Efficient Transfection of Primary Cells in a Canine Hemophilia B Model Using Adenovirus–Polylysine–DNA Complexes

Abstract

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 β-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/10⁶ 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.

Overview summary

Adenovirus–polylysine–DNA molecular conjugates have been used to transfect and express various reporter genes in primary cells and cell lines from various mammalian species. Lozier et al., have successfully utilized molecular conjugates containing canine factor IX DNA to transfect primary cells from canines with hemophilia B in vitro, and have demonstrated efficient transfection and expression of factor IX. Expression of canine factor IX in skin fibroblasts and bone marrow stromal cells continues for 2 weeks or more after transfection. The results of these experiments in primary cells, along with other data obtained from transfection of a canine epithelial cell line, suggest the feasibility of somatic cell gene therapy using molecular conjugates for in vivo or in vitro transfection of cells from canines with hemophilia B.