This review focuses on recent progress and novel strategies to improve the efficiency of in vivo non-viral gene delivery. Examples of the most promising attempts to overcome specific barriers are presented in fuller detail. Current research into several of the most difficult steps in the gene delivery pathway is discussed including particle stabilization, targeting, cytoplasmic entry and access to the nucleus. The impact of recent reports on our current understanding of the true limitations to in vivo delivery is also discussed. The importance of preclinical animal models for the development of clinical applications of gene therapy is noted.