Nontargeted stable integration of recombinant adeno-associated virus into human leukemia and lymphoma cell lines as evaluated by fluorescence in situ hybridization.
A number of studies on human epithelial cells of varying origin have demonstrated integration of recombinant adeno-associated virus (AAV) vectors into a variety of chromosomes compared with the site-specific integration on chromosome 19 predominantly observed for wild-type (wt) AAV. We have constructed a recombinant AAV (rAAV) vector and tested the integration into hematopoietic cells, using the human acute myeloid leukemia cell line AML5 and the human non-Hodgkin's lymphoma cell line OCI-LY18 as targets. The integration sites were visualized by fluorescence in situ hybridization (FISH). Positive signals were observed for chromosomes 1, 2, 3, 8, 14, 15, 19, and Y. The majority of cells demonstrated integration into one specific site. A minority showed simultaneous integration into more than one chromosome. The frequency of observed integrations was not uniformly distributed among chromosomes; for instance, in AML5 chromosome 2 seemed to be favored. Colony-derived AML5 clones bore unique integration patterns indicating successful transduction of clonogenic progenitor cells with high proliferative potential. The integration was stable and observed for more than 12 months after transduction. FISH has been shown to be a powerful tool for detailed analyses of rAAV integration patterns and can be used to evaluate targets and transduction conditions.
Omori
F
F
Department of Medicine and Medical Biophysics, University of Toronto and Ontario Cancer Institute, Canada.
Messner
H A
HA
Ye
C
C
Gronda
M V
MV
O'Neill
J P
JP
Atkins
H
H
Heng
H H
HH
D000208:Acute Disease; D001483:Base Sequence; D015139:Blotting, Southern; D002999:Clone Cells; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000473:pathology / Q000821:virology; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics* / Q000473:pathology / Q000821:virology; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D016662:Virus Integration
D017931:DNA Primers