Autor: |
Harvey, Scott J., Zheng, Keqin, Jefferson, Barbara, Moak, Peter, Sado, Yoshikazu, Naito, Ichiro, Ninomiya, Yoshifumi, Jacobs, Robert, Thorner, Paul S. |
Zdroj: |
American Journal of Pathology; March 2003, Vol. 162 Issue: 3 p873-885, 13p |
Abstrakt: |
X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5gene, which encodes the α5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant α5(IV) collagen in vitroand in vivo. A full-length cDNA-encoding canine α5(IV) collagen was cloned and expressed in vitroby transfection of HEK293 cells that synthesize the α1(IV) and α2(IV), but not the α3(IV) to α6(IV) collagen chains. By Northern blotting, an α5(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 α1(IV) and α2(IV) chains but the recombinant α5(IV) chain was not incorporated. These findings are consistent with the concept that the α5(IV) chain requires one or more of the α3(IV), α4(IV), or α6(IV) chains for triple helical assembly. In vivostudies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the α5(IV) transgene was injected into bladder smooth muscle that lacks both the α5(IV) and α6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the α5(IV) and α6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant α5(IV) chain is capable of restoring expression of a second α(IV) chain that requires the presence of the α5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome. |
Databáze: |
Supplemental Index |
Externí odkaz: |
|