An inherent difficulty in the field of gene therapy is the delivery of the gene of interest to a specific target cell population. The ideal system should be both efficient and accurate.1 The use of retroviral vectors has proved both inefficient and non-specific in its quest to deliver genes to targets such as hematopoietic progenitor cell lines.2 A more efficient gene transfer system has been described in which a vector conjugated to a ligand can target cells expressing specific receptors to that ligand on their surface.3 This method of gene transfer utilizes the mechanism of receptor-mediated endocytosis to allow for the specific introduction of the therapeutic gene into a target cell population.
|Figure 1. Targeted gene transfer with an SCF-targeted vector. The targeted vector is constructed by condensing DNA with polylysine covalently linked to adenovirus and covalently linked to streptavidin. The biotinylated SCF Fluorokine binds to streptavidin on the vector. Expression of c-kit on hematopoietic progenitor cells facilitates binding of the SCF-targeted vector. Figure adapted from Schwarzenberger et al. (1996) Blood 87:472.
Schwarzenberger et al.4 utilized the SCF (stem cell factor/steel factor) R&D Systems' Fluorokine to aid in this receptor-mediated transfer process [R&D Systems' Fluorokine kits are labeled cytokines used to identify cell surface receptors (i.e. typically, in flow cytometry applications)]. Hematopoietic stem cells are candidates of choice for the treatment of genetic diseases of the hematopoietic system.5 These progenitor cells express the receptor to SCF known as c-kit on their surface.
The targeted vector was constructed by incubating polylysine-conjugated adenovirus and polylysine-conjugated streptavidin with plasmid DNA containing the gene of interest. Polylysine binds DNA electrostatically, the replication-incompetent adenovirus prevents the degradation of the vector DNA by lysosomes and streptavidin binds the biotinylated protein. Incubation with biotinylated-SCF results in a molecular conjugate vector capable of specifically targeting hematopoietic progenitor cells expressing c-kit (see Figure 1). The presence of the streptavidin molecule lends versatility to the system, as other biotinylated ligands can be substituted for the biotinylated SCF. The inclusion of a luciferase reporter gene into the plasmid DNA provides a means for monitoring successful transfection.
The authors found that transfected MO-7e cell lines expressing c-kit produce increased luciferase gene expession over control values. Specificity was shown by blocking the activity with the incubation of the cells with excess SCF or with antibodies directed toward c-kit. This system has been successfully used with biotinylated ligands to the asialo-orosomucoid receptor3 and transferrin receptors of hepatocytes,6 and immunoglobulin receptor of respiratory epithelial cells.7
- Miller, N. and R. Vile. (1995) FASEB J. 9:190.
- Schuening, F.G. et al. (1991) Blood 78:2568.
- Wu, G.Y. and C.H. Wu. (1987) J. Biol. Chem. 262:4429.
- Schwarzenberger, P. et al. (1996) Blood 87:472.
- Karlsson, S. (1991) Blood 78:2481.
- Schwarzenberger, P. et al. (1996) Blood 87:2723.
- Ferkol, T. et al. (1995) J. Clin. Invest. 95:493.