Artificial cells as a novel approach to gene therapy - Artificial Cells, Cell Engineering and Therapy - page 272

physiological levels of transferring (Seligman, 1983). The mouse monoclonal
antibody OX26 (against rat TfR) binds to a different site on the TfR from
transferrin so it is not subject to competitive binding with transferrin (Jefferies et
al., 1984). An OX26 single chain Fv antibody-streptavidin fusion protein has
been used to selectively target blood±brain barrier endothelial cells (Li et al.,
1999), thus demonstrating the feasibility of targeted delivery of biotinylated
drugs. While the OX26 monoclonal antibody does not recognize the mouse TfR,
analogous anti-mouse TfR monoclonal antibodies have been developed and
tested in vivo. The 8D3 monoclonal antibody has been shown to selectively
target the mouse brain via the TfR and could be used as a carrier protein in
mouse studies (Lee et al., 2000).
Melanotransferrin is a glycoprotein first discovered in malignant melanoma
cells (originally called P97) (Brown et al., 1981). The gene for human P97 is on
chromosome 3q21 and codes for a 738 amino acid protein (Rose et al., 1986) that
is processed to a 694 amino acid mature protein. The mature form is membrane
bound by a glycosyl phosphatidylinositol (GPI) anchor (Alemany et al., 1993),
but a less well-characterized soluble form of P97 has also been described (Food et
al., 1994) that lacks this GPI anchor. P97 has been shown to reversibly bind iron
(Baker et al., 1992), but the exact function of the protein is unknown. One
property of the protein that has been explored is its transcytosis into the brain,
both in situ and in an in vitro blood±brain barrier model. The model system used
bovine brain capillary endothelial cells grown in co-culture with newborn rat
astrocytes in monolayers to examine the transcytosis across the monolayer. The
putative receptor for melanotransferrin is the lipoprotein receptor-related protein
(LRP) (Demeule et al., 2003), which is known to rapidly internalize the
urokinase : plasminogen activator inhibitor : urokinase receptor complex (Conese
et al., 1995). Internalization of melanotransferrin by LRP has been shown to be
14-fold higher than internalization of transferrin by TfR with no intra-endothelial
degradation (Demeule et al., 2002), although RAP seems to have even higher
transcytosis (Pan et al., 2004). We have constructed a fusion protein of a marker
enzyme, firefly luciferase and P97. C2C12 cells transfected with this construct
were encapsulated and implanted into the peritoneal cavity of Balb/c mice.
Tissue homogenates showed elevated levels of luciferase activity, including the
brain (Li, Hou, Shen & Potter, unpublished observation). While more study is
needed before any of these approaches can be used clinically, the preliminary
reports discussed here are encouraging that the blood±brain barrier may be
broached without resorting to direct implantation of artificial cells in the brain.
10.7 Conclusions
Immuno-isolation of genetically modified cells is a platform technology,
potentially offering a high degree of flexibility and efficacy towards treatments
of many disorders ± from classical Mendelian single-gene diseases to multi-