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. 2015 Jun 2;10(6):e0129013.
doi: 10.1371/journal.pone.0129013. eCollection 2015.

Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 Onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants

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Free PMC article

Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 Onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants

Hua Li et al. PLoS One. .
Free PMC article

Abstract

The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Construction of the FLAG-VP2 fusion expression plasmid.
(A) The vector map of pCMV-3×FLG-VP2. (B) Identification of FLAG-VP2 expression in 293 cells by western blot analysis using primary anti-FLAG antibody and IR700 labeled fluorescence secondary antibody at 24 h post transfection. (C) Immuno-staining of FLAG-VP2 in CHO cells transfected with pCMV-3×FLG-VP2 at 48 h using an anti-FLAG polyclonal antibody.
Fig 2
Fig 2. Preparation and characterization of recombinant AAV2 with FLAG-tagged capsid proteins.
(A) Scheme of the AAV packaging system by cotransfection of 4 plasmids in 293T cells. (B) Comparison of the viral package yields of the 4-plasmid system (pAV-Luc:pAAV-RC:pAV-Helper:pCMV-FLG-VP2 at 1:1:1:10) with those from the conventional 3-plasmid approach (pAV-Luc:pAV-RC:pAV-Helper of equal amounts) to produce conventional viruses. The design of specific primers and their qPCR products in 2% gel electrophoresis were shown. (NTC: no template control) (C) Determination of the FLAG-VP2 incorporation in the purified FL-AV2 by western blot with the B1 anti-VP antibody (green) and anti-FLAG antibody (red). (D) Comparison of recombinant FL-AV2.Luc with regular AV2.Luc of the abilities to transduce HeLa cells (left panel) or 3T3-L1 cells (right panel). The infection doses from 1×101 to 1×105 virions per cell were used. The chemiluminescence measurements were carried out at 48 h after infections.
Fig 3
Fig 3. Transduction of immobilized FL-AV2 with a FLAG antibody in 2D HeLa cell cultures.
ELISA plates were pre-coated with bovine type I collagen overnight. Anti-FLAG antibody or non-specific IgG mix was cross-linked collagen by incubation with using activated EDAC and SPDP. The plates were loaded with FL-AV2.GFP overnight and washed 3 times with PBS before use. HeLa cells (5×104 cells/well in 48-well plates) were seeded into the plates and continuingly cultured for 48 h. Fluorescent images were acquired using a Leica DMIRB microscope. (B) Experiments under same conditions of (A) were performed using FL-AV2.Luc viruses for immobilization and infections. The quantitative chemo-luminescence was determined by luciferase assays. Measurements from 6 independent samples were averaged and presented and shown as mean+/-SE.
Fig 4
Fig 4. Transduction of immobilized FL-AV2 with FLAG antibodies in 3D cell cultures in vitro.
Tissue engineering scaffolds of PLGA (A) and gelatin sponge (C) were pre-coated with bovine type I collagen. Anti-FLAG antibody or mixed IgG was cross-linked to the collagen coating. FL-AV2.LacZ was immobilized to the scaffolds overnight and clean washed with PBS. HeLa cells of 1×106 cells in 1 mL were seeded and cultured for 48 h prior to ß-gal staining was performed. The 10× phase-contract images were acquired under a Leica DMIRB microscope. Randomly selected 6 non-overlapped microscopic fields were analyzed for the area percentages of β-gal stained regions(B and D), and the values were used as a semi-quantitative indication of viral transduction efficiencies.
Fig 5
Fig 5. Long term transduction of antibody immobilized FL-AV2 in 3D cell cultures in vivo.
PLGA scaffolds immobilized with FLAG antibodies were loaded with FL-AV2.LacZ and used as the 3D culture matrix for HeLa cells. After 48 h of culture, the seeded cells in scaffolds were implanted into the subcutaneous tissue under the neck skin of nude mice. After 4 weeks, the tissues surround the implanted regions were removed and subjected for histological examinations. Images of frozen sections stained with a β-galactosidase staining kit (A and B) or with H.E. staining (D and E) were obtained using a Leica DM 5000 B microscope. Randomly selected 9 non-overlapped microscopic fields were analyzed for the area percentages of β-gal stained regions(C), and the values were used as a semi-quantitative indication of viral transduction efficiencies.

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This study was supported by Chinese National Science Foundation (NSFC 30970161 and 81372284). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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