Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations

Rafał Hołubowicz(Wrocław University of Science and Technology), Samuel W. Du(University of California, Irvine), Jiin Felgner(University of California, Irvine), Roman Šmidák(University of California, Irvine), Elliot H. Choi(University of California, Irvine), Grażyna Palczewska(University of California, Irvine), Carolline Rodrigues Menezes(University of California, Irvine), Zhiqian Dong(University of California, Irvine), Fangyuan Gao(University of California, Irvine), Omar Medani(University of California, Irvine), Alexander L. Yan(Amherst College), Maria W. Hołubowicz(University of California, Irvine), Paul Chen(Broad Institute), Marco Bassetto(University of California, Irvine), Eleonora Risaliti(University of California, Irvine), David Salom(University of California, Irvine), J. Noah Workman(Johns Hopkins University), Philip D. Kiser(University of California, Irvine), Andrzej T. Foik(Institute of Physical Chemistry), David C. Lyon(University of California, Irvine), Gregory A. Newby(Broad Institute), David R. Liu(Broad Institute), Philip L. Felgner(University of California, Irvine), Krzysztof Palczewski(University of California, Irvine)
Nature Biomedical Engineering
November 28, 2024
10.1038/s41551-024-01296-2
Cited by 70Open Access
Full Text

Abstract

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.

Related Papers

The VideoToolbox software for visual psychophysics: transforming numbers into movies
Denis G. Pelli|Spatial Vision|1997|11.6k
The Psychophysics Toolbox.
David H. Brainard|PubMed|1997|9.2k
Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo
John A. Zuris, David B. Thompson, Yilai Shu et al.|Nature Biotechnology|2014|1.5k
Effect of Genome Size on AAV Vector Packaging
Zhijian Wu, Hongyan Yang, Peter Colosi|Molecular Therapy|2009|969
Prevalence of Serum IgG and Neutralizing Factors Against Adeno-Associated Virus (AAV) Types 1, 2, 5, 6, 8, and 9 in the Healthy Population: Implications for Gene Therapy Using AAV Vectors
Sylvie Boutin, Virginie Monteilhet, P. Véron et al.|Human Gene Therapy|2010|941