*6.6. Gene Therapy*

In 2010, an esoteric approach was explored based on the observation that plasma levels of fetuin-A in patients with PXE as well as in *Abcc6*−/<sup>−</sup> mice are reduced by up to 30%. Fetuin-A is a circulating "hepatokine" predominantly synthesized in the liver, which possesses diverse physiological functions, including bone metabolism regulation, vascular calcification, and insulin resistance. Because fetuin-A is able to form complexes with calcium and phosphate ions, it acts as an inhibitor of ectopic calcification. Mice deficient in this glycoprotein show systemic calcification of soft tissues [134]. In this study [135], a fetuin-A cDNA was transiently expressed in the liver of *Abcc6*−/<sup>−</sup> mice and resulted in an approximately 70% reduction of whisker calcification. However, the positive effects on calcification were not persistent.

More recently, the transient expression of the human *ABCC6* in mouse liver was shown to have a positive effect on DCC [13], while permanent expression via transgenes produced remarkable effects on ectopic calcification in several tissues (whiskers, kidneys, heart), despite having only modest effects on plasma PPi [18].

A more elaborate methodology was explored with the intravenous administration to *Abcc6*−/<sup>−</sup> mice of a recombinant adenovirus carrying a cDNA encoding the normal human ABCC6 [136]. The mice showed high-level expression of the human ABCC6 in liver for several weeks post-delivery. This resulted in the normalization of plasma pyrophosphate levels. For sustained expression up to three repeated adenovirus injections 4 weeks apart were also tested with success. At the time of sacrifice the mice were 4–5 months old and showed very limited signs of mineralization in vibrissae. By contrast, treatments applied to older mice (11 months old) had no effect on existing mineralization. The human species C adenovirus serotype 5 (Ad5) used in this study is one the most frequently used gene delivery systems in animal and clinical studies [189] and presents a high predilection for liver transduction, which is the primary site of expression of ABCC6 [79,81]. This proof-of-concept study suggested that adenovirus-mediated *ABCC6* gene delivery may be possible to treat PXE and ABCC6-related GACI patients, when gene therapy, especially one targeting the liver, has reached sufficient maturity for clinical use.
