Rationally Engineering Pro‐Proteins and Membrane‐Penetrating α‑Helical Polypeptides for Genome Editing Toward Choroidal Neovascularization Treatment

Abstract Ribonucleoprotein (RNP)‐based CRISPR/Cas9 genome editing holds great potential for the treatment of choroidal neovascularization (CNV), which however, is challenged by the lack of efficient cytosolic protein delivery tools. Herein, reversibly‐phosphorylated pro‐proteins (P‐proteins) with conjugated adenosine triphosphate (ATP) tags are engineered and coupled with a membrane‐penetrating, guanidine‐enriched, α‐helical polypeptide ( L GP) to mediate robust and universal cytosolic delivery. L GP forms salt‐stable nanocomplexes (NCs) with P‐proteins via electrostatic interaction and salt bridging, and the helix‐assisted, strong membrane activities of L GP enabled efficient cellular internalization and endolysosomal escape of NCs. Therefore, this approach allows efficient cytosolic delivery of a wide range of protein cargoes and maintains their bioactivities due to endolysosomal acidity‐triggered traceless restoration of P‐proteins. Notably, intravitreally delivered L GP/P‐RNP NCs targeting hypoxia‐inducible factor‐1α (HIF‐1α) induce pronounced gene disruption to downregulate pro‐angiogenic factors and alleviate subretinal fibrosis, ultimately provoking robust therapeutic efficacy in CNV mice. Such a facile and versatile platform provides a powerful tool for cytosolic protein delivery and genome editing, and it holds promising potential for the treatment of CNV‐associated diseases, such as age‐related macular degeneration.