Pharmacological treatments designed to reactivate fetal γ globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired as such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop a novel chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human γ globin promoter, and reactivates this normally dormant transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. Novel in vitro and in vivo DNA binding assays in conjunction with live cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific g promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest, and are of direct relevance to clinical applications that would benefit from such a need.