Ad vectors can efficiently transfect a large number of organs or tissues in vivo, including liver (Jaffe et ah, 1992), kidney (Moullier et al, 1994), skin (Setoguchi et al., 1994b), brain (Akli et al., 1993; Doran et al, 1997), skeletal muscle (Ragot et al., 1993), heart (Strat-ford-Perricaudet et al, 1992), lung epithelium (Rosenfeld et al,
1991), and several ocular tissues (Mashhour et al., 1994) (for further review, see Ali et al., 1994, or Kozarsky and Wilson, 1993). This property allows protein function to be investigated in vivo without generating transgenic animals.
Adenoviral gene delivery is particularly useful if a large number of variant proteins are to be evaluated. For example, to identify the domains of the closely related lipoprotein lipase and hepatic lipase proteins responsible for determining their substrate specificity in vivo, Kobayashi et al. (1996) made several chimeric proteins by exchanging domains between the two and cloned the resulting genes into El-deleted Ad vectors. They systemically administered these viruses to hepatic lipase-deficient mice and examined changes in serum lipids. These experiments showed that the greater ability of hepatic lipase to hydrolyze phospholipids and to reduce total serum cholesterol is largely determined by the "lid" domain of the proteins, which is thought to restrict access to the active site of the enzyme.
Cytokine and chemokine functions in vivo have been studied using Ad vectors to locally produce factors, including GM-CSF, RANTES, and IL-6 in rat lung (Xing et al., 1994, 1996; Braciak et al., 1996). Expression of these molecules in a defined tissue has allowed investigation of their roles on inflammatory processes and recruitment of cells such as monocytes and T lymphocytes. Local expression of Ad-delivered genes may be more relevant to their normal physiological roles than systemic administration of the purified protein.
Systemically administered Ad vectors can also provide high-level temporary expression of a protein that is secreted into the bloodstream. Much of this work has focused on preclinical applications, as in expression of factor VIII for the treatment of hemophilia A (Connelly et al., 1996) or of erythropoietin to stimulate erythro-poiesis (Setoguchi et al., 1994a), but this property can also be useful in understanding the in vivo roles of secreted proteins. For example, rats treated intravenously with a viral vector directing expression of the rat leptin protein exhibited reduced food intake and weight gain, as well as a disappearance of fat deposits (Chen et al., 1996). Intraperitoneal administration of an Ad vector containing the gene encoding HST-l/FGF-4 to mice stimulated platelet production (Sakamoto et al., 1994). This group then used the same viral vector in an in vitro study to demonstrate that the increased platelet count was due to FGF-4 stimulation of megakaryocyte maturation (Konishi et al., 1996).
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