The developed disease and mutations in treatment need emerging strategies to tackle physiological and biopharmaceutical challenges. Many drugs/therapies are available for symptomatic relief; however, they are sometimes only partially effective and have a number of dangerous adverse effects. New therapeutic approaches must therefore be developed to overcome complications and target permanent cures for specific diseases. Permanent and targeted clinical care has been significantly impacted by the production of nanotechnology-based products. The use of nanotechnology has aided in the creation of extremely disease-selective gene therapy systems, which will be able to deliver the gene in the body to correct the abnormality and recover the genetic function. Carriers like liposomes, dendrimers, and polymer nanomaterials have recently gained attention as possible drug delivery systems for various complex physiological diseases, decreasing toxicity and increasing biocompatibility. PEGylated nanocarriers are significantly the most cutting-edge techniques for non-invasive gene delivery technologies. When compared to non-PEGylated complexes, PEGylated nanocarriers showed superior biodistribution, enhanced safety profiles, and effective gene transfer in vector-based nanoformulations. Additionally, compared to polymeric systems PEGylation improves the gene expression and stability in a system that has been progressively studied in pre-clinical settings. In the present chapter, we compiled the utilization of PEGylated nanocarriers along with different formulation approaches for gene therapy in an extensive set of disease conditions and barriers.