Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations
Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.
Authors
- Carolline Rodrigues Menezes
- Jiin Felgner
- Philip L. Felgner (ORCID: https://orcid.org/0000-0002-4117-8505)
- Alexander L. Yan (ORCID: https://orcid.org/0000-0002-8594-7994)
- Samuel W. Du (ORCID: https://orcid.org/0000-0002-9655-3659)
- David Salom (ORCID: https://orcid.org/0000-0002-3208-1509)
- Elliot H. Choi (ORCID: https://orcid.org/0000-0001-8762-5473)
- Rafał Hołubowicz (ORCID: https://orcid.org/0000-0002-2555-2069)
- Grażyna Palczewska (ORCID: https://orcid.org/0000-0003-4030-4685)
- Gregory A. Newby (ORCID: https://orcid.org/0000-0001-7869-2615)
- Roman Šmidák (ORCID: https://orcid.org/0000-0001-8737-7596)
- David C. Lyon (ORCID: https://orcid.org/0000-0002-4367-5636)
- Krzysztof Palczewski (ORCID: https://orcid.org/0000-0002-0788-545X)
- Zhiqian Dong (ORCID: https://orcid.org/0000-0002-8748-4532)
- David R. Liu (ORCID: https://orcid.org/0000-0002-9943-7557)
- Fangyuan Gao (ORCID: https://orcid.org/0000-0002-1270-8781)
- Andrzej T. Foik (ORCID: https://orcid.org/0000-0003-1110-9223)
- J. Noah Workman (ORCID: https://orcid.org/0000-0002-0921-1803)
- Philip D. Kiser (ORCID: https://orcid.org/0000-0003-1184-9539)
- Marco Bassetto (ORCID: https://orcid.org/0000-0003-0048-0203)
- Paul Chen (ORCID: https://orcid.org/0000-0001-5261-1610)
- Omar Medani
- Maria W. Hołubowicz
- Eleonora Risaliti
Institutions
- Broad Institute (US)
- Wrocław University of Science and Technology (PL)
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- Johns Hopkins University (US)
- Amherst College (US)
- University of California, Irvine (US)
- Johns Hopkins Medicine (US)
- Harvard University Press (US)
- Vision Eye Institute (AU)
- Tibor Rubin VA Medical Center (US)
- Institute of Physical Chemistry (PL)
- Massachusetts Institute of Technology (US)
- AGH University of Krakow (PL)
- Polish Academy of Sciences (PL)
Publication Details
- Journal
- Nature Biomedical Engineering
- Published
- 2024-11-28
- DOI
- https://doi.org/10.1038/s41551-024-01296-2
- Citations
- 59
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 13.80
Funders
- Howard Hughes Medical Institute
- U.S. Department of Veterans Affairs
- Knights Templar Eye Foundation
- Fundacja na rzecz Nauki Polskiej
- Narodowe Centrum Nauki
- National Institutes of Health
- Defense Threat Reduction Agency
- University of California, Los Angeles
- California NanoSystems Institute