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1209. Targeting Adenoviral Vectors with a Reovirus Spike Fusion Protein
AD VECTOR TARGETING 1206. Quantifying Retrograde Transport of AAV Vectors in the CNS Brian K. Kaspar,1 Nushin Sherkat,1 Melanie Lucero,1 Lindsay Frost,1 Lindsey Christian,1 Fred H. Gage.1 1 Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA. Development of gene therapy vectors for therapeutic applications has been hampered by the ability to effectively deliver genes to target tissues and cells. The central nervous system has proven difficult to easily target large numbers of cells, and the spinal cord presents a significant challenge to effectively target motor neurons. We have previously reported that the non-lytic, AAV-2 vector is capable of retrograde transport by infecting presynaptic axonal terminals projecting from various brain regions including the substantia nigra and entorhinal cortex. In the present study we show that AAV vectors have the capacity for retrograde transport over long distances. By single injections into peripheral muscles such as the quadriceps, AAV virus infects innervating axon terminals and is transported in a retrograde manner to the spinal cord. We present here for the first time, quantitative data on the number and percentage of AAV particles that are retrogradely transported. The current data suggests a titer threshold for retrograde transport and is increased in a dose dependent manner. Furthermore, we present data on the time course for transport to occur and provide insight into the mechanisms underlying the viral infection pathway. Finally, we show that we can target large numbers of motorneurons by retrograde transport infection based upon GFP and Bgalactosidase expression.
AD VECTOR TARGETING 1207. dl-VSVG-LacZ, a VSV-G Epitope Incorporated Adenovirus Exhibit Marked Enhancement in Gene Transduction Efficiency E. A. Cho,1 J. J. Song,1 D. B. Kang,1 E. Kim,1 T. Y. Ha,1 J. H. Sohn,2 C. O. Yun,1 J. H. Kim.1,2 1 BK21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea; 2Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea. Recombinant adenovirus has emerged as the choice of vector system for cancer gene therapy. However, the utility of adenoviral vectors is limited due to the low efficiency of adenovirus-mediated gene transfer to cancer cells, which inherently express low level of coxackie and adenovirus receptor (CAR) on their surface. Development of novel strategies to achieve adenovirus infection via CAR-independent pathway may help to overcome this limitation. To this end, we have generated a novel dl-VSVG-LacZ that contains fiber knob with intact CAR-entry capability and an additional PSentry capability by incorporating the VSV-G epitope onto the Cterminus of the fiber knob. VSV-G is an envelope protein of Vesicular Stomatitis virus that facilitates the specificity for the binding of the virus to the phosphatidyl serine (PS)-moiety on the cellular plasma membrane surface. In this study, we have explored the utility of a retargeted adenovirus vector containing the VSV-G epitope in the terminus of the fiber knob to improve the transduction efficiency. dl-VSVG-LacZ showed a remarkable improvement (300%-2000%) in the delivery of LacZ to a variety of mammalian cells including those that are CAR-deficient. Largest improvement in gene transfer was observed for cells that were difficult to transduce with an untargeted Ad (wild type fiber). In addition, we demonstrate that viral propagation of dl-VSVG-LacZ was greatly improved by enhanced infectivity. Furthermore, treatment with dl-VSVG-LacZ significantly enhanced gene transfer in vivo when compared to control Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
adenovirus that lack the VSV-G epitope. Taken together, these studies demonstrate that a strategy to extend adenovirus tropism may greatly improve utilities of adenovirus for gene therapy application.
1208. HIV Tat PTD-Modified Adenovirus: Improving Gene Transfer to CAR Negative Cells Haibin Xia,1 Qinwen Mao,1 Beverly L. Davidson.1,3 1 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA. Adenovirus (Ad) is widely used for gene delivery in therapies for inherited diseases and cancers. The efficiency of gene transfer with Ad5 based vectors depends on the levels of coxsackie-adenovirus receptor (CAR) expression. Capsid modifications are required to accomplish gene transfer to tissues expressing low levels of CAR. We hypothesized that the protein transduction domain (PTD) from HIV tat could allow for CAR-independent entry into cells. The HIV tat PTD consists of an epitope shown to allow for protein delivery to cells in culture and in vivo. To test our hypothesis, PTD.AdeGFP was constructed by modifying the HI-loop of the Ad5 fiber to contain sequences encoding this epitope. PTD.AdeGFP was then tested in CAR-positive (A549) and CAR-low (T24, NIH 3T3, CHO, HUVEC) cell lines. Our results show that PTDAdeGFP infected A549 cells less efficiently than AdeGFP, but unlike AdeGFP, infection was independent of CAR. In CAR-low cell lines, PTD.AdeGFP infection was significantly increased over AdeGFP. To test that gene transfer was not due to increased chargedirected binding between the virus (the tat motif is highly positively charged) and the cell surface, cells were treated with Heparinase I prior to virus exposure. Heparinase I treatement did not decrease PTD.AdeGFP infection. In additional studies we tested the tempature dependence of infection, and the role of dynamin in the entry pathway. The infection efficiency was not significantly reduced when cells were lowered to 4°C, or when cells were expressing a dominant negative form of dynamin. Additional EM studies on the virus indicate that the purified particles exist as clumps rather than independent virions. The PTD motif may act to concentrate the virus onto cells, allowing for CAR- and dynamin-independent gene transfer.
1209. Targeting Adenoviral Vectors with a Reovirus Spike Fusion Protein Frederik H. E. Schagen,1 Jan E. Carette,1 Terence S. Dermody,2 Winald R. Gerritsen,1 Victor W. van Beusechem.1 1 Medical Oncology, VU University Medical Center, Amsterdam, Netherlands; 2Microbiology & Immunology, Vanderbilt-Ingram Cancer Center, Nashville, TN. The affinity of reovirus for cancer cells and the close resemblance of the reovirus spike protein with the adenovirus fiber protein raises the question whether the reovirus spike protein can be applied for targeting of adenovirus vectors (AdV) towards cancer cells. Such a targeting strategy is supported by the fact that the reovirus spike protein imposes less restrains on genetic modifications than does the adenoviral fiber. In contrast to the adenoviral fiber, the tropismdetermining and trimerization functions of the reovirus spike reside in distinct domains of the molecule. In this regard, it has already been shown that the tropism-determining domain can be replaced for an irrelevant peptide without loss of trimerization and capsid incorporation functions. This suggests that by replacing the tropismdetermining domain of reovirus spike for a tumor targeting ligand, tumor-selectivity can be obtained. Before the possibility of this targeting strategy could be evaluated, we first investigated if the reovirus spike protein can be adapted for incorporation into the adenovirus capsid and whether AdV carrying the adapted spike protein can be propagated in the absence of adenovirus fiber protein. S467
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
AD VECTOR TARGETING 1207. dl-VSVG-LacZ, a VSV-G Epitope Incorporated Adenovirus Exhibit Marked Enhancement in Gene Transduction Efficiency E. A. Cho,1 J. J. Song,1 D. B. Kang,1 E. Kim,1 T. Y. Ha,1 J. H. Sohn,2 C. O. Yun,1 J. H. Kim.1,2 1 BK21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea; 2Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea. Recombinant adenovirus has emerged as the choice of vector system for cancer gene therapy. However, the utility of adenoviral vectors is limited due to the low efficiency of adenovirus-mediated gene transfer to cancer cells, which inherently express low level of coxackie and adenovirus receptor (CAR) on their surface. Development of novel strategies to achieve adenovirus infection via CAR-independent pathway may help to overcome this limitation. To this end, we have generated a novel dl-VSVG-LacZ that contains fiber knob with intact CAR-entry capability and an additional PSentry capability by incorporating the VSV-G epitope onto the Cterminus of the fiber knob. VSV-G is an envelope protein of Vesicular Stomatitis virus that facilitates the specificity for the binding of the virus to the phosphatidyl serine (PS)-moiety on the cellular plasma membrane surface. In this study, we have explored the utility of a retargeted adenovirus vector containing the VSV-G epitope in the terminus of the fiber knob to improve the transduction efficiency. dl-VSVG-LacZ showed a remarkable improvement (300%-2000%) in the delivery of LacZ to a variety of mammalian cells including those that are CAR-deficient. Largest improvement in gene transfer was observed for cells that were difficult to transduce with an untargeted Ad (wild type fiber). In addition, we demonstrate that viral propagation of dl-VSVG-LacZ was greatly improved by enhanced infectivity. Furthermore, treatment with dl-VSVG-LacZ significantly enhanced gene transfer in vivo when compared to control Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
adenovirus that lack the VSV-G epitope. Taken together, these studies demonstrate that a strategy to extend adenovirus tropism may greatly improve utilities of adenovirus for gene therapy application.
1208. HIV Tat PTD-Modified Adenovirus: Improving Gene Transfer to CAR Negative Cells Haibin Xia,1 Qinwen Mao,1 Beverly L. Davidson.1,3 1 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA. Adenovirus (Ad) is widely used for gene delivery in therapies for inherited diseases and cancers. The efficiency of gene transfer with Ad5 based vectors depends on the levels of coxsackie-adenovirus receptor (CAR) expression. Capsid modifications are required to accomplish gene transfer to tissues expressing low levels of CAR. We hypothesized that the protein transduction domain (PTD) from HIV tat could allow for CAR-independent entry into cells. The HIV tat PTD consists of an epitope shown to allow for protein delivery to cells in culture and in vivo. To test our hypothesis, PTD.AdeGFP was constructed by modifying the HI-loop of the Ad5 fiber to contain sequences encoding this epitope. PTD.AdeGFP was then tested in CAR-positive (A549) and CAR-low (T24, NIH 3T3, CHO, HUVEC) cell lines. Our results show that PTDAdeGFP infected A549 cells less efficiently than AdeGFP, but unlike AdeGFP, infection was independent of CAR. In CAR-low cell lines, PTD.AdeGFP infection was significantly increased over AdeGFP. To test that gene transfer was not due to increased chargedirected binding between the virus (the tat motif is highly positively charged) and the cell surface, cells were treated with Heparinase I prior to virus exposure. Heparinase I treatement did not decrease PTD.AdeGFP infection. In additional studies we tested the tempature dependence of infection, and the role of dynamin in the entry pathway. The infection efficiency was not significantly reduced when cells were lowered to 4°C, or when cells were expressing a dominant negative form of dynamin. Additional EM studies on the virus indicate that the purified particles exist as clumps rather than independent virions. The PTD motif may act to concentrate the virus onto cells, allowing for CAR- and dynamin-independent gene transfer.
1209. Targeting Adenoviral Vectors with a Reovirus Spike Fusion Protein Frederik H. E. Schagen,1 Jan E. Carette,1 Terence S. Dermody,2 Winald R. Gerritsen,1 Victor W. van Beusechem.1 1 Medical Oncology, VU University Medical Center, Amsterdam, Netherlands; 2Microbiology & Immunology, Vanderbilt-Ingram Cancer Center, Nashville, TN. The affinity of reovirus for cancer cells and the close resemblance of the reovirus spike protein with the adenovirus fiber protein raises the question whether the reovirus spike protein can be applied for targeting of adenovirus vectors (AdV) towards cancer cells. Such a targeting strategy is supported by the fact that the reovirus spike protein imposes less restrains on genetic modifications than does the adenoviral fiber. In contrast to the adenoviral fiber, the tropismdetermining and trimerization functions of the reovirus spike reside in distinct domains of the molecule. In this regard, it has already been shown that the tropism-determining domain can be replaced for an irrelevant peptide without loss of trimerization and capsid incorporation functions. This suggests that by replacing the tropismdetermining domain of reovirus spike for a tumor targeting ligand, tumor-selectivity can be obtained. Before the possibility of this targeting strategy could be evaluated, we first investigated if the reovirus spike protein can be adapted for incorporation into the adenovirus capsid and whether AdV carrying the adapted spike protein can be propagated in the absence of adenovirus fiber protein. S467
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