Ocular gene therapy may offer new hope for severe eye diseases. Many of these ocular diseases are due to a gene defect in the retina, a multi-layered sensory tissue that lines the back of the eye. However, it is well known that the blood–retina barrier and sclera prevent hydrophilic and high molecular weight drugs to reach the retina after systemic or topical application. Therefore, intravitreal injection of non-viral nucleic acid nanoparticles has been considered as a safe and promising approach in ocular gene transfer. However, after intravitreal injection the non-viral nucleic acid nanoparticles should be stable and mobile in the vitreous. In this overview we focus on the behavior of non-viral nucleic acid nanoparticles (lipoplexes) in vitreous and on PEGylation strategies that improve their behavior in vitreous, but that do not affect their transfection capacity.