Faculty Collaboration Database

A bicistronic therapeutic retroviral vector enables sorting of transduced CD34+ cells and corrects the enzyme deficiency in cells from Gaucher patients. Blood 1996 Mar 01;87(5):1754-62

Date

03/01/1996

Pubmed ID

8634421

DOI

10.1182/blood.v87.5.1754.1754

Scopus ID

2-s2.0-0030028313 (requires institutional sign-in at Scopus site)   39 Citations

Abstract

Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral-producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models.

Author List

Medin JA, Migita M, Pawliuk R, Jacobson S, Amiri M, Kluepfel-Stahl S, Brady RO, Humphries RK, Karlsson S

Author

Jeffrey A. Medin PhD Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

3T3 Cells
Animals
Antigens, CD
Antigens, CD34
B-Lymphocytes
CD24 Antigen
Cell Line, Transformed
Cell Separation
Cells, Cultured
DNA, Complementary
Fibroblasts
Flow Cytometry
Gaucher Disease
Genetic Therapy
Genetic Vectors
Glucosylceramidase
Hematopoietic Stem Cells
Humans
Membrane Glycoproteins
Mice
Moloney murine leukemia virus
Retroviridae
Selection, Genetic
Transfection