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Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

ABSTRACT: In central nervous system (CNS)-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad) vectors across the blood-brain barrier (BBB) in vitro models. As a proof-of-principle ligand, maleimide-activated full-length human transferrin (hTf) was covalently attached to cysteine-modified Ad serotype 5 vectors either to its fiber or hexon protein. In transcytosis experiments, hTf-coupled vectors were shown to be redirected across the BBB models, the transcytosis activity of the vectors being dependent on the location of the capsid modification and the in vitro model used. The transduction efficiency of hTf-targeted vectors decreased significantly in confluent, polarized cells, indicating that the intracellular route of the vectors differed between unpolarized and polarized cells. After transcellular delivery the majority of the hTf-modified vectors remained intact and partly capable of gene transfer. Altogether, our results demonstrate that i) covalent attachment of a ligand to Ad capsid can mediate transcellular targeting across the cerebral endothelium in vitro, ii) the attachment site of the ligand influences its transcytosis efficiency and iii) combined genetic/chemical modification of Ad vector can be used as a versatile platform for the development of Ad vectors for transcellular targeting.

SUBMITTER: Laakkonen JP

PROVIDER: S-EPMC3459940 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

Laakkonen Johanna P JP Engler Tatjana T Romero Ignacio A IA Weksler Babette B Couraud Pierre-Olivier PO Kreppel Florian F Kochanek Stefan S

PloS one 20120927 9

In central nervous system (CNS)-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad) vectors across the blood-brain barrier (BBB) in vitro models. As a ...[more]

PMID: 23029348

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Project description:ObjectiveTo evaluate the effects of fiber-modified hexon-chimeric recombinant oncolytic adenovirus targeting cancer associated fibroblasts (CAFs) on the gastric CAFs and the transplantation tumor mice model of gastric carcinoma (GC).ResultsCompared with BJ cells and GPFs, the reproduction and infectivity of P9, P9-4C or GP adenoviruses were markedly higher in gastric CAFs. In addition, P9, P9-4C or GP had a significantly relatively more killing effect on gastric CAFs compared with GPFs, and have less oncolytic effect in BJ cells. Furthermore, in transplantation tumor mice model of GC we found significantly higher hexon protein expression in tumor tissues, more decreasing tumor growth and increasing inhibitory rates after treatment of P9, P9-4C or GP adenoviruses compared with Ad adenovirus.Materials and methodsBased on the construction of the recombinant oncolytic adenoviruses pRCAdHVR48-SDF1p-Ad/EGFP (Ad, as control) with the E1A gene transcription regulated by stromal-derived factor 1 (SDF1) promoter and the hexon replaced by hexon-chimeric (H5HVR48) gene, three fiber-modified hexon-chimeric oncolytic adenovirus through the modification fiber protein by insertion of different short peptides specifically binding to fibroblast activation protein (FAP), including pRCAdHVR48-SDF1p-FAP-P9/EGFP (P9), pRCAdHVR48-SDF1p-FAP-P9-4C/EGFP (P9-4C), pRCAdHVR48-SDF1p-FAP-GP/EGFP (GP), and their corresponding replication-defective adenovirus in parallel were reconstructed. Then the reproduction, infectivity and killing ability of the four above recombinant adenoviruses were evaluated in gastric CAFs compared with gastric para-mucosa fibroblasts (GPFs) and neonatal human foreskin fibroblasts (BJ). Furthermore, transplantation tumor mice model of GC was established, and then treated by the four above recombinant adenoviruses. Tumor size and tumor growth inhibitory rates were calculated, and histomorphology by HE staining and hexon expressions by immunohistochemistry were evaluated in tumor tissues.ConclusionsThe fiber-modified hexon-chimeric recombinant oncolytic adenovirus targeting CAFs can relatively specifically kill gastric CAFs and inhibit GC cells growth in vivo.

Project description:Following receptor binding and internalization, intracellular trafficking of adenovirus (Ad) among subgroups B and C is different, with significant amounts of Ad serotype 7 (Ad7) (subgroup B) virions found in cytoplasm during the initial hours of infection while Ad5 (subgroup C) virions rapidly translocate to the nucleus. To evaluate the role of the fiber in these differences, we examined intracellular trafficking of Ad5, Ad7, and Ad5f7 (a chimeric vector composed of the Ad5 capsid with the fiber replaced by the Ad7 fiber) by conjugating Ad capsids directly with Cy3 fluorescent dye, permitting the trafficking of the capsids to be examined by fluorescence microscopy. The human lung carcinoma cell line A549 was infected with Cy3-conjugated viruses for 10 min followed by a 1-h incubation. Ad5 virions rapidly translocated to the nucleus (within 1 h of infection), while Ad7 virions were widely distributed in the cytoplasm at the same time point. Interestingly, chimeric Ad5f7 virions behaved similarly to Ad7 but not Ad5. In this regard, the percentages of nuclear localization of Ad5, Ad7, and Ad5f7 at 1 h following infection were 72% +/- 4%, 32% +/- 6%, and 38% +/- 2%, respectively. Consistent with these observations, fluorescence in situ hybridization demonstrated that most of the Ad5 DNA was detected at the nucleus after 1 h, but at the same time point, DNA of Ad7 and Ad5f7 was distributed in both the nucleus and cytoplasm. Quantification of the kinetics of Ad genomic DNA delivery to the nucleus using a fluorogenic probe-based PCR assay (TaqMan PCR) demonstrated that the percentages of nuclear association of Ad5 DNA and Ad5f7 DNA at 1 h postinfection were 80% +/- 13% and 43% +/- 1%, respectively. Although it has been generally accepted that Ad fiber protein mediates attachment of virions to cells and that fibers dissociate during endocytic uptake, these data suggest that in addition to mediating binding to the cell surface, fiber likely modulates intracellular trafficking as well.

Project description:BackgroundTrypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.Methodology/principal findingsThe "antigen capsid-incorporation" strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5) vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83). This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.Conclusions/significanceThis data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes. Taken together, these novel results show that the recombinant Ad5 presenting T. cruzi gp83 antigen is a useful candidate for the development of a vaccine against Chagas disease.

Dataset Information

Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

Publications

Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

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