A capsid-modified helper-dependent adenovirus vector containing the beta-globin locus control region displays a nonrandom integration pattern and allows stable, erythroid-specific gene expression.
Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the beta-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated virus (AAV) inverted terminal repeats (Ad.LCR-beta-GFP). This vector possesses the adenovirus type 35 fiber knob that allows efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth factor-dependent CD34(+) erythroleukemic cell line, and in cord blood-derived human CD34(+) cells. Stable transduction of MO7e cells with Ad.LCR-beta-GFP was more efficient and less subject to position effects and silencing than transduction with a vector that did not contain the beta-globin LCR. Analysis of integration sites indicated that Ad.LCR-beta-GFP integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed integration sites were within the chromosomal beta-globin LCR. None of the Ad.LCR-beta-GFP integrations occurred in exons. The integration pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the beta-globin LCR-containing vector. Infection of primary CD34(+) cells with Ad.LCR-beta-GFP did not affect the clonogenic capacity of CD34(+) cells. Transduction of CD34(+) cells with Ad.LCR-beta-GFP resulted in vector integration and erythroid lineage-specific GFP expression.
Wang
Hongjie
H
Division of Medical Genetics, University of Washington, Seattle, 98195, USA.
Shayakhmetov
Dmitry M
DM
Leege
Tobias
T
Harkey
Michael
M
Li
Qiliang
Q
Papayannopoulou
Thalia
T
Stamatoyannopolous
George
G
Lieber
André
A
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D018952:Antigens, CD34; D002213:Capsid / Q000378:metabolism; D002478:Cells, Cultured; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology; D005914:Globins / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D019901:Locus Control Region / Q000235:genetics*; D014161:Transduction, Genetic; D016662:Virus Integration
D018952:Antigens, CD34; D049452:Green Fluorescent Proteins; D005914:Globins