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(American Journal of Pathology. 2003;162:873-885.)
© 2003 American Society for Investigative Pathology

Transfer of the 5(IV) Collagen Chain Gene to Smooth Muscle Restores in Vivo Expression of the 6(IV) Collagen Chain in a Canine Model of Alport Syndrome

Scott J. Harvey^*, Keqin Zheng^*, Barbara Jefferson, Peter Moak, Yoshikazu Sado, Ichiro Naito^¶, Yoshifumi Ninomiya^||, Robert Jacobs and Paul S. Thorner^*

From the Division of Pathology,^* Hospital for Sick Children, and the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; the Department of Pathobiology, University of Guelph, Guelph, Canada; the Divisions of Immunology and Ultrastructural Biology,^¶ Shigei Medical Research Institute, Okayama, Japan; and the Department of Molecular Biology and Biochemistry,|| Okayama University Medical School, Okayama, Japan

X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the 5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant 5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine 5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the 1(IV) and 2(IV), but not the 3(IV) to 6(IV) collagen chains. By Northern blotting, an 5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the 1(IV) and 2(IV) chains but the recombinant 5(IV) chain was not incorporated. These findings are consistent with the concept that the 5(IV) chain requires one or more of the 3(IV), 4(IV), or 6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the 5(IV) transgene was injected into bladder smooth muscle that lacks both the 5(IV) and 6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the 5(IV) and 6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant 5(IV) chain is capable of restoring expression of a second (IV) chain that requires the presence of the 5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome.

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