stability.

    • Main Results:

    • BKV-derived episomes replicated extrachromosomally in HT-1376 cells, while EBV replicons were non-functional.
    • Stable transfectants maintained approximately 150 copies of BKV episomes per cell without genomic integration.
    • BKV episomal transfectants showed 20-fold higher neomycin resistance gene mRNA expression compared to integrated controls.
    • BKV episomes were stably inherited across cell generations and maintained copy number after selection withdrawal.

    Conclusions:

    • BKV-derived episomes are efficient replicons in bladder carcinoma cells, facilitating high-level, stable gene expression.
    • These episomes can be maintained without integration, offering a valuable tool for gene therapy applications in transitional carcinoma.