Preclinical assessment of human hematopoietic progenitor cell transduction in long-term marrow cultures

Ian D. Dubé, Steven Kruth, Anthony Abrams-Ogg, Suzanne Kamel-Reid, Carolyn Lutzko, Shaherose Nanji, Christine Ruedy, Roshni Singaraja, Anthony Wild, Phyllis Krygsman, Peter Chu, Hans Messner, Vijay Reddy, Gerard McGarrity, A. Keith Stewart

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neo(r)-containing vectors, the average G418 resistance was 28% of 1393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4-5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer.

Original languageEnglish (US)
Pages (from-to)2089-2100
Number of pages12
JournalHuman gene therapy
Volume7
Issue number17
DOIs
StatePublished - Nov 10 1996

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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    Dubé, I. D., Kruth, S., Abrams-Ogg, A., Kamel-Reid, S., Lutzko, C., Nanji, S., Ruedy, C., Singaraja, R., Wild, A., Krygsman, P., Chu, P., Messner, H., Reddy, V., McGarrity, G., & Stewart, A. K. (1996). Preclinical assessment of human hematopoietic progenitor cell transduction in long-term marrow cultures. Human gene therapy, 7(17), 2089-2100. https://doi.org/10.1089/hum.1996.7.17-2089