Self-assembling protein nanoparticles for cytosolic delivery of nucleic acids and proteins

Feyisayo Eweje^#^{1

2

3

4}, Vanessa Ibrahim¹, Aram Shajii¹, Michelle L Walsh^{1

2

3}, Kiran Ahmad¹, Assma Alrefai¹, Dominie Miyasato¹, Jessie R Davis^{5

6

7}, Hyunok Ham^{1

4}, Kaicheng Li^{1

4}, Michael Roehrl⁸, Carolyn A Haller^{1

4}, David R Liu^{5

6

7}, Jiaxuan Chen^#^{9

10}, Elliot L Chaikof^{11

12}

Affiliations

¹ Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
² Harvard and MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA.
³ Harvard/MIT MD-PhD Program, Boston, MA, USA.
⁴ Wyss Institute of Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
⁵ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
⁶ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁷ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁸ Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
⁹ Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. jchen13@bidmc.harvard.edu.
¹⁰ Wyss Institute of Biologically Inspired Engineering, Harvard University, Boston, MA, USA. jchen13@bidmc.harvard.edu.
¹¹ Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. echaikof@bidmc.harvard.edu.
¹² Wyss Institute of Biologically Inspired Engineering, Harvard University, Boston, MA, USA. echaikof@bidmc.harvard.edu.

^# Contributed equally.

PMID: 40374955
DOI: 10.1038/s41587-025-02664-2

Self-assembling protein nanoparticles for cytosolic delivery of nucleic acids and proteins

Feyisayo Eweje et al. Nat Biotechnol. 2025.

. 2025 May 15.

doi: 10.1038/s41587-025-02664-2. Online ahead of print.

Authors

Affiliations

¹ Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
² Harvard and MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA.
³ Harvard/MIT MD-PhD Program, Boston, MA, USA.
⁴ Wyss Institute of Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
⁵ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
⁶ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁷ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁸ Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
⁹ Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. jchen13@bidmc.harvard.edu.
¹⁰ Wyss Institute of Biologically Inspired Engineering, Harvard University, Boston, MA, USA. jchen13@bidmc.harvard.edu.
¹¹ Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. echaikof@bidmc.harvard.edu.
¹² Wyss Institute of Biologically Inspired Engineering, Harvard University, Boston, MA, USA. echaikof@bidmc.harvard.edu.

^# Contributed equally.

PMID: 40374955
DOI: 10.1038/s41587-025-02664-2

Abstract

Intracellular delivery of biomacromolecules is hampered by low efficiency and cytotoxicity. Here we report the development of elastin-based nanoparticles for therapeutic delivery (ENTER), a recombinant elastin-like polypeptide (ELP)-based delivery system for effective cytosolic delivery of biomacromolecules in vitro and in vivo. Through iterative design, we developed fourth-generation ELPs fused to cationic endosomal escape peptides (EEPs) that self-assemble into pH-responsive micellar nanoparticles and enable cytosolic entry of cargo following endocytic uptake. In silico screening of α-helical peptide libraries led to the discovery of an EEP (EEP13) with 48% improved protein delivery efficiency versus a benchmark peptide. Our lead ELP-EEP13 showed similar or superior performance compared to lipid-based transfection reagents in the delivery of mRNA-encoded, DNA-encoded and protein-form Cre recombinase and CRISPR gene editors as well as short interfering RNAs to multiple cell lines and primary cell types. Intranasal administration of ELP-EEP13 combined with Cre protein achieved efficient editing of lung epithelial cells in reporter mice.

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Conflict of interest statement

Competing interests: F.E., J.C. and E.L.C. are inventors on a pending patent related to this work filed by the Beth Israel Deaconess Medical Center (PCT/US2024/038614). D.R.L. is a consultant and equity holder of Nvelop Medicine, Prime Medicine, Beam Therapeutics, Pairwise Plants and Chroma Medicine, companies that use or deliver gene editing or genome engineering agents. The other authors declare no competing interests.

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Self-assembling protein nanoparticles for cytosolic delivery of nucleic acids and proteins

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Self-assembling protein nanoparticles for cytosolic delivery of nucleic acids and proteins

Authors

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Abstract

Conflict of interest statement

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