319. Improvement of Transgene Expression through the Direct Intrahepatic Injection of Adenoviral Vectors

Abstract

Top of pageAbstract Numerous diseases could be potentially treated using gene therapy based strategies. An ideal gene therapy system should lead to a safe, low toxicity, long termed and high-level of expression of the therapeutic protein. However, the scaling up of the production for its application to general recombinant viral vector clinical treatment in humans presents a big problem for some of them, for example the third generation adenovirus. Focusing on the improvement of the high-level transgene expression of an adenoviral vector, we characterized a simple and efficient technique to transduce the liver.First and third generation adenovirus were used in mice to characterize the administration of the virus by direct intrahepatic injection. First and third generation adenovirus expressing human interleukin twelve (hIL12), as reporter gene, were assayed in mice by intravenous (IV) and intrahepatic (IH) administration. The expression of the transgene in the first generation adenovirus is under control of the CMV promoter. The third generation adenovirus, contains the hIL-12 gene under the control of an antiprogesterone receptor promoter system. In order to study the relevance of Kupffer cells in the adenovirus liver transduction, we injected both vectors after a clodronate containing liposomes injection. The IH injection of 1×1010 pfu of the first generation adenovirus Ad-IL-12 shows protein levels 2–3 times higher than the administration of the same dose by conventional IV administration. Il-12 expression it is not detected in mice receiving 109 pfu of the Ad-hIL-12 I.V., however the same dose administerred I.H. resulted in clearly detectable levels of the transgene in serum. That should result in an at least 2–3 times lower dose of virus needed to get an identical expression level. The previous injection of clodronate-liposomes improves substantially the expression in both routes and maintains the previously described effect. Moreover the kinetic of the transgene expression is different depending on the route of administration, after IH. administration the highest expression level of the transgene is detected at early time points and an abrupt progressive decrease, on the other hand adenovirus IV administerred showed the peak of transgene expression 48 hours after viral injection. The third generation adenovirus injected intrahepatically offers a better increase in protein expression, 3–4 times, respect to the IV injection. However the empty-liposome preinjection decreases this improvement and the clodronate-liposomes administration inverts this tendency, being the IV better than the IH, in this case. This method evades the phagocytosis by the liver resident macrophages and permits a good expression at low virus doses. Empty-liposomes poorly and the clodronate-liposomes clearly deplete the Kupffer cells and eliminate this difference. This novel technique of gene therapy administration offers several advantages. It makes feasible the administration of less adenovirus that expresses high levels of transgene. It presents a quick and easy treatment method for humans and superior mammals, especially for third generation adenovirus.