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Aerosol delivery of nucleic acids to the lung bears great potential for the treatment of inherited genetic and acquired pulmonary diseases. We examined factors affecting critical parameters of aerosol delivery of DNA-loaded nanoparticles both in vitro and in vivo. The efficiency of aerosol delivery of nanometric DNA-loaded particles to the lungs of mice depends on various factors such as i) the cationic polymer used for DNA condensation, ii) protection against destructive shearing forces, iii) choice of the nebulizer and nebulization device, and iv) solvents used for gene vector particle formulation. All of these factors have to be considered to successfully develop novel strategies to improve aerosol gene delivery by using nanometric DNA-loaded particles.