Substitution of Hexon Hypervariable Region 5 of Adenovirus Serotype 5 Abrogates Blood Factor Binding and Limits Gene Transfer to Liver

Abstract

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

Efficient delivery of angiostatin K1-5 into tumors following insertion of an NGR peptide into adenovirus capsid

“…After PBS-5% milk-0.05% IgePal (Sigma) blockage of free sites, the membrane, was incubated with 10 11 vp of different Ad in PBS-0.5% milk-0.05% IgePal, for 4 h at room temperature and processed as described previously. 36 …”

“…High b-gal activity was documented in liver lysates of Adwt-and AdFNGR-injected mice, but not in AdHGNR-injected mice (Supplementary Figure 1a-b), as we previously observed for different hexon-modified Ads. 36 AdHNGR was shown to bind to immobilized blood factors FIX and FX, but was unable to use FX to Figure 1c-d). In tumor lysates, low b-gal activity was measured with no difference between groups (data not shown), thus suggesting that Ad liver uptake precludes efficient targeting.…”

The Role of Liver Sinusoidal Cells in Hepatocyte-Directed Gene Transfer

“…Indeed, only factor X can rescue liver transduction in warfarin-anticoagulated mice. 22 Recently, it has been shown that the ␥-carboxyglutamic acid domain of factor X binds in a calcium-dependent manner to hexon protein in Ad5 8,22,23 and that this binding occurs at the cup formed by the center of each hexon trimer. Moreover, Ads with a high affinity for factor X, such as the species C serotypes Ad2 and Ad5, have been shown to efficiently transduce hepatocytes following i.v.…”

Coagulation factor X mediates adenovirus type 5 liver gene transfer in non-human primates (Microcebus murinus)

“…[4][5][6] Several studies have documented the role of blood coagulation factors in mediating liver gene transfer in vivo and the mechanism underlying Ad5 hepatocyte transduction. [7][8][9][10][11][12] We demonstrated that coagulation factor X (FX) binds to the hexon protein of Ad5 at high affinity (B2 nM) and that this interaction mediates hepatocyte transduction following i.v. delivery in mice and rats.…”