研究目的
To design hydrogel contact lenses that can act as platforms for the controlled delivery of viral vectors (recombinant adeno-associated virus, rAAV) to the eye for gene therapy, improving patient compliance compared to eye drops and ocular injections.
研究成果
HEMA hydrogels functionalized with APMA can effectively load and release rAAV vectors for corneal gene therapy, demonstrating controlled delivery and transduction efficacy in hMSCs and bovine cornea. This approach offers a patient-friendly alternative to traditional ocular gene therapy methods.
研究不足
The study is a proof-of-principle and may require further optimization for clinical application. The stability of viral vectors at physiological conditions and the scalability of hydrogel production are potential limitations.
1:Experimental Design and Method Selection
Hydrogels of hydroxyethyl methacrylate (HEMA) with aminopropyl methacrylamide (APMA) or without were synthesized, sterilized, and tested for gene therapy using rAAV vectors. The hydrogels were evaluated for light transparency, oxygen permeability, and swelling.
2:Sample Selection and Data Sources
Human bone marrow derived mesenchymal stem cells (hMSCs) and bovine cornea were used to test transduction efficacy and gene delivery.
3:List of Experimental Equipment and Materials
Hydrogels (HEMA with APMA), rAAV vectors (rAAV-lacZ, rAAV-RFP, rAAV-hIGF-I), culture medium, fluorescence microscopy, ELISA kits, and immunohistochemistry tools.
4:Experimental Procedures and Operational Workflow
Loading of viral vectors at 4°C, release monitoring in culture medium, transduction efficacy tests in hMSCs and bovine cornea, and evaluation of cell proliferation and gene expression.
5:Data Analysis Methods
Fluorescence measurements, ELISA for viral vector quantification, X-Gal staining for β-galactosidase detection, and immunohistochemistry for IGF-I expression analysis.
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HEMA
Merck
Structural monomer for hydrogel synthesis
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APMA
Polysciences Inc.
Functional monomer for hydrogel synthesis
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EGDMA
Sigma-Aldrich
Crosslinking agent for hydrogel synthesis
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AIBN
Acros
Initiator for polymerization
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Cy3 Ab Labeling Kit
Amersham/GE Healthcare
Labeling of rAAV vectors for fluorescence monitoring
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AAV Titration ELISA
Progen
Quantification of rAAV vectors
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Beta-Glo Assay System
Promega
Quantification of β-galactosidase activity
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WST-1 Cell Proliferation Reagent
Roche Applied Science
Cell viability and proliferation assay
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