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

Rafał Hołubowicz; Samuel W Du; Jiin Felgner; Roman Smidak; Elliot H Choi; Grazyna Palczewska; Carolline Rodrigues Menezes; Zhiqian Dong; Fangyuan Gao; Omar Medani; Alexander L Yan; Maria W Hołubowicz; Paul Z Chen; Marco Bassetto; Eleonora Risaliti; David Salom; J Noah Workman; Philip D Kiser; Andrzej T Foik; David C Lyon; Gregory A Newby; David R Liu; Philip L Felgner; Krzysztof Palczewski

doi:10.1038/s41551-024-01296-2

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

Nat Biomed Eng. 2024 Nov 28. doi: 10.1038/s41551-024-01296-2. Online ahead of print.

Authors

Rafał Hołubowicz^#^{1

2}, Samuel W Du^#^{1

3}, Jiin Felgner^#⁴, Roman Smidak¹, Elliot H Choi¹, Grazyna Palczewska¹, Carolline Rodrigues Menezes^{1

3}, Zhiqian Dong¹, Fangyuan Gao¹, Omar Medani¹, Alexander L Yan^{1

5}, Maria W Hołubowicz¹, Paul Z Chen^{6

7

8

9}, Marco Bassetto^{1

3

10}, Eleonora Risaliti^{1

3}, David Salom¹, J Noah Workman¹¹, Philip D Kiser^{1

3

10

12}, Andrzej T Foik^{13

14}, David C Lyon¹⁵, Gregory A Newby^{6

7

8

11}, David R Liu^{16

17

18}, Philip L Felgner¹⁹, Krzysztof Palczewski^{20

21

22

23}

Affiliations

¹ Gavin Herbert Eye Institute - Center for Translational Vision Research, Department of Ophthalmology, University of California, Irvine, CA, USA.
² Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland.
³ Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA, USA.
⁴ Adeline Yen Mah Vaccine Center, Department of Physiology and Biophysics, University of California, Irvine, CA, USA.
⁵ Program in Neuroscience, Amherst College, Amherst, MA, USA.
⁶ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
⁷ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
⁸ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁹ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
¹⁰ Research Service, Tibor Rubin VA Long Beach Medical Center, Long Beach, CA, USA.
¹¹ Department of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA.
¹² Department of Clinical Pharmacy Practice, School of Pharmacy and Pharmaceutical Sciences, University of California, Irvine, CA, USA.
¹³ International Centre for Translational Eye Research (ICTER), Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland.
¹⁴ Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland.
¹⁵ Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA, USA.
¹⁶ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA. [email protected].
¹⁷ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. [email protected].
¹⁸ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. [email protected].
¹⁹ Adeline Yen Mah Vaccine Center, Department of Physiology and Biophysics, University of California, Irvine, CA, USA. [email protected].
²⁰ Gavin Herbert Eye Institute - Center for Translational Vision Research, Department of Ophthalmology, University of California, Irvine, CA, USA. [email protected].
²¹ Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA, USA. [email protected].
²² Department of Chemistry, University of California, Irvine, CA, USA. [email protected].
²³ Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA. [email protected].

^# Contributed equally.

PMID: 39609561
DOI: 10.1038/s41551-024-01296-2

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.

Abstract

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