A multi-domain protein for β1 integrin-targeted DNA delivery

Fortunati, Elisabetta; Ehlert, Ehrich M. E.; Loo, Nicki; Wyman, Claire; Eble, Johannes A.; Grosveld, Frank; Scholte, Bob J.

doi:10.1038/sj.gt.3301258

Cited by 22 publications

(11 citation statements)

References 38 publications

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“…[5][6][7][8][9] However, current gene therapy techniques have the need for vehicles that can provide selective, efficient and targeted gene delivery in a nontoxic, noninflammatory and nonimmunogenic manner. [10][11][12][13][14][15][16][17][18][19] Our vehicle for plasmid DNA transfection is a commercially available two component fibrin glue, which is known to be well degradated by the organism, not immunogenic, and causing no inflammatory response to the tissue.…”

Section: Introductionmentioning

confidence: 99%

Plasmid Gene Delivery to Human Keratinocytes Through a Fibrin-Mediated Transfection System

et al. 2001

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We have developed a matrix-mediated transfection system to deliver plasmids to human keratinocytes. The matrix is a soluble, self-hardening fibrin matrix (Tissucol), Baxter) that has been used clinically. Recently it has been shown that full thickness burn wounds can be successfully treated with a keratinocyte fibrin glue suspension. Further, it has been demonstrated that hEGF transfected cells accelerate wound healing. In this study, we inoculated the matrix with the hEGF expression plasmid and resuspended the matrix with either cultured or noncultured human keratinocytes. We obtained successful transfection rates of these cells (up to a 100-fold increase compared to controls containing no EGF expression plasmid) in vitro. After transplantation to full thickness wounds on athymic mice we were able to show a 180-fold increase in EGF concentration compared to controls, which persisted over the entire 7-day monitored period, decreasing from 180 to 20 pg/mL at day seven. This unique approach indicates the possible utility to combine a matrix for cell transplantation with a transfection system to release therapeutic proteins in vitro and in vivo.

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Section: Introductionmentioning

confidence: 99%

Plasmid Gene Delivery to Human Keratinocytes Through a Fibrin-Mediated Transfection System

et al. 2001

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“…This has important consequences for the engineering of tissue supports that, to date, generally involves RGD conjugation to polymers in a random distribution or non-specific adsorption of collagen or fibronectin (Rowley et al 1999;Tan et al 2001;Yang et al 2001). Similarly, recombinant proteins and polypeptides binding integrin for targeted gene delivery via receptor-mediated endocytosis have focussed on monovalent RGD and invasin ligands, extended by cationic polypeptides binding DNA (Harbottle et al 1998;Fortunati et al 2000;Kunath et al 2003). As our understanding of integrin-ligand binding advances, it would appear timely to explore alternative polypeptide templates facilitating a clustered organisation of RGD ligands.…”

Section: Introductionmentioning

confidence: 99%

A tetravalent RGD ligand for integrin-mediated cell adhesion

Watson

Duncan

Annan

et al. 2006

Journal of Pharmacy and Pharmacology

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Monovalent RGD (arginine-glycine-aspartic acid) peptides or polymers furnished with RGD in random distributions are employed as cell-scaffolds and gene delivery vehicles. However, integrin binding to RGD is dependent on the spatial distribution (clustering) of the ligand and intrinsic integrin affinity via conformational changes (avidity). Here we have designed and expressed a polypeptide consisting of a tetrameric coiled coil and spacer facilitating polyvalent (clustered) display of integrin ligands; the RGD motif was used as proof of principle. Size-exclusion chromatography and circular dichroism showed that the polypeptide self assembled as a tetramer in solution with a defined secondary structure. Cell adhesion to surfaces coated with the polypeptide was up to 3-fold greater than that for (monovalent) RGDS peptide at equivalent concentrations. Moreover, the polypeptide in solution at concentrations ‡ 1 M inhibited cell adhesion to fibronectin-coated surfaces, while RGDS peptide in solution at concentrations up to 500 M did not. These cell data demonstrate that the polypeptide bound integrin receptors in a polyvalent manner. The polypeptide will therefore be of use in the engineering of tissue-culture scaffolds with increased cell adhesion activity, or to targeted gene delivery vehicles, and could incorporate protein ligands in place of the RGD motif.

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“…Specific receptor clustering seems to be required for other bacterial-ligand receptor interactions (4, 10); therefore, our strategy has the potential to apply to the functional analysis for other bacterial entries. Furthermore, our system is able to be adapted to molecular delivery to mammalian epithelial cells expressing integrin β1, which is a recent topic of therapeutic utilization of bacterial proteins (5,6,8,9). INTRACELLULAR …”

Section: Discussionmentioning

confidence: 99%

Observation of the Intracellular Behavior of Recombinant Yersinia pseudotuberculosis Invasin Protein

Koga

Izawa

Araki

et al. 2005

Microbiology and Immunology

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In this study, we observed the intracellular behavior of recombinant invasin, a 103‐kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST‐fused invasin C‐terminal portion protein (GST‐INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin‐mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, 125I‐uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA‐insoluble (polymerized‐actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial‐free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.

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A multi-domain protein for β1 integrin-targeted DNA delivery

Cited by 22 publications

References 38 publications

Plasmid Gene Delivery to Human Keratinocytes Through a Fibrin-Mediated Transfection System

Plasmid Gene Delivery to Human Keratinocytes Through a Fibrin-Mediated Transfection System

A tetravalent RGD ligand for integrin-mediated cell adhesion

Observation of the Intracellular Behavior of Recombinant Yersinia pseudotuberculosis Invasin Protein

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