Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34+ cells emphasize limited transduction at low receptor levels

Peter Kurre, J. Morris, A. D. Miller, H. P. Kiem

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Successful gene therapy for the treatment of heritable or acquired diseases typically requires high efficiency gene transfer and sustained transgene expression. Indirect evidence on the basis of RNA analysis and in vivo competitive repopulation experiments in animal models suggests a correlation between transduction efficiency and the abundance of retrovirus receptors on the hematopoietic target cell. However, transduction by oncoretroviral vectors is also subject to other factors such as target cell cycle status and the composition of the virus-containing medium, making it difficult to determine the level of receptor expression required for efficient transduction. In the present study we investigated the impact of receptor expression level on transduction by a vector with a gibbon ape leukemia virus (GALV) envelope protein in a tetracycline-inducible tissue culture model that allowed for the cell cycle-independent, regulated expression of the GALV receptor (Pit1) in otherwise non-susceptible NIH 3T3 cells. Up-regulation of receptor RNA expression by 4.5-fold resulted in a mean 150-fold increase in transduction efficiency. We then analyzed cell surface expression of the Pit1 receptor using a fusion protein consisting of GALV SU portion of the viral envelope protein linked to the human IgG Fc. These experiments showed that tetracycline-regulated receptor induction resulted in a dose-dependent increase in binding of fusion protein. At maximum induction fusion protein binding increased up to five-fold which paralleled the increase in RNA expression, and correlated with the improved transduction efficiency. Finally, studies of pseudotype-specific fusion protein binding to human CD34-enriched cells revealed increased expression of retrovirus receptors after cytokine stimulation, although overall receptor expression in CD34+ cells remained lower than in fibroblast cell lines efficiently transduced by amphotropic and GALV vectors.

Original languageEnglish (US)
Pages (from-to)593-599
Number of pages7
JournalGene Therapy
Volume8
Issue number8
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Gibbon ape leukemia virus
Retroviridae
Protein Binding
Viral Envelope Proteins
RNA
Tetracycline
Cell Cycle
NIH 3T3 Cells
Cytokine Receptors
Transgenes
Genetic Therapy
Carrier Proteins
Up-Regulation
Animal Models
Fibroblasts
Immunoglobulin G
Viruses
Cell Line
Genes
Proteins

Keywords

  • Fusion protein
  • Pit1
  • Pit2
  • Retrovirus receptor
  • Transduction

ASJC Scopus subject areas

  • Genetics

Cite this

Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34+ cells emphasize limited transduction at low receptor levels. / Kurre, Peter; Morris, J.; Miller, A. D.; Kiem, H. P.

In: Gene Therapy, Vol. 8, No. 8, 2001, p. 593-599.

Research output: Contribution to journalArticle

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