Adenovirus-platelet interaction in blood causes virus sequestration to the reticuloendothelial system of the liver

Abstract

Intravenous (i.v.) delivery of recombinant adenovirus serotype 5 (Ad5) vectors for gene therapy is hindered by safety and efficacy problems. We have discovered a new pathway involved in unspecific Ad5 sequestration and degradation. After i.v. administration, Ad5 rapidly binds to circulating platelets, which causes their activation/aggregation and subsequent entrapment in liver sinusoids. Virus-platelet aggregates are taken up by Kupffer cells and degraded. Ad sequestration in organs can be reduced by platelet depletion prior to vector injection. Identification of this new sequestration mechanism and construction of vectors that avoid it could improve levels of target cell transduction at lower vector doses.

FIG. 1.

Ad association in blood. hCD46 transgenic mice were injected i.v. with 10¹¹ VP of an Ad5 vector. (A) Blood extracted 5 min after injection was immediately separated into blood cell and serum fractions. Total DNA was extracted from blood cell and serum fractions along with carrier DNA and analyzed for the presence of Ad genomes by quantitative PCR. (B) Platelets purified from blood 10 min after Ad delivery were subjected to TEM analysis. Virus particles associated with the external platelet membrane and membranes of the open canalicular system are shown (arrows). (C) Levels of soluble CD62p in sera were measured by ELISA 5 min and 6 h after Ad delivery. (D) Levels of D-dimer fibrinogen breakdown products in sera were measured by ELISA 5 min and 6 h after Ad delivery. Four mice per group were used.

FIG. 2.

Ad-platelet deposition in liver. hCD46 transgenic mice were injected i.v. with 10¹¹ VP of an Ad5 vector. (A) Total genomic DNA was extracted from organs 5 min after Ad injection. A total of 10 μg was digested with EcoRI and subjected to Southern blot analysis with an Ad5 fiber-specific probe, and signals were quantified by a phosphorimager. (B and C) Livers from phosphate-buffered saline (PBS) (B)- or Ad5 (C)-injected mice were taken 5 min after delivery and stained with antibodies against platelets (mCD41) (red), KCs (F4/80) (green), and Ad capsid (hexon) (blue). Areas labeled with hexon plus F4/80 (arrowheads) or hexon plus F4/80 plus mCD41 (arrows) are shown. (D) Quantification of double- or triple-labeled regions in sections from livers of Ad5-injected mice. Total hepatic sinusoidal regions with hexon-CD41-F4/80 and hexon-F4/80 colabeling were counted for five fields of view. (E and F) Immunohistochemistry for the granulocyte/monocyte marker Gr-1 (red) on liver sections from PBS (E)- or Ad5 (F)-injected animals 30 min p.i. (G to L) Livers from Ad5-injected mice were analyzed by TEM 5 min p.i., and representative images are shown. High-power composite images are shown to the right. VP (arrows) and degranulated platelet/endothelial cell interactions (arrowheads) are shown. D, Disse spaces; DeP, degranulated platelet; E, erythrocyte; EC, endothelial cell; H, hepatocyte; Ito, Ito cell; P, platelet.

FIG. 3.

Effect of platelet depletion on sequestration to blood cells and liver. hCD46 transgenic mice were platelet depleted using MWReg30 antibody, and after 3 h, the mice received 10¹¹ VP of Ad5 vector. Blood samples, platelets, and livers were harvested 5 min after virus injection. Equalized amounts of isolated DNA were digested with EcoRI and subjected to Southern blot analysis with an Ad5 fiber-specific probe. Southern blot signals were quantified by a phosphorimager and are shown for blood cells and sera (A), platelets (B), or organs (C). Four mice per group were used. IgG1, immunoglobulin G1.