- Email: [email protected]
125. Generation of an Adenovirus Vector with the Reovirus Attachment Protein Sigma1 for Novel Tropism
TARGETING AD VECTORS AND HOST RESPONSE cells. The analysis of Ad5ΔRGD and Ad5/35LΔRGD infectivity (at high MOIs) on HeLa, A549 and MO7e cells demonstrated that they transduced cells with equal efficiencies, as compared to Ad5 and Ad5/35L. However, the rate of internalization was significantly lower for RGD motif-deleted vectors, compared to RGD-motif containing variants. By analyzing Ad5ΔRGD and Ad5/35LΔRGD infectivity at low MOIs using plaque forming assays on AE25 cells (A549 derivative cell line expressing Ad5 E1 proteins), we found that the infectivity of RGD-motif deleted vectors was more than 10 times lower compared to vectors containing intact an RGD motif, when vectors were allowed to internalize for 5 min only. Moreover, both the rates of internalization and endosome escape (assessed by virus neutralization with ammonium chloride) were significantly decreased for Ad5ΔRGD and Ad5/35LΔRGD vectors. In conclusion, our data demonstrate that for both Ad5 and capsid-modified Ad5/ 35L–based vectors, the interaction with cellular integrins are important for virus internalization. The penton base RGD motif cellular integrin interaction plays an essential role in capsid-modified virus escape from the endosomal environment, and is required for a high infection efficiency.
124. Characterization of a Chimeric Xeno-Knob Adenovirus Vector for Novel Tropism Joel N. Glasgow,1 Victor N. Krasnykh,1,2,3 Akseli Hemminki,4 Eric J. Kremer,5 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, United States; 4 Rational Drug Design Program, Department of Oncology, University of Helsinki, Helsinki, Finland; 5Institut de Genetique Moleculaire de Montpellier, Montpellier, France. 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. Early gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Hence, a fundamental requirement for the achievement of somatic cell gene therapy is the exploitation of advanced molecular biology and cellular physiology to design targetable gene delivery vectors. Based on a clear understanding of native Ad cell recognition, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism. Many clinically relevant tissues are refractory to Ad5 infection, including several cancer tissue types, due to reduced CAR levels. Thus, development of additional chimeric-knob Ad vectors could lead to discovery of novel CAR-independent tropism leading directly to therapeutic gain. In this regard, we hypothesized that replacement of the Ad5 knob with Ad knobs of non-human origin (xeno-knobs) would provide a paradigm of novel tropism discovery. Many xeno-Ads have been characterized to date, including several murine, bovine, canine, porcine and ovine serotypes. Other than the canine Ad type 2 (CAd2), cell entry of these xeno-Ads is CAR-independent. We constructed an E1-deleted, luciferase transgene Ad5 vector encoding the knob domain from the canine adenovirus type 2 (Ad5Luc1CK), utilizing knob-switching technology established in our laboratory. Viral particles were harvested and purified by standard procedures and yielded titers in excess of 2x10e11 vp/mL. Characterization of Ad5Luc1-CK vector infectivity in CAR-deficient RD rabdomyosarcoma cells and MG118 glioma cells showed 15-to21 fold greater luciferase reporter gene activities versus the isogenic AdLuc1 control vector, suggesting CAR-independent entry biology. Indeed, competitive inhibitory knob blocking assays using Ad5 and Ad3 recombinant knob proteins demonstrated these increases to be independent of CAR or Ad3 receptor binding. Further, FACS Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
analysis of Ad5Luc1-CK binding to U118MG cells showed dramatically increased cell-surface binding versus Ad5Luc1; functionally linking increased Ad5Luc1-CK transgene expression to CAR-independent cell surface interaction. Herein, we have demonstrated the construction, rescue, purification, and tropism characterization of a novel xeno-knob chimeric Ad vector. The activity strongly suggests that our genetic xeno-knob paradigm alters Ad vector tropism, a key gene therapy vector design principle.
125. Generation of an Adenovirus Vector with the Reovirus Attachment Protein Sigma1 for Novel Tropism Yuko Tsuruta,1 Joel N. Glasgow,1 Victor N. Krasnykh,1,2,3 Cindy L. Luongo,4 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; 4Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL. 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. Early gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Many clinically relevant tissues are refractory to Ad5 infection, including several cancer tissue types, due to negligible CAR levels. Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Based on a clear understanding of native Ad cell recognition, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism. Our group previously created an Ad5 vector expressing a chimeric fiber displaying the Ad serotype 3 knob domain (Ad5/3) that demonstrated the same coxsackie and adenovirus receptor (CAR) -independent cell recognition as does Ad3. This vector provided ample gene transfer into cancer cells previously refractory to Ad5 infection, linking rational genetic Ad modification to functional Ad retargeting. Serotype three reoviruses (T3) utilize a fiber-like attachment protein, Sigma1, to infect human cells. The Sigma1 molecule is comprised of an N-terminal tail region of alpha helix and eight beta-repeats, and a C-terminal globular head domain. Sigma1 is known to contain two receptor-binding domains (RBDs): one within the fibrous tail that binds sialic acid, and the other in the globular head that binds to the junction adhesion molecule (JAM). The head RBD plays a key role in determining the tropism of T3 reoviruses, which have CAR-independent tropism. Based on these observations, we hypothesized that replacement of the Ad5 fiber with reovirus attachment protein Sigma1, a fiber-like molecule, would result in an Ad5 vector with a novel, CAR-independent tropism, and would provide a paradigm of tropism discovery. We constructed E1-deleted, luciferase transgene Ad5 vectors encoding a chimeric fiber comprised of the tail domain of Ad5 fiber fused to the T3 reovirus Sigma1 alone or co-expressed with the wild-type Ad5 fiber. Following sequencing, recombinant Sigma1-Ad5 tail genomes with or without wild type Ad5 fiber were transfected into 293 cells. Western Blot analysis confirmed expression of the Sigma1-Ad5 tail chimeric fiber as a trimer. Ad genomes with the chimeric fiber, with or without wild type Ad5 fiber, replicated in 293 cells. Rescue, purification and characterization of these chimeric fiber- containing Ad5 vectors is ongoing. Herein, we have demonstrated the construction of a Sigma1 chimeric fiber containing Ad5 genome in addition to a mosaic genome that also incorporates the wild-type fiber. Furthermore, we have shown that the chimeric fiber is capable of trimerization, a critical biophysical outcome. By addressing a key issue that limits the S49
TARGETING AD VECTORS AND HOST RESPONSE infectivity of Ad5 in gene therapy strategies, the development of such a vector system would promise its utilization in future clinical trials, particularly for cancer gene therapy.
127. Mosaic Adenoviruses Expressing Two Different Fiber Types: Increased Possibilities for Improved Infection Efficiency and Specificity
126. Genetic Targeting of Adenoviral Vectors with Knobless Fibers for Tumor-Specific Gene Transfer
Marianne G. Rots,1 Igor P. Dmitriev,2 Willemijn M. Gommans,1 Elena A. Kashentseva,2 David T. Curiel,2 Hidde J. Haisma.1 1 Therapeutic Gene Modulation, University Center for Pharmacy, Groningen, Netherlands; 2Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL.
Frederik H. E. Schagen,1 Jan E. Carette,1 Petra D. van den Doel,1 Winald R. Gerritsen,1 Victor W. van Beusechem.1 1 Medical Oncology, VU University Medical Center, Amsterdam, Netherlands. Tumor-specific infection by recombinant adenoviruses seems elementary for cancer gene therapy, in terms of efficacy, toxicity and safety. Nowadays, several strategies are available to target adenoviral vectors (AdV) or conditionally replicative adenoviruses (CRAds) to the cell type of choice. In this regard, methods that combine ablation of native tropism with addition of target specificity can be considered as most promising. Our strategy to accomplish this is based on replacement of the fiber knob by an alpha-helical coiled-coil motif of the Mo-MuLV envelope glycoprotein, which compensates for the loss of trimerisation caused by the removal of the knob domain. The required target specificity is introduced through attachment of targeting ligands at the carboxy-terminus of the knobless fiber molecule. As we reported previously, the resulting chimeric knobless fiber is incorporated in the viral capsid of mosaic virions and, in this context, mediates targeted infection (van Beusechem et al., Gene Ther. 2000; 7: 1940). The subsequent move towards a truly targeted virus required deletion of the wild type fiber from the adenoviral genome. To that end, and to ensure correct timing and extent of knobless fiber expression, we replaced the endogenous fiber gene by knobless counterparts with targeting peptides for integrins or the Epithelial Cell Adhesion Molecule. These truly targeted vectors could only be propagated on adenovirus packaging-cells if wild type fiber was complemented during production. This suggested an important role for the fiber knob during adenovirus replication and demanded restoration of the lost function to regain efficient virus propagation. The latter is of particular importance for application of this targeting strategy in CRAds, since the oncolytic effect of this promising class of anticancer agents depends on efficient replication. Towards establishing this without compromising target specificity, we evaluated the role of the fiber knob domain in adenovirus propagation. To this end, we constructed a recombinant knob molecule carrying the SV40 large T nuclear localization signal (NLS), to compensate for the lost nuclear localization function normally residing in the fiber tail domain. Proper nuclear import of NLS-knob was confirmed by immunocytochemical analysis. To test if NLS-knob could support AdV propagation, it was used to complement the production of the fiberless Ad5.βgal.ΔF in trans (Von Seggern et al. J. Virol. 1999, 73: 1601). Expression of NLS-knob did not improve the production of infectious Ad5.βgal.ΔF compared to non-complemented controls, whereas complementation with wild type fiber did enhance production. This finding suggests that the knob in itself is not sufficient to rescue efficient production of the fiberless AdV. Further studies are needed to clarify the cause of the hampered propagation of AdV with knobless fibers.
Low efficiency of gene transfer remains one of the main problems in cancer gene therapy. A promising approach in solving this problem is by introducing restricted replication characteristics allowing the virus to multiply only within the tumor mass, thus killing cancer cells. Unfortunately, also in this approach the infection efficiency of (2nd or later round) infection is limited by expression of the primary receptor for adenoviruses on the tumor cells. It has been shown for several (primary) tumor cells that the receptor for adenovirus type 3 (Ad3) is favorably expressed over the Ad5 receptor. To circumvent the variable expression of the receptors reported for primary tumor cells, we broadened the infection spectrum by constructing an adenovirus expressing two different types of fibers. One fiber is the wildtype Ad5 fiber, the second type consists of the tail of Ad5 and the shaft and the knob of Ad3. Both fibers are under the control of the major late promoter. In the E1 region, the genes encoding for luciferase and green fluorescent protein were cloned. We could successfully rescue the recombinant mosaic viruses and western blot demonstrated the expression of the two different fiber types. Subsequently, we blocked infection through Ad3 (or Ad5) receptor using recombinant adenoviral knob 3 (or knob 5) protein on cell lines expressing differential levels of Ad3 and Ad5 receptors. Furthermore, the infectivity efficiency was tested on cell lines expressing low levels of Ad5 receptor and high levels of the Ad3 receptor (head and neck-, ovarium and glioma cancer cell lines). The infection by the mosaic virus was similar or better compared to the control Ad5 virus. We conclude that construction of a mosaic virus expressing two distinct types of fibers is feasible and provides a way to increase infection efficiency while allowing targeting devices to introduce specificity. Especially in combination with the conditional replication, these characteristic might prove very efficient.
128. RGD Modification in the HI Loop of the Fiber Knob Diminishes Immunogenicity of Type 5 Adenoviral Vectors Minghui Wang, Akseli Hemminki, Ronald D. Alvarez, Mack N. Barnes, Gene P. Siegal, Victor Krasnykh, David T. Curiel. 1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 2Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 3Division of Gynecological Ongology, University of Alabama at Birmingham, Birmingham, AL, United States; 4Division of Gynecological Ongology, University of Alabama at Birmingham, Birmingham, AL, United States; 5Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, United States; 6Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 7Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States. Host immune response is a major safety issue in the context of adenoviral mediated human gene therapy. Various approaches including manipulation of vector design have been endeavored to decrease the host immune response to adenoviral vectors. We have developed a type 5 adenovirus with an RGD modification in the HI loop of the fiber knob. Previous work demonstrated this RGD
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Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
124. Characterization of a Chimeric Xeno-Knob Adenovirus Vector for Novel Tropism Joel N. Glasgow,1 Victor N. Krasnykh,1,2,3 Akseli Hemminki,4 Eric J. Kremer,5 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, United States; 4 Rational Drug Design Program, Department of Oncology, University of Helsinki, Helsinki, Finland; 5Institut de Genetique Moleculaire de Montpellier, Montpellier, France. 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. Early gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Hence, a fundamental requirement for the achievement of somatic cell gene therapy is the exploitation of advanced molecular biology and cellular physiology to design targetable gene delivery vectors. Based on a clear understanding of native Ad cell recognition, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism. Many clinically relevant tissues are refractory to Ad5 infection, including several cancer tissue types, due to reduced CAR levels. Thus, development of additional chimeric-knob Ad vectors could lead to discovery of novel CAR-independent tropism leading directly to therapeutic gain. In this regard, we hypothesized that replacement of the Ad5 knob with Ad knobs of non-human origin (xeno-knobs) would provide a paradigm of novel tropism discovery. Many xeno-Ads have been characterized to date, including several murine, bovine, canine, porcine and ovine serotypes. Other than the canine Ad type 2 (CAd2), cell entry of these xeno-Ads is CAR-independent. We constructed an E1-deleted, luciferase transgene Ad5 vector encoding the knob domain from the canine adenovirus type 2 (Ad5Luc1CK), utilizing knob-switching technology established in our laboratory. Viral particles were harvested and purified by standard procedures and yielded titers in excess of 2x10e11 vp/mL. Characterization of Ad5Luc1-CK vector infectivity in CAR-deficient RD rabdomyosarcoma cells and MG118 glioma cells showed 15-to21 fold greater luciferase reporter gene activities versus the isogenic AdLuc1 control vector, suggesting CAR-independent entry biology. Indeed, competitive inhibitory knob blocking assays using Ad5 and Ad3 recombinant knob proteins demonstrated these increases to be independent of CAR or Ad3 receptor binding. Further, FACS Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
analysis of Ad5Luc1-CK binding to U118MG cells showed dramatically increased cell-surface binding versus Ad5Luc1; functionally linking increased Ad5Luc1-CK transgene expression to CAR-independent cell surface interaction. Herein, we have demonstrated the construction, rescue, purification, and tropism characterization of a novel xeno-knob chimeric Ad vector. The activity strongly suggests that our genetic xeno-knob paradigm alters Ad vector tropism, a key gene therapy vector design principle.
125. Generation of an Adenovirus Vector with the Reovirus Attachment Protein Sigma1 for Novel Tropism Yuko Tsuruta,1 Joel N. Glasgow,1 Victor N. Krasnykh,1,2,3 Cindy L. Luongo,4 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; 4Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL. 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. Early gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Many clinically relevant tissues are refractory to Ad5 infection, including several cancer tissue types, due to negligible CAR levels. Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Based on a clear understanding of native Ad cell recognition, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism. Our group previously created an Ad5 vector expressing a chimeric fiber displaying the Ad serotype 3 knob domain (Ad5/3) that demonstrated the same coxsackie and adenovirus receptor (CAR) -independent cell recognition as does Ad3. This vector provided ample gene transfer into cancer cells previously refractory to Ad5 infection, linking rational genetic Ad modification to functional Ad retargeting. Serotype three reoviruses (T3) utilize a fiber-like attachment protein, Sigma1, to infect human cells. The Sigma1 molecule is comprised of an N-terminal tail region of alpha helix and eight beta-repeats, and a C-terminal globular head domain. Sigma1 is known to contain two receptor-binding domains (RBDs): one within the fibrous tail that binds sialic acid, and the other in the globular head that binds to the junction adhesion molecule (JAM). The head RBD plays a key role in determining the tropism of T3 reoviruses, which have CAR-independent tropism. Based on these observations, we hypothesized that replacement of the Ad5 fiber with reovirus attachment protein Sigma1, a fiber-like molecule, would result in an Ad5 vector with a novel, CAR-independent tropism, and would provide a paradigm of tropism discovery. We constructed E1-deleted, luciferase transgene Ad5 vectors encoding a chimeric fiber comprised of the tail domain of Ad5 fiber fused to the T3 reovirus Sigma1 alone or co-expressed with the wild-type Ad5 fiber. Following sequencing, recombinant Sigma1-Ad5 tail genomes with or without wild type Ad5 fiber were transfected into 293 cells. Western Blot analysis confirmed expression of the Sigma1-Ad5 tail chimeric fiber as a trimer. Ad genomes with the chimeric fiber, with or without wild type Ad5 fiber, replicated in 293 cells. Rescue, purification and characterization of these chimeric fiber- containing Ad5 vectors is ongoing. Herein, we have demonstrated the construction of a Sigma1 chimeric fiber containing Ad5 genome in addition to a mosaic genome that also incorporates the wild-type fiber. Furthermore, we have shown that the chimeric fiber is capable of trimerization, a critical biophysical outcome. By addressing a key issue that limits the S49
TARGETING AD VECTORS AND HOST RESPONSE infectivity of Ad5 in gene therapy strategies, the development of such a vector system would promise its utilization in future clinical trials, particularly for cancer gene therapy.
127. Mosaic Adenoviruses Expressing Two Different Fiber Types: Increased Possibilities for Improved Infection Efficiency and Specificity
126. Genetic Targeting of Adenoviral Vectors with Knobless Fibers for Tumor-Specific Gene Transfer
Marianne G. Rots,1 Igor P. Dmitriev,2 Willemijn M. Gommans,1 Elena A. Kashentseva,2 David T. Curiel,2 Hidde J. Haisma.1 1 Therapeutic Gene Modulation, University Center for Pharmacy, Groningen, Netherlands; 2Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL.
Frederik H. E. Schagen,1 Jan E. Carette,1 Petra D. van den Doel,1 Winald R. Gerritsen,1 Victor W. van Beusechem.1 1 Medical Oncology, VU University Medical Center, Amsterdam, Netherlands. Tumor-specific infection by recombinant adenoviruses seems elementary for cancer gene therapy, in terms of efficacy, toxicity and safety. Nowadays, several strategies are available to target adenoviral vectors (AdV) or conditionally replicative adenoviruses (CRAds) to the cell type of choice. In this regard, methods that combine ablation of native tropism with addition of target specificity can be considered as most promising. Our strategy to accomplish this is based on replacement of the fiber knob by an alpha-helical coiled-coil motif of the Mo-MuLV envelope glycoprotein, which compensates for the loss of trimerisation caused by the removal of the knob domain. The required target specificity is introduced through attachment of targeting ligands at the carboxy-terminus of the knobless fiber molecule. As we reported previously, the resulting chimeric knobless fiber is incorporated in the viral capsid of mosaic virions and, in this context, mediates targeted infection (van Beusechem et al., Gene Ther. 2000; 7: 1940). The subsequent move towards a truly targeted virus required deletion of the wild type fiber from the adenoviral genome. To that end, and to ensure correct timing and extent of knobless fiber expression, we replaced the endogenous fiber gene by knobless counterparts with targeting peptides for integrins or the Epithelial Cell Adhesion Molecule. These truly targeted vectors could only be propagated on adenovirus packaging-cells if wild type fiber was complemented during production. This suggested an important role for the fiber knob during adenovirus replication and demanded restoration of the lost function to regain efficient virus propagation. The latter is of particular importance for application of this targeting strategy in CRAds, since the oncolytic effect of this promising class of anticancer agents depends on efficient replication. Towards establishing this without compromising target specificity, we evaluated the role of the fiber knob domain in adenovirus propagation. To this end, we constructed a recombinant knob molecule carrying the SV40 large T nuclear localization signal (NLS), to compensate for the lost nuclear localization function normally residing in the fiber tail domain. Proper nuclear import of NLS-knob was confirmed by immunocytochemical analysis. To test if NLS-knob could support AdV propagation, it was used to complement the production of the fiberless Ad5.βgal.ΔF in trans (Von Seggern et al. J. Virol. 1999, 73: 1601). Expression of NLS-knob did not improve the production of infectious Ad5.βgal.ΔF compared to non-complemented controls, whereas complementation with wild type fiber did enhance production. This finding suggests that the knob in itself is not sufficient to rescue efficient production of the fiberless AdV. Further studies are needed to clarify the cause of the hampered propagation of AdV with knobless fibers.
Low efficiency of gene transfer remains one of the main problems in cancer gene therapy. A promising approach in solving this problem is by introducing restricted replication characteristics allowing the virus to multiply only within the tumor mass, thus killing cancer cells. Unfortunately, also in this approach the infection efficiency of (2nd or later round) infection is limited by expression of the primary receptor for adenoviruses on the tumor cells. It has been shown for several (primary) tumor cells that the receptor for adenovirus type 3 (Ad3) is favorably expressed over the Ad5 receptor. To circumvent the variable expression of the receptors reported for primary tumor cells, we broadened the infection spectrum by constructing an adenovirus expressing two different types of fibers. One fiber is the wildtype Ad5 fiber, the second type consists of the tail of Ad5 and the shaft and the knob of Ad3. Both fibers are under the control of the major late promoter. In the E1 region, the genes encoding for luciferase and green fluorescent protein were cloned. We could successfully rescue the recombinant mosaic viruses and western blot demonstrated the expression of the two different fiber types. Subsequently, we blocked infection through Ad3 (or Ad5) receptor using recombinant adenoviral knob 3 (or knob 5) protein on cell lines expressing differential levels of Ad3 and Ad5 receptors. Furthermore, the infectivity efficiency was tested on cell lines expressing low levels of Ad5 receptor and high levels of the Ad3 receptor (head and neck-, ovarium and glioma cancer cell lines). The infection by the mosaic virus was similar or better compared to the control Ad5 virus. We conclude that construction of a mosaic virus expressing two distinct types of fibers is feasible and provides a way to increase infection efficiency while allowing targeting devices to introduce specificity. Especially in combination with the conditional replication, these characteristic might prove very efficient.
128. RGD Modification in the HI Loop of the Fiber Knob Diminishes Immunogenicity of Type 5 Adenoviral Vectors Minghui Wang, Akseli Hemminki, Ronald D. Alvarez, Mack N. Barnes, Gene P. Siegal, Victor Krasnykh, David T. Curiel. 1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 2Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 3Division of Gynecological Ongology, University of Alabama at Birmingham, Birmingham, AL, United States; 4Division of Gynecological Ongology, University of Alabama at Birmingham, Birmingham, AL, United States; 5Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, United States; 6Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 7Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States. Host immune response is a major safety issue in the context of adenoviral mediated human gene therapy. Various approaches including manipulation of vector design have been endeavored to decrease the host immune response to adenoviral vectors. We have developed a type 5 adenovirus with an RGD modification in the HI loop of the fiber knob. Previous work demonstrated this RGD
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Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy