Efficient transfection of primary cells in a canine hemophilia B model using adenovirus-polylysine-DNA complexes.
We have used molecular conjugates containing combinations of DNA, adenovirus, polylysine, and transferrin to transfect primary cells derived from canines with hemophilia B (factor IX deficiency), as well as a canine epithelial cell line. Transfection of canine hemophilia B fibroblasts with molecular conjugates resulted in efficient transfection and expression of luciferase DNA-adenovirus-polylysine (AdpL) conjugates or luciferase DNA-adenovirus-polylysine-transferrin (hTfpL/AdpL) conjugates. No expression in canine hemophilia B fibroblasts was evident after exposure to DNA alone, or DNA conjugated with polylysine and transferrin. Transfection efficiencies of 50% or more could be demonstrated in cells transfected with a beta-galactosidase reporter gene as part of an hTfpL/AdpL molecular conjugate. Transfection with canine factor IX AdpL conjugates or canine factor IX hTfpL/AdpL conjugates resulted in factor IX expression for more than 2 weeks in vitro in hemophilia B canine fibroblasts. Maximum levels of expression of over 700 ng of canine factor IX/10(6) cells/24 hr were demonstrated in fibroblasts after transfection with canine factor IX hTfpL/AdpL conjugates. Similar conjugates were used to transfect hemophilia B canine bone marrow stromal cells and Madin-Darby canine kidney cells that also expressed canine factor IX. The use of molecular conjugates to transfect primary cells may be feasible as a means of in vitro or in vivo gene therapy for hemophilia B, and can be tested in the canine hemophilia B model.
University of North Carolina at Chapel Hill 27599-7035.
J N
JN
Lozier
A R
AR
Thompson
P C
PC
Hu
M
M
Read
K M
KM
Brinkhous
K A
KA
High
D T
DT
Curiel
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D003239:Connective Tissue Cells; D004247:DNA / Q000008:administration & dosage / Q000235:genetics*; D004283:Dog Diseases / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics; D005347:Fibroblasts; D005822:Genetic Vectors; D002836:Hemophilia B / Q000235:genetics / Q000473:pathology / Q000628:therapy / Q000662:veterinary*; D008668:Metallothionein / Q000235:genetics; D009928:Organ Specificity; D011107:Polylysine; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012137:Respiratory System / Q000166:cytology; D014162:Transfection; D014168:Transferrin
D011993:Recombinant Fusion Proteins; D014168:Transferrin; D011107:Polylysine; D005164:Factor IX; D004247:DNA; D008668:Metallothionein