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499. Chemically Inactivated Adenoviral Vectors with Conditional and Targetable Infectivity: A Strategy for Safer Delivery of Adenoviral Vectors
NOVEL AD VECTORS AND APPLICATIONS with the transgene cassette required. Given the large genome size that can thus be packaged, HD provide not only a safer vector, but allows to encode very large transgenic sequences. To improve currently available plasmids for HD construction we have engineerd a novel HD plasmid using sequences from human chromosome 22 as ‘stuffer’ sequences. Additionally, we inserted all cis elements necessary for replication and packaging of the adenovirus (ITRs, and Y), as well as the E4 promoter, which has been shown by Sandig et al., 2000, to increase the packaging efficiency of HD vectors. The length of ‘stuffers’ used was between 7-9 kb. To select suitable sequences we used the following criteria: absence of CpG islands, minimal number of inverted repeats, and no- or potentially no coding sequences. The overall CG composition achieved using these sequences is near the DNA composition of the adenovirus type 5 (aprox. 55%). In this way we generated a backbone similar in DNA composition to wild type adenovirus 5, which would also minimize immune responses. To assses the performance of the HD plasmid an mCMV lacZ WRPE cassette was cloned nearby the E4 promoter. The HD plasmid containing the lacZ cassette achieved good level of expression (~50% of transfected cells) after transfection into 293 cells. To rescue the HD vector, we used the Flpe/frt system previously developed in our lab [1]. The vector system is now being characterized both in vitro and in the brain in vivo. Its performance, packaging efficiency, and expression levels will be compared to that obtained with HD vectors constructed using different available vector backbones. [1] Umana, P., Gerdes, C., Stone, D., Davis, J., Ward, D., Castro, M. and Lowenstein, P. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nat.Biotech.19:582-5, 2001.
498. A Chimeric Adenovirus Vector Expressing an Ovine Adenovirus Fiber Has CAR-Independent Tropism Joel N. Glasgow,1 Gerald W. Both,4 Victor N. Krasnykh,1,2,3 David T. Curiel.1,2,3 1 Division of Human Gene Therapy; 2Departments of Medicine, Pathology and Surgery; 3The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 4Molecular Science, Commonwealth Scientific and Industrial Research Organization, North Ryde, New South Wales, Australia. Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles, particularly in the context of cancer gene therapy. However, many clinically important tissues are refractory to Ad5 infection, including numerous cancer tissue types, due to reduced levels of the coxsackie and adenovirus receptor (CAR). Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Ovine adenovirus type 7, isolate 287 (OAd287), has recently been described as the prototype of the new genus Atadenovirus, which contains several animal Ads. OAd287 encodes a fiber molecule containing 25 repeating units, and has a unique C-terminal knob domain of 121 amino acids. OAd287 abortively infects several human, rodent and rabbit cell lines in vitro, and efficiently transduces murine cells in vivo. Unlike Ad5, however, systemically administered OAd-based vectors do not predominately localize to the liver in rodents, but are distributed more evenly among the major organs. Based on these observations, we hypothesized that replacement of the Ad5 fiber with the non-human OAD287 fiber would result in an Ad5 vector with a novel, CARindependent, tropism in vitro and liver “detargeting” in vivo. We constructed an E1-deleted, luciferase transgene Ad5 vector encoding a chimeric fiber from ovine adenovirus 287 that includes the Ad5 tail region (Ad5Luc1-OV), utilizing fiber-switching technology established in our laboratory. Viral particles were harvested from Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
911 cells, purified by standard cesium chloride gradient centrifugation and yielded titers in excess of 2x10e11 vp/mL. Characterization of Ad5Luc1-OV vector infectivity in CAR-deficient RD human rabdomyosarcoma cells and low-CAR MG118 human glioma cells showed 13-to-25 fold greater luciferase reporter gene activities versus the isogenic AdLuc1 control vector. Furthermore, competitive inhibitory knob blocking assays in high-CAR cells using Ad5 recombinant knob protein demonstrated these increases to be independent of CAR receptor binding. Herein, we have demonstrated the construction, rescue, purification, and initial infectivity characterization of a novel CAR-independent, infectivity enhanced chimeric fiber Ad vector. These data strongly suggest that our genetic xeno-fiber paradigm alters Ad vector tropism, a key gene therapy vector design principle.
499. Chemically Inactivated Adenoviral Vectors with Conditional and Targetable Infectivity: A Strategy for Safer Delivery of Adenoviral Vectors Mark W. Pandori,1 Takeshi Sano.1 Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States. 1
We have developed strategies for the creation of adenoviral vectors with conditional and targetable infectivity. Chemical modification (biotinylation) of the surface of adenoviral particles under controlled conditions resulted in nearly total ablation of the infectivity of the viral vectors. These modified viral vectors possessed a severely limited ability to infect even highly permissive target cells. However, when these modified viral vectors were attached to the surfaces of streptavidin-coated silica microbeads (specific gravity, 1.95 g/ml) and the resulting conjugates were delivered to target cells, the modified viral vectors showed very high infectivity, which was equal to or even greater than that of the parental unmodified adenoviral vectors used free in solution. This result suggests that the infectivity of delivered adenoviral particles can be controlled in a site-specific manner, because only those cells that are in contact with the adenovirus-microbead conjugates are transduced. The infection of target cells by these adenovirus-microbead conjugates was minimally affected by the presence of a monoclonal antibody against the coxsackie-adenovirus receptor, which effectively blocked infection by free, unmodified adenoviral vectors. Thus, these conjugates have a lesser dependence on the coxsackie-adenovirus receptor for infection of target cells. When the biotinylated, uninfectious adenoviral vectors were conjugated to the surfaces of streptavidincoated polystyrene microbeads (specific gravity, 1.05 g/ml) and the resulting conjugates were applied to target cells, the infectivity was not “rescued” as found with silica microbeads. However, when the biotinylated adenoviral vectors were attached to polystyrene microbeads, on which lectins were co-immobilized, the uninfectious adenoviral particles on the microbead surface regained their infectious capacity to the levels of the unmodified adenoviral vectors. This suggests that the ability of lectins on the microbead surface allows the conjugates to be brought into contact with target cells, resulting in the infection of the cells by viral particles on the microbeads. These methods of creating adenoviral vectors with conditional infectivity possess potential as strategies for safer, controlled delivery of viral vectors for gene therapy applications, since they allow for rigorous spatial control of tissue transduction by applied viral vectors. In particular, the almost completely ablated infectivity of these modified adenoviral vectors, when used free in solution, implies minimal uncontrolled transduction of non-target tissues in cases where viral particles are somehow released from the surface of the microbeads.
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498. A Chimeric Adenovirus Vector Expressing an Ovine Adenovirus Fiber Has CAR-Independent Tropism Joel N. Glasgow,1 Gerald W. Both,4 Victor N. Krasnykh,1,2,3 David T. Curiel.1,2,3 1 Division of Human Gene Therapy; 2Departments of Medicine, Pathology and Surgery; 3The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 4Molecular Science, Commonwealth Scientific and Industrial Research Organization, North Ryde, New South Wales, Australia. Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles, particularly in the context of cancer gene therapy. However, many clinically important tissues are refractory to Ad5 infection, including numerous cancer tissue types, due to reduced levels of the coxsackie and adenovirus receptor (CAR). Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Ovine adenovirus type 7, isolate 287 (OAd287), has recently been described as the prototype of the new genus Atadenovirus, which contains several animal Ads. OAd287 encodes a fiber molecule containing 25 repeating units, and has a unique C-terminal knob domain of 121 amino acids. OAd287 abortively infects several human, rodent and rabbit cell lines in vitro, and efficiently transduces murine cells in vivo. Unlike Ad5, however, systemically administered OAd-based vectors do not predominately localize to the liver in rodents, but are distributed more evenly among the major organs. Based on these observations, we hypothesized that replacement of the Ad5 fiber with the non-human OAD287 fiber would result in an Ad5 vector with a novel, CARindependent, tropism in vitro and liver “detargeting” in vivo. We constructed an E1-deleted, luciferase transgene Ad5 vector encoding a chimeric fiber from ovine adenovirus 287 that includes the Ad5 tail region (Ad5Luc1-OV), utilizing fiber-switching technology established in our laboratory. Viral particles were harvested from Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
911 cells, purified by standard cesium chloride gradient centrifugation and yielded titers in excess of 2x10e11 vp/mL. Characterization of Ad5Luc1-OV vector infectivity in CAR-deficient RD human rabdomyosarcoma cells and low-CAR MG118 human glioma cells showed 13-to-25 fold greater luciferase reporter gene activities versus the isogenic AdLuc1 control vector. Furthermore, competitive inhibitory knob blocking assays in high-CAR cells using Ad5 recombinant knob protein demonstrated these increases to be independent of CAR receptor binding. Herein, we have demonstrated the construction, rescue, purification, and initial infectivity characterization of a novel CAR-independent, infectivity enhanced chimeric fiber Ad vector. These data strongly suggest that our genetic xeno-fiber paradigm alters Ad vector tropism, a key gene therapy vector design principle.
499. Chemically Inactivated Adenoviral Vectors with Conditional and Targetable Infectivity: A Strategy for Safer Delivery of Adenoviral Vectors Mark W. Pandori,1 Takeshi Sano.1 Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States. 1
We have developed strategies for the creation of adenoviral vectors with conditional and targetable infectivity. Chemical modification (biotinylation) of the surface of adenoviral particles under controlled conditions resulted in nearly total ablation of the infectivity of the viral vectors. These modified viral vectors possessed a severely limited ability to infect even highly permissive target cells. However, when these modified viral vectors were attached to the surfaces of streptavidin-coated silica microbeads (specific gravity, 1.95 g/ml) and the resulting conjugates were delivered to target cells, the modified viral vectors showed very high infectivity, which was equal to or even greater than that of the parental unmodified adenoviral vectors used free in solution. This result suggests that the infectivity of delivered adenoviral particles can be controlled in a site-specific manner, because only those cells that are in contact with the adenovirus-microbead conjugates are transduced. The infection of target cells by these adenovirus-microbead conjugates was minimally affected by the presence of a monoclonal antibody against the coxsackie-adenovirus receptor, which effectively blocked infection by free, unmodified adenoviral vectors. Thus, these conjugates have a lesser dependence on the coxsackie-adenovirus receptor for infection of target cells. When the biotinylated, uninfectious adenoviral vectors were conjugated to the surfaces of streptavidincoated polystyrene microbeads (specific gravity, 1.05 g/ml) and the resulting conjugates were applied to target cells, the infectivity was not “rescued” as found with silica microbeads. However, when the biotinylated adenoviral vectors were attached to polystyrene microbeads, on which lectins were co-immobilized, the uninfectious adenoviral particles on the microbead surface regained their infectious capacity to the levels of the unmodified adenoviral vectors. This suggests that the ability of lectins on the microbead surface allows the conjugates to be brought into contact with target cells, resulting in the infection of the cells by viral particles on the microbeads. These methods of creating adenoviral vectors with conditional infectivity possess potential as strategies for safer, controlled delivery of viral vectors for gene therapy applications, since they allow for rigorous spatial control of tissue transduction by applied viral vectors. In particular, the almost completely ablated infectivity of these modified adenoviral vectors, when used free in solution, implies minimal uncontrolled transduction of non-target tissues in cases where viral particles are somehow released from the surface of the microbeads.
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