Recombinant human adenovirus: Targeting to the human transferrin receptor improves gene transfer to brain microcapillary endothelium

Some inborn errors of metabolism due to deficiencies of soluble lysosomal e nzymes cause global neurodegenerative disease. Representative examples incl ude the infantile and late infantile forms of the ceroid lipofuscinoses (CL N1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (NI PS VII), a deficiency of beta -glucuronidase. Treatment of the central nerv ous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of br ain microcapillary endothelium (BME) with recombinant viral vectors, with s ecretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the B ME. This requires (i) identification of a BME-resident target receptor, (ii ) identification of motifs targeted to that molecule, (iii) the constructio n of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of princi pal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was pa nned for motifs which could bind hTfR. Forty-three clones were sequenced, m ost of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides rep resentative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bin d transferrin receptor, and seven allowed for recombinant adenovirus produc tion. Importantly, the fiber-modified viruses facilitated increased gene tr ansfer (2- to 34-fold) to hTfR expressing cell lines and human brain microc apillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tro pism to the hTfR.