| 1. |
- Magnusson, Maria K, 1972, et al.
(författare)
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Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu.
- 2007
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Ingår i: Cancer gene therapy. - : Springer Science and Business Media LLC. - 0929-1903 .- 1476-5500. ; 14:5, s. 468-79
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Tidskriftsartikel (refereegranskat)abstract
- In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.
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| 2. |
- Myhre, Susanna, 1974, et al.
(författare)
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Decreased immune reactivity towards a knobless, affibody-targeted adenovirus type 5 vector
- 2007
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Ingår i: Gene Therapy. - : Springer Science and Business Media LLC. - 0969-7128 .- 1476-5462. ; 14:4, s. 376-381
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Tidskriftsartikel (refereegranskat)abstract
- In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (>= 1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.
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| 3. |
- Myhre, Susanna, 1974, et al.
(författare)
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Re-targeted adenovirus vectors with dual specificity; binding specificities conferred by two different Affibody molecules in the fiber.
- 2009
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Ingår i: Gene therapy. - : Springer Science and Business Media LLC. - 1476-5462 .- 0969-7128. ; 16:2, s. 252-61
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Tidskriftsartikel (refereegranskat)abstract
- Vectors based on Adenovirus type 5 (Ad5) are among the most common vectors in cancer gene therapy trials to date. However, for increased efficiency and safety, Ad5 should be de-targeted from its native receptors and re-targeted to a tumor antigen. We have described earlier an Ad5 vector genetically re-targeted to the tumor antigen HER2/neu by a dimeric version of the Affibody molecule ZH inserted in the HI-loop of the fiber knob of a coxsackie and adenovirus receptor-binding ablated fiber. This virus showed almost wild-type growth characteristics and infected cells through HER2/neu. Here we generate vectors with double specificity by incorporating two different Affibody molecules, ZH (HER2/neu-binding) and ZT (Taq polymerase-binding), at different positions relative to one another in the HI-loop. Receptor-binding studies together with viral production and gene transfer assays showed that the recombinant fiber with ZT in the first position and ZH in the second position (ZTZH) bound to both its targets, whereas surprisingly, the fiber with ZHZT was devoid of binding to HER2/neu. Hence, it is possible to construct a recombinant adenovirus with dual specificity after evaluating the best position for each ligand in the fiber knob.
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| 4. |
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| 5. |
- Myhre, Susanna, 1974
(författare)
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Genetic re-targeting and de-targeting of adenovirus type 5 in order to create vectors for gene therapy
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Gene therapy has been considered to be a revolutionary development in medicine, whereby the cause and not the symptoms of the disease would be treated. These expectations have as yet not been realized, mainly due to lack of suitable vectors. Adenoviruses type 5 (Ad5) are the most commonly used vectors for gene therapy and have a great potential in this field. The main aim of this thesis was to generate genetically re-targeted and de-targeted Ad5 in order to create suitable vectors for gene therapy. To re-target the Ad5 vector ligands have been incorporated into the C-terminus at the pIX protein and at different positions in the fiber protein. Ligands that have been used are affibody molecules, with specificity for Taq DNA polymerase and for the tumour antigen HER2/neu, and a hyperstable single chain antibody, scFv, directed against ?-galactosidase. In order to generate a virus with double specificity and determine which position is best suited for ligand?target cell interaction a re-targeted Ad5 vector with two different affibody molecules in the same genome was constructed. The re-targeted vectors were evaluated for growth, infectivity and specificity. For de-targeting assessments the neutralizing antibody reactivity in blood donor sera have been tested against a recombinant Ad5 vector with a shortened knobless fiber and a new cell binding ligand and a re-targeted vector with three different de-targeting steps have been evaluated for vector characterizations, tissue distribution and interaction with blood cells. Ligands that are to be used for re-targeting of Ad must be able to fold correctly and stably in the reducing milieu of the eukaryotic cytoplasm which is not conducive to the formation of disulphide bonds. Both the affibody molecules and the scFv did fulfill these criteria and could be rescued into functional Ad. Incorporation of ligands in the HI-loop of the fiber knob was shown to be superior to ligand insertions into truncated knobless fibers in terms of growth characteristics. It was possible to incorporate the scFv at the pIX protein with retained antigen binding when loaded on Ad5 virions, however transduction experiments could not be performed because a suitable cell line was not avalible. It was shown that generation of a vector with dual specificity was feasible, on the other hand it was important to evaluate which positions is best suited for efficient binding to target cell. The virus with a truncated knobless fiber and a new cellular ligand showed a much-reduced sensitivity to human pre-formed antibodies compared to wild type (WT) Ad5. The re- and de-targeted Ad vectors did not bind to normal tissues in mice as much as WT Ad5 and the association with human blood cells was much decreased for the recombinant vectors when compared to WT. In conclusion, both the affibody molecules and the scFv evaluated in this thesis can be used for genetic re-targeting of Ad5. Re-targeted viruses with ligands in the fiber often suffer from low growth rate, high infectivity indexes (PP/pfu) and low fiber content. However virus with ligand incorporation in the HI-loop does largely overcome those obstacles and the virus with ligand specificity for the tumour antigen HER2/neu may have relevance as a clinical vector. The adenovirus minor capsid protein IX can function as an anchor protein for relatively large ligand insertions, which is promising for future de-targeting purposes. Generation of recombinant viruses with double specificity may exploit the possibility to target several tumour antigens. The reduced neutralizing activity against the short knobless fiber and the reduced binding to normal tissues and interaction with blood cells of the re-targeted viruses with additional de-targeting steps represents, to this author, an important step towards the construction of ?stealth? adenoviruses for gene therapy.
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| 6. |
- Olofsson, Sven-Olof, 1947, et al.
(författare)
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The formation of lipid droplets: possible role in the development of insulin resistance/type 2 diabetes.
- 2011
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Ingår i: Prostaglandins, leukotrienes, and essential fatty acids. - : Elsevier BV. - 1532-2823 .- 0952-3278. ; 85:5, s. 215-8
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Tidskriftsartikel (refereegranskat)abstract
- Neutral lipids are stored in so-called lipid droplets, which are formed as small primordial droplets at microsomal membranes and increase in size by a fusion process. The fusion is catalyzed by the SNARE proteins SNAP23, syntaxin-5 and VAMP4. SNAP23 is involved in the insulin dependent translocation of GLUT4 to the plasma membrane, and has an important role in the development of insulin resistance. Thus fatty acids relocalize SNAP23 from the plasma membrane (and the translocation of GLUT 4) to the interior of the cell giving rise to insulin resistance. Moreover this relocalization is seen in skeletal muscles biopsies from patients with type 2 diabetes compared to matched control. Thus a missorting of SNAP23 is essential for the development of insulin resistance.
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| 7. |
- Vellinga, J, et al.
(författare)
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Efficient incorporation of a functional hyper-stable single-chain antibody fragment protein-IX fusion in the adenovirus capsid.
- 2007
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Ingår i: Gene therapy. - : Springer Science and Business Media LLC. - 0969-7128 .- 1476-5462. ; 14:8, s. 664-70
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant adenoviruses are frequently used as gene transfer vehicles for therapeutic gene delivery. Strategies to amend their tropism include the incorporation of polypeptides with high affinity for cellular receptors. Single-chain antibodies have a great potential to achieve such cell type specificity. In this study, we evaluated the efficiency of incorporation of a single-chain antibody fused with the adenovirus minor capsid protein IX in the capsid of adenovirus type 5 vectors. To this end, the codons for the single-chain antibody fragments (scFv) 13R4 were fused with those encoding of pIX via a 75-Angstrom spacer sequence. The 13R4 is a hyper-stable single-chain antibody directed against beta-galactosidase, which was selected for its capacity to fold correctly in a reducing environment such as the cytoplasm. A lentiviral vector was used to stably express the pIX.flag.75.13R4.MYC.HIS fusion gene in 911 helper cells. Upon propagation of pIX-gene deleted human adenovirus-5 vectors on these cells, the pIX-fusion protein was efficiently incorporated in the capsid. Here, the 13R4 scFv was functional as was evident from its capacity to bind its ligand beta-galactosidase. These data demonstrate that the minor capsid protein IX can be used as an anchor for incorporation of single-chain antibodies in the capsids of adenovirus vectors.
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