Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34+ cells.
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.
Dando
J S
JS
San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy.
Aiuti
A
A
Deola
S
S
Ficara
F
F
Bordignon
C
C
D016475:3T3 Cells; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000502:physiology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection / Q000379:methods*
D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins