| 1. |
- Lindholm, L, et al.
(author)
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Genetic re-targeting of adenovirus using a hyperstable scFv domain and an affibody (R) molecule against Her2/neu
- 2004
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In: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 9, s. S250-S250
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Journal article (peer-reviewed)abstract
- One important goal in gene therapy is to develop adenovirus (Ad) vectors that are genetically de-targeted as well as re-targeted. Genetic re-targeting of Ad using complex cell-binding ligands has previously not been possible. We have previously demonstrated that ligands for genetic re-targeting of adenoviruses must be able to fold correctly in the cytoplasm of virus producing cells, a milieu that is not conducive to the formation of disulphide bonds. Here, we describe functional Ad5 viruses with fibers and pIX capsid proteins genetically modified to contain two types of complex ligands. One is affibody® molecules, corresponding to small (6 kDa) binding proteins developed by combinatorial protein engineering using a single three-helix bundle staphylococcal protein A domain. The other type is hyperstable antibody scFv domains. The affibody molecule used here (ZH2N) is directed against Her2/neu. Recombinant viruses were constructed with ZH2N in three different positions: (i) at the C-terminus of shaft repeat 7 of de-knobbed fibers; (ii) at the C-terminus of pIX; and (iii) in the HI-loop of the fiber knob. Each of the viruses exhibited a characteristic phenotype regarding fiber content, growth and ability to infect Her2/neu expressing cells. In order to test the potentials of scFv liganded Ad vectors, a hyperstable antibody scFv against b-galactosidase was genetically incorporated into knobless fibers, in tandem with a mutated protein A domain reactive with IgG1 Fc that targeted the virus to Fc-expressing 293 cells. These fibers could be rescued into viable virions that retained the original antigen binding specificity of the scFv, demonstrating the basic potential of hyperstable scFvs for genetic re-targeting of Ad. Quite unexpectedly, the fiber content of Ad with knobless, scFv containing fibers was close to normal in contrast to other Ad with knobless fibers that generally has a much reduced fiber content. The hyperstable scFv was further fused to the C-terminus of the capsid protein pIX. The recombinant molecules could be rescued into viable viruses with wt fibers. The scFv retained its binding-specificity on the recombinant virions. The results demonstrate that, contrary to current beliefs, it is possible to construct Ad that genetically incorporates functional scFvs and other complex ligands into the virus fiber and pIX. The feasibility is demonstrated by the creation of different viruses that are re-targeted to Her2/neu. These viruses are currently in pre-clinical development.
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| 2. |
- Henning, Petra, 1974, et al.
(author)
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Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from staphylococcal protein A.
- 2002
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In: Human gene therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 13:12, s. 1427-39
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Journal article (peer-reviewed)abstract
- The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.
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| 3. |
- Hong, Saw See, et al.
(author)
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Adenovirus stripping: a versatile method to generate adenovirus vectors with new cell target specificity.
- 2003
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In: Molecular therapy : the journal of the American Society of Gene Therapy. - 1525-0016. ; 7:5 Pt 1, s. 692-9
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Journal article (peer-reviewed)abstract
- We developed a new type of adenovirus type 5 (Ad5)-derived vector with genetically modified fiber proteins whose knob domains could be stripped off due to the insertion of a single Factor Xa cleavage site in the fiber shaft, between a cellular ligand and the knob domain. This Ad vector did not require a specific cell line for propagation and could be grown in HEK-293 cells. Stripping off the knob domains removed the endogenous cell-binding moiety of Ad but retained the new cell ligand for retargeting purposes. As experimental models for cell ligands, we used two peptides with different sequence complexities: (i) the integrin-binding tripeptide RGD and (ii) a 58-residue oligopeptide termed affibody (Zwt). Zwt binds specifically to the human IgG1 Fc domain or to its Fc3(1) homolog. The modified fibers were efficiently encapsidated into virions, and the Factor Xa sites were fully accessible to proteolysis. In vitro binding assays using recombinant Fc3(1) protein and Ad5-mediated gene transduction of Fc3(1)-expressing cells demonstrated that the proteolytically deknobbed Ad5-Zwt vector was functional and specific for receptor targeting.
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| 4. |
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| 5. |
- Magnusson, Maria K, 1972
(author)
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Genetic re-targeting of adenoviral vectors for gene therapy applications
- 2002
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Doctoral thesis (other academic/artistic)abstract
- The adenovirus (Ad) is a frequent cause of upper respiratory tract infections, enteritis and conjunctivitis. Since its first isolation in 1953, the study of Ad has contributed greatly to the understanding of intracellular events and has also served as a model system for many viruses. Today, Ad is one of the most commonly used vectors for gene therapy and is considered to have considerable potential within this field. The main aim of this thesis was to develop a technology, based on genetic engineering of the virus fiber, to create genetically re-targeted adenovirus type 5 (Ad5) constructs that could be used as in vivo gene therapy vectors. To achieve this aim, the knob and the 15 terminal shaft repeats of the virus fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand. Different polypeptide ligands for cell-surface molecules, such as single-chain antibodies, epidermal growth factor and affibody-like molecules (see below) were cloned into the recombinant fibers. Phenotypic analysis of the fiber constructs was performed, and the ability of Ad5 to encapsidate the new ligand-bound fibers to yield functional re-targeted viruses was studied. Recombinant viruses were characterized with regard to cell specificity, fiber content, growth rate and infectivity. Recombinant fibers retained the basic biological functions of the native fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding, on the condition that the inserted ligand folds correctly within the cellular cytoplasm. Correct cytoplasmic folding of the ligand is the major limiting factor in the ability of recombinant fibers to be rescued into viable recombinant Ad5. Therefore, ligands that depend on disulphide bond formation for stable folding are not functional within this context, since the reducing milieu in the cytoplasm is not conducive to the formation of disulphide bonds. We used a new class of cell-surface-binding ligands, which is based on a triple a-helix framework from one of the Ig-binding domains of staphylococcal protein A. This Ig-binding domain folds correctly in the cytosol and can be mutated at certain positions so that it retains its a-helical framework. Ligands that are generated by this technique have new specificities so-called affibodies, and are well suited to the construction of Ads with novel tropisms. It has been demonstrated that recombinant viruses expressing affibody-like ligands or the RGD motif infected cells through binding of the new ligand to cellular receptors and had lost the binding properties of the native Ad5 that were associated with the fiber knob. It was also shown that viruses with manipulated fibers had diminished growth rates and infectivity and had reduced fiber content. The reasons for these defects have not been fully elucidated.The results of the work described in this thesis show that it is possible to generate viable, recombinant, knobless Ad5 with new cellular specificities by deleting the fiber knob and replacing it with a new cell-binding ligand and a non-native trimerization motif. Previously, a major constraint on the ligands that could be used for genetic re-targeting of Ad was the ability to fold correctly in the cellular cytoplasm. We have identified a new class of ligands that meet this criterion. Fundamental techniques for the construction of genetically re-targeted Ads for human gene therapy are described. However, recombinant viruses that have been generated according to the above strategy are less infectious and contain fewer fibers per virion compared to wild-type Ad5. It will be necessary to optimize the technology to overcome these limitations before recombinant targeted viruses can be applied to human gene therapy.
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| 6. |
- Magnusson, Maria K, 1972, et al.
(author)
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Genetic retargeting of adenovirus: novel strategy employing "deknobbing" of the fiber.
- 2001
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In: Journal of virology. - 0022-538X. ; 75:16, s. 7280-9
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Journal article (peer-reviewed)abstract
- For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.
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