Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34+ cells

Jonathan S. Dando, Alessandro Aiuti, Sara Deola, Francesca Ficara, Claudio Bordignon

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Background Clinically applicable protocols for ex vivo modification of human CD34+ hematopoietic stem/progenitor cells rely on incubation of the target cell with supernatant containing recombinant retroviral particles. Although components of the supernatant may have a profound impact on both preclinical and clinical outcome, to date supernatant production has not been properly addressed with regard to CD34+ cells. We wanted to investigate and optimise production conditions for this target using simple, reproducible and clinically applicable procedures and reagents. Methods Retroviral supernatant was obtained from producer cell GP+Am12 under various production conditions and tested for bulk transduction efficiency and endpoint titre on murine and human cell lines. Gene transfer efficiency into CD34+ cells from mobilised peripheral blood, after a single exposure to retroviral supernatant, was measured by transgene expression, colony forming assay and long-term culture colony forming assay. Results Bulk gene transfer or endpoint titre values obtained on cell lines for the different production conditions were not predictive of gene transfer efficiency into hematopoietic progenitors. Time of virus production appeared to have the greatest impact on gene transfer, peaking at 6 h and decreasing 2-3-fold at longer time points. Neither the culture vessel used nor the temperature for virus production had any significant effect on gene transfer into CD34+ cells. Supernatant could be produced under defined serum-free conditions as efficiently as serum containing conditions for CD34+ cell gene transfer. Conclusions The present data provide important implications for the establishment of quality controls for small- and large-scale clinical grade supernatant production for gene transfer into human hematopoietic stem/ progenitor cells.

Original languageEnglish
Pages (from-to)219-227
Number of pages9
JournalJournal of Gene Medicine
Volume3
Issue number3
DOIs
Publication statusPublished - 1 May 2001
Externally publishedYes

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Medical Genetics
Hematopoietic Stem Cells
Genes
Viruses
Cell Line
Serum
Transgenes
Quality Control
Temperature

Keywords

  • CD34 cells
  • Quality control
  • Retroviral supernatant

ASJC Scopus subject areas

  • Genetics

Cite this

Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34+ cells. / Dando, Jonathan S.; Aiuti, Alessandro; Deola, Sara; Ficara, Francesca; Bordignon, Claudio.

In: Journal of Gene Medicine, Vol. 3, No. 3, 01.05.2001, p. 219-227.

Research output: Contribution to journalArticle

Dando, Jonathan S. ; Aiuti, Alessandro ; Deola, Sara ; Ficara, Francesca ; Bordignon, Claudio. / Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34+ cells. In: Journal of Gene Medicine. 2001 ; Vol. 3, No. 3. pp. 219-227.
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