Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.
Previous studies have shown that the choice of envelope protein (pseudotype) can have a significant effect on the efficiency of retroviral gene transfer into hematopoietic stem cells. This study used a competitive repopulation assay in the dog model to evaluate oncoretroviral vectors carrying the envelope protein of the endogenous feline virus, RD114. CD34-enriched marrow cells were divided into equal aliquots and transduced with vectors produced by the RD114-pseudotype packaging cells FLYRD (LgGLSN and LNX) or by the gibbon ape leukemia virus (GALV)-pseudotype packaging cells PG13 (LNY). A total of 5 dogs were studied. One dog died because of infection before sustained engraftment could be achieved, and monitoring was discontinued after 9 months in another animal that had very low overall gene-marking levels. The 3 remaining animals are alive with follow-ups at 11, 22, and 23 months. Analyses of gene marking frequencies in peripheral blood and marrow by polymerase chain reaction revealed no significant differences between the RD114 and GALV-pseudotype vectors. The LgGLSN vector also contained the enhanced green fluorescent protein (GFP), enabling us to monitor proviral expression by flow cytometry. Up to 10% of peripheral blood cells expressed GFP shortly after transplantation and approximately 6% after the longest follow-up of 23 months. Flow cytometric analysis of hematopoietic subpopulations showed that most of the GFP-expressing cells were granulocytes, although GFP-positive lymphocytes and monocytes were also detected. In summary, these results show that RD114-pseudotype oncoretroviral vectors are able to transduce hematopoietic long-term repopulating cells and, thus, may be useful for human stem cell gene therapy.
Goerner
M
M
Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Horn
P A
PA
Peterson
L
L
Kurre
P
P
Storb
R
R
Rasko
J E
JE
Kiem
H P
HP
D026962:Amino Acid Transport System ASC / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D001773:Blood Cells / Q000378:metabolism; D001854:Bone Marrow Cells / Q000276:immunology* / Q000378:metabolism; D016026:Bone Marrow Transplantation / Q000379:methods*; D002415:Cats; D019070:Cell Lineage / Q000235:genetics*; D004285:Dogs; D020077:Endogenous Retroviruses / Q000235:genetics; D005500:Follow-Up Studies; D015870:Gene Expression; D005822:Genetic Vectors / Q000592:standards*; D006085:Graft Survival / Q000235:genetics; D049452:Green Fluorescent Proteins; D006410:Hematopoiesis; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism; D012191:Retroviridae Proteins / Q000235:genetics / Q000378:metabolism; D015996:Survival Rate; D014161:Transduction, Genetic / Q000379:methods* / Q000592:standards
D026962:Amino Acid Transport System ASC; D018952:Antigens, CD34; D008164:Luminescent Proteins; D011991:Receptors, Virus; D012191:Retroviridae Proteins; C067981:leukemia virus receptor, gibbon ape; D049452:Green Fluorescent Proteins