Adeno-Associated Viral Vector Immobilization and Local Delivery from Bare Metal Surfaces.
Methods Mol Biol
; 2394: 601-616, 2022.
Article
em En
| MEDLINE
| ID: mdl-35094349
ABSTRACT
Spatially and temporally controlled delivery of biologicals, including gene vectors, represents an unmet need for regenerative medicine and gene therapy applications. Here we describe a method of reversible attachment of serotype 2 adeno-associated viral vectors (AAV2) to metal surfaces. This technique enables localized delivery of the vector to the target cell population in vitro and in vivo with the subsequent effective transduction of cells adjacent to the metal substrate. The underlying bioengineering approach employs coordination chemistry between the bisphosphonic groups of polyallylamine bisphosphonates and the metal atoms on the surface of metallic samples. Formation of a stable polybisphosphonate monolayer with plentiful allyl-derived amines allows for further chemical modification to consecutively append thiol-modified protein G, an anti-AAV2 antibody, and AAV2 particles. Herein we present a detailed protocols for the metal substrate modification, for the visualization of the metal surface-immobilized vector using direct and indirect fluorescent AAV2 labeling and scanning electron microscopy, for quantification of the surface-immobilized vector load with RT-PCR, and for the localized vector transduction in vitro and in vivo.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Dependovirus
/
Vetores Genéticos
Tipo de estudo:
Guideline
/
Risk_factors_studies
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article