RESUMEN
Nonviral gene therapy utilizing plasmid DNA (pDNA) complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has demonstrated considerable potential for the treatment of a variety of diseases. However, progress toward clinical application is often delayed by the lack of reliable and scalable mixing of components sufficient to guarantee consistent performance in vivo. Attempts to improve and standardize mixing have been limited by the sensitivity of pDNA to shear-related degradation. Here we describe a simple pneumatic mixing device that enables the rapid and reproducible production of large volumes of nonviral gene therapy formulations and demonstrate its suitability for use with shear-sensitive pDNA.
Asunto(s)
ADN/administración & dosificación , Terapia Genética/instrumentación , Plásmidos/administración & dosificación , Animales , Cationes/química , ADN/química , ADN/genética , Diseño de Equipo , Expresión Génica , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Plásmidos/química , Plásmidos/genéticaRESUMEN
We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.
