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

Xun Liu; Ziyin Zhao; Wei Li; Mengyao Ren; Haoyu Zhang; Desheng Cao; Yehan Wang; He Yang; Yajie Li; Manhui Zhu; Laiqing Xie; Lichen Yin

doi:10.1002/adma.202412366

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

Adv Mater. 2024 Dec 24:e2412366. doi: 10.1002/adma.202412366. Online ahead of print.

Authors

Xun Liu^{1

2}, Ziyin Zhao², Wei Li², Mengyao Ren², Haoyu Zhang², Desheng Cao², Yehan Wang², He Yang¹, Yajie Li², Manhui Zhu³, Laiqing Xie⁴, Lichen Yin^{1

2}

Affiliations

¹ Department of Thoracic Surgery, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, China.
² Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory of Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, 215123, China.
³ Department of Ophthalmology, Lixiang Eye Hospital of Soochow University, Suzhou, 215123, China.
⁴ Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, China.

PMID: 39718218
DOI: 10.1002/adma.202412366

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 (_LGP) to mediate robust and universal cytosolic delivery. _LGP forms salt-stable nanocomplexes (NCs) with P-proteins via electrostatic interaction and salt bridging, and the helix-assisted, strong membrane activities of _LGP 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 _LGP/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.

Keywords: choroidal neovascularization; cytosolic protein delivery; genome editing; pro‐protein; α‐helical polypeptide.

Abstract

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