Methods and devices for delivering aerosolized formulations containing polynucleotides to specified regions within a subject's respiratory tract are disclosed. The methods find use in the delivery of ribozymes, antisense polynucleotides, and DNA and RNA expression vectors into airway epithelial cells, alveoli, pulmonary macrophages and other cells in the respiratory tract (including the oropharynx, nose, nasopharynx). These methods may be used for optimization of transfection efficiency and expression in vivo, and for in vivo expression, for example for generating an immune response, or inducing immunological tolerance.