Table of Contents
ISRN Hematology
Volume 2012 (2012), Article ID 212586, 12 pages
http://dx.doi.org/10.5402/2012/212586
Research Article

Cotransduction with MGMT and Ubiquitous or Erythroid-Specific GFP Lentiviruses Allows Enrichment of Dual-Positive Hematopoietic Progenitor Cells In Vivo

1Division of Infectious Diseases, Department of Pediatrics, The University of Alabama at Birmingham, Birmingham, AL 35294, USA
2Division of Hematology Oncology, Case Comprehensive Cancer Center and The Center for Stem Cell and Regenerative Medicine, Cleveland, OH 44106, USA
3H. San Raffaele-Telethon Institute for Gene Therapy (HSR-TIGET), 20132 Milan, Italy

Received 24 April 2012; Accepted 29 May 2012

Academic Editors: S. J. Brandt and H. Knecht

Copyright © 2012 Justin C. Roth et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The P140K point mutant of MGMT allows robust hematopoietic stem cell (HSC) enrichment in vivo. Thus, dual-gene vectors that couple MGMT and therapeutic gene expression have allowed enrichment of gene-corrected HSCs in animal models. However, expression levels from dual-gene vectors are often reduced for one or both genes. Further, it may be desirable to express selection and therapeutic genes at distinct stages of cell differentiation. In this regard, we evaluated whether hematopoietic cells could be efficiently cotransduced using low MOIs of two separate single-gene lentiviruses, including MGMT for dual-positive cell enrichment. Cotransduction efficiencies were evaluated using a range of MGMT : GFP virus ratios, MOIs, and selection stringencies in vitro. Cotransduction was optimal when equal proportions of each virus were used, but low MGMT : GFP virus ratios resulted in the highest proportion of dual-positive cells after selection. This strategy was then evaluated in murine models for in vivo selection of HSCs cotransduced with a ubiquitous MGMT expression vector and an erythroid-specific GFP vector. Although the MGMT and GFP expression percentages were variable among engrafted recipients, drug selection enriched MGMT-positive leukocyte and GFP-positive erythroid cell populations. These data demonstrate cotransduction as a mean to rapidly enrich and evaluate therapeutic lentivectors in vivo.