Increased Resistance and Selection in Paclitaxel for Peripheral Blood Progenitor Cells Following Transduction with the MDR1 Gene

Paclitaxel-Resistenz und Selektion nach Transfer des MDR1-Gens in periphere Blutvorläuferzellen.

Einleitung: Die Technik des retroviralen Gentransfers erlaubt die Integration und stabile Expression therapeutischer Gene in hämatopoetischen Zellen. Zu den klinischen Anwendungsmöglichkeiten gehören die Markierung von Stammzellentransplantaten aus Knochenmark oder peripherem Blut, die Korrektur monogenetischer Erkrankungen und die Therapie viraler Infektionskrankheiten. Eine für Krebspatienten spezifische therapeutische Option besteht im Transfer von Genen, die transplantierbare hämatopoetische Zellen im Rahmen von Hochdosis-Chemotherapieprotokollen gegenüber Zytostatika resistent machen. Methode: Der Effekt des Transfers der MDR1 cDNA wurde mit Hilfe retroviraler Vektoren in prästimulierte CD34-angereicherte primäre hämatopoetische Zellen des peripheren Blutes untersucht. Ergebnisse: Mit Hilfe der PCR, die spezifisch für provirale Sequenzen im Genom der Zielzelle war, wurde eine Transfereffizienz von 70 % in klonogene Vorläuferzellen nachgewiesen. Die Effizienz für unreifere hämatopoetische Subpopulationen war jedoch deutlich niedriger. Beim Vergleich MDR1-transduzierter Zellen mit negativen Kontrollzellen waren zusätzlich 14,8 ± 2,6 % klonogene Vorläuferzellen resistent gegenüber Paclitaxel. MDR1-transduzierte Zellen und Kontrollzellen wurden in vitro in Paclitaxel selektioniert und dann weiter expandiert. Die Anzahl der kernhaltigen Zellen und der klonogenen Vorläuferzellen wurden miteinander verglichen und zeigten einen zweifachen Selektionsvorteil für klonogene Vorläuferzellen, die von der MDR1-transduzierten Zellpopulation abstammten. Die Proliferationskapazität war für MDR1-transduzierte Zellen und Kontrollzellen gleich. Schlußfolgerung: Eine vermehrte Resistenz gegenüber Paclitaxel sowie einen Selektionsvorteil für in vitro expandierte hämatopoetische Vorläuferzellen nach MDR1-Gentransfer wurde beobachtet. Diese Resultate können bei der Entwicklung zukünftiger Gentherapie-Protokolle Verwendung finden.

Introduction: The technique of retrovirally mediated gene transfer allows the introduction and stable expression of therapeutic genes in hematopoietic cells. Clinical applications include the marking of bone marrow and peripheral blood stem cell transplants, the correction of monogenetic disorders, and the inhibition of viral infections. A specific therapeutic option for the treatment of cancer patients consists in the transfer of drug resistance genes rendering transplantable hematopoietic cells resistant to the cytotoxic drugs used in high-dose chemotherapy protocols. Methods: We have investigated the effects of MDR1 gene transfer via a retroviral vector into prestimulated CD34-enriched primary hematopoietic cells from human peripheral blood. Results: Using a PCR to detect proviral sequences within the DNA of the target cells we observed a transfer efficiency of 70 % into clonogenic progenitor cells with a markedly decreasing efficiency into more immature subpopulations. Clonogenic progenitors could be rendered resistant to paclitaxel at a frequency of 14.8 ± 2.6 % when comparing MDR1 transduced cells to a mock control. Retrovirally transduced cells and mock control cells were selected in vitro for seven days in paclitaxel and then further expanded. The numbers of nucleated cells and progenitor cells were compared and revealed a twofold selective advantage for progenitors derived from MDR1-transduced cells. The proliferative capacity did not differ between MDR1-transduced and mock cells. Conclusion: Increased paclitaxel resistance and a selective advantage for in vitro-expanded hematopoietic progenitors were detectal. This observation may be helpful for the design of future gene therapy trials.

Schlüsselwörter:

Multidrug Resistance - Gentherapie - Retrovirus - Hämatopoetische Stammzelle

Key words:

Multidrug resistance - Gene therapy - Retroviral vector - Hematopoietic stem cells

References

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