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1210. Targeting Adenoviral Vectors Via Modification of Minor Capsid Protein IX
AD VECTOR TARGETING To this end, we exchanged the reovirus spike anchoring-domain, which attaches the spike to the reovirus virion, for its adenovirus fiber analog, the tail domain. The resulting spike/fiber fusion protein was tested for its capacity to complement the production of fiberless AdV by transfecting a fusion-protein expression plasmid into 293T cells and infecting the cells subsequently with the fiberless Ad5.βgal.ΔF (Von Seggern et al. J. Virol. 1999, 73: 1601). Progeny virus was harvested and its infection efficiency was determined on 293T cells. In contrast to production of Ad5.βgal.ΔF without complementation or production of Ad5.βgal.ΔF complemented with the native reovirus spike, the fusion protein facilitated efficient production of infectious Ad5.βgal.ΔF, at titers similar to the production of Ad5.βgal.ΔF complemented with the native adenovirus fiber. Moreover, specific inhibition of the fiber-CAR interaction with neutralizing anti-fiber antibodies decreased the infection efficiency of the adenovirus-fiber complemented Ad5.βgal.ΔF, whereas the infection efficiency of fusion-protein complemented Ad5.βgal.ΔF remained unaltered. The nature of the AdV tropism, which is defined by the spike/fiber fusion protein is currently under investigation. In conclusion, these findings strongly suggest efficient AdV production and incorporation of the spike/fiber fusion protein into the adenovirus capsid, supporting further exploration of AdV with chimeric reovirus spike proteins for selective cancer treatment.
1210. Targeting Adenoviral Vectors Via Modification of Minor Capsid Protein IX Jort Vellinga,1 Martijn J. Rabelink,1 Diana J. Van den Wollenberg,1 Steve J. Cramer,1 Rob C. Hoeben.1 1 Dept. of Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands. Adenovirus vectors are among the most efficient vectors for gene transfer. Nevertheless, the vectors used to date still need improvement with respect to their cell-type specificity. Genetic modifications, to modify the tropism of adenoviral vectors, have relied on recruiting receptors different than its natural receptor CAR. Peptide ligands have been inserted into the fiber-knob or the hexon. Although effective, this approach is hampered by the limited tolerance of these capsid proteins toward insertion of longer peptides, frustrating the targeting of many candidate receptors. Also the fibers of the CAR binding adenoviruses have been replaced by fibers from adenoviruses recognizing other receptors. This approach is limited to the available repertoire of adenovirus fibers variants. Our strategy to modify the tropism of adenoviral vectors relies on fusing ligands to minor capsid protein IX. Protein IX (pIX) is a small, 14.3 kDa component of the capsid. Four pIX trimers are located within each group-of-nine hexons (GONs), and 240 molecules pIX are present per virion. The four trimers are embedded in the large cavities of the GONs and ‘cement’ the GONs into a highly stable assembly. Hence, virions devoid of pIX are more heatsensitive than wild-type particles. Despite its function, pIX is dispensable: mutants lacking pIX can be grown to titers similar to wild-type viruses. Constitutive expression of pIX in helper cell lines can complement the pIX deficiency. We have developed a panel of C-terminal Ad5-pIX variants, which all carry an integrin-binding RGD or a Myc tag motive. These motives are separated from the body of pIX by alpha-helical spacers with predicted lengths up to 75 Angstrom. A pIX-deficient adenovirus, carrying a GFP gene as reporter, was grown on helper cells that express the different pIX-fusion proteins. Analyses of protein lysates of the producer cells and isolated viruses show that these spacers do not impede pIX incorporation into the Ad5 capsid. We showed that the modified pIX proteins stabilize the capsids. Transductions of endothelial cells, which have a low CAR expression, show that transduction efficiency improves with spacer length. Electron-microscopy analyses using gold-labeled antibodies S468
confirm that the longer spacers increase the accessibility of the epitopes. Our data clearly show that alpha-helical spacers augment the presentation of ligands linked to the C-terminus of pIX. Presumably the spacer lifts the ligand out of the hexon cavity, and increases the accessibility for their cognate receptors. Our data provide a new avenue for ‘pseudotyping’ adenovirus vectors.
1211. Engineering Viral Vectors with Smooth Muscle Cell Targeting Peptides Lorraine M. Work,1 Stuart A. Nicklin,1 Nick J. R. Brain,1 Dan J. Von Seggern,2 Michael Hallek,3 Hildegard Buening,3 Andrew H. Baker.1 1 Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom; 2Department of Immunology, Scripps Research Institute, San Diego, CA, United States; 3Gene Centre, Ludwig-Maximilians University, Munich, Germany. Local delivery of gene therapy vectors to the vessel wall holds potential for the treatment of late vein graft failure and restenosis. However, existing vectors such as adenovirus (Ad) and adenoassociated virus (AAV) have a poor relative tropism for smooth muscle cells (SMC) necessitating the use of high viral titers. This in turn may lead to local toxicity and/or systemic leakage which compromises the safety of such an approach clinically, especially if cytotoxic transgenes are used. Gene delivery in this context would benefit from the availability of more efficient and selective vectors. Here, we sought to identify novel targeting peptides that selectively bind human saphenous vein SMC by biopanning with phage display libraries expressing linear 7mer peptides. We performed 4 sequential rounds of biopanning on SMC with a pre-clearing step on nontarget cells, thus removing phage displaying peptides that bind to common receptors. Phage (2x1011 pfu) displaying linear 7mer peptides were incubated with SMC at 4ºC for 1 hour in order to isolate peptides which would selectively improve Ad/AAV transduction of SMC. Cells were washed and weakly associated phage dissociated by acid elution. Cells were lysed and tightly associated phage harvested. After 4 rounds we sequenced 96 phage DNAs to identify the encoded peptide insert. Analysis of peptide sequences resulted in the identification of 9 novel candidate consensus tripeptide motifs and 2 consensus peptide sequences. 2 consensus peptides (GLA1 and GLA2) were incorporated into the HI loop of Ad5 with additional ablation of native tropism for the coxsackie adenovirus receptor (the KO1 mutation). Short exposures (up to one hour) of the modified virus to SMC significantly improved reporter gene expression in the absence of EC transduction for GLA1 but not GLA2. We additionally incorporated GLA1 into the VP3 capsid protein of AAV after residue 587. Virus titers equivalent to non-modified controls were obtained. A significant enhancement in reporter gene expression with the modified AAV compared to nonmodified control was observed. Again, this effect was observed at short exposures to AAV. Identification of SMC targeting peptides by phage display and their subsequent incorporation into viral vectors has important implications for the treatment of late vein graft failure and post-angioplasty restenosis by enabling selective and efficient gene delivery to SMC.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
1210. Targeting Adenoviral Vectors Via Modification of Minor Capsid Protein IX Jort Vellinga,1 Martijn J. Rabelink,1 Diana J. Van den Wollenberg,1 Steve J. Cramer,1 Rob C. Hoeben.1 1 Dept. of Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands. Adenovirus vectors are among the most efficient vectors for gene transfer. Nevertheless, the vectors used to date still need improvement with respect to their cell-type specificity. Genetic modifications, to modify the tropism of adenoviral vectors, have relied on recruiting receptors different than its natural receptor CAR. Peptide ligands have been inserted into the fiber-knob or the hexon. Although effective, this approach is hampered by the limited tolerance of these capsid proteins toward insertion of longer peptides, frustrating the targeting of many candidate receptors. Also the fibers of the CAR binding adenoviruses have been replaced by fibers from adenoviruses recognizing other receptors. This approach is limited to the available repertoire of adenovirus fibers variants. Our strategy to modify the tropism of adenoviral vectors relies on fusing ligands to minor capsid protein IX. Protein IX (pIX) is a small, 14.3 kDa component of the capsid. Four pIX trimers are located within each group-of-nine hexons (GONs), and 240 molecules pIX are present per virion. The four trimers are embedded in the large cavities of the GONs and ‘cement’ the GONs into a highly stable assembly. Hence, virions devoid of pIX are more heatsensitive than wild-type particles. Despite its function, pIX is dispensable: mutants lacking pIX can be grown to titers similar to wild-type viruses. Constitutive expression of pIX in helper cell lines can complement the pIX deficiency. We have developed a panel of C-terminal Ad5-pIX variants, which all carry an integrin-binding RGD or a Myc tag motive. These motives are separated from the body of pIX by alpha-helical spacers with predicted lengths up to 75 Angstrom. A pIX-deficient adenovirus, carrying a GFP gene as reporter, was grown on helper cells that express the different pIX-fusion proteins. Analyses of protein lysates of the producer cells and isolated viruses show that these spacers do not impede pIX incorporation into the Ad5 capsid. We showed that the modified pIX proteins stabilize the capsids. Transductions of endothelial cells, which have a low CAR expression, show that transduction efficiency improves with spacer length. Electron-microscopy analyses using gold-labeled antibodies S468
confirm that the longer spacers increase the accessibility of the epitopes. Our data clearly show that alpha-helical spacers augment the presentation of ligands linked to the C-terminus of pIX. Presumably the spacer lifts the ligand out of the hexon cavity, and increases the accessibility for their cognate receptors. Our data provide a new avenue for ‘pseudotyping’ adenovirus vectors.
1211. Engineering Viral Vectors with Smooth Muscle Cell Targeting Peptides Lorraine M. Work,1 Stuart A. Nicklin,1 Nick J. R. Brain,1 Dan J. Von Seggern,2 Michael Hallek,3 Hildegard Buening,3 Andrew H. Baker.1 1 Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom; 2Department of Immunology, Scripps Research Institute, San Diego, CA, United States; 3Gene Centre, Ludwig-Maximilians University, Munich, Germany. Local delivery of gene therapy vectors to the vessel wall holds potential for the treatment of late vein graft failure and restenosis. However, existing vectors such as adenovirus (Ad) and adenoassociated virus (AAV) have a poor relative tropism for smooth muscle cells (SMC) necessitating the use of high viral titers. This in turn may lead to local toxicity and/or systemic leakage which compromises the safety of such an approach clinically, especially if cytotoxic transgenes are used. Gene delivery in this context would benefit from the availability of more efficient and selective vectors. Here, we sought to identify novel targeting peptides that selectively bind human saphenous vein SMC by biopanning with phage display libraries expressing linear 7mer peptides. We performed 4 sequential rounds of biopanning on SMC with a pre-clearing step on nontarget cells, thus removing phage displaying peptides that bind to common receptors. Phage (2x1011 pfu) displaying linear 7mer peptides were incubated with SMC at 4ºC for 1 hour in order to isolate peptides which would selectively improve Ad/AAV transduction of SMC. Cells were washed and weakly associated phage dissociated by acid elution. Cells were lysed and tightly associated phage harvested. After 4 rounds we sequenced 96 phage DNAs to identify the encoded peptide insert. Analysis of peptide sequences resulted in the identification of 9 novel candidate consensus tripeptide motifs and 2 consensus peptide sequences. 2 consensus peptides (GLA1 and GLA2) were incorporated into the HI loop of Ad5 with additional ablation of native tropism for the coxsackie adenovirus receptor (the KO1 mutation). Short exposures (up to one hour) of the modified virus to SMC significantly improved reporter gene expression in the absence of EC transduction for GLA1 but not GLA2. We additionally incorporated GLA1 into the VP3 capsid protein of AAV after residue 587. Virus titers equivalent to non-modified controls were obtained. A significant enhancement in reporter gene expression with the modified AAV compared to nonmodified control was observed. Again, this effect was observed at short exposures to AAV. Identification of SMC targeting peptides by phage display and their subsequent incorporation into viral vectors has important implications for the treatment of late vein graft failure and post-angioplasty restenosis by enabling selective and efficient gene delivery to SMC.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy