Genome Editing in the Mouse Brain with Minimally Immunogenic Cas9 RNPs

Overview

Journal Mol Ther

Publisher Cell Press

Specialties Molecular Biology
Pharmacology

Date 2023 Jul 5

PMID 37403358

Authors

Elizabeth C Stahl

Jennifer K Sabo

Min Hyung Kang

Ryan Allen

Elizabeth Applegate

Shin Eui Kim

Yoonjin Kwon

Anmol Seth

Nicholas Lemus

Viviana Salinas-Rios

Katarzyna M Soczek

Marena Trinidad

Linda T Vo

Chris Jeans

Anna Wozniak

Timothy Morris

Athen Kimberlin

Thomas Foti

David F Savage

Jennifer A Doudna

Affiliations

Soon will be listed here.

Abstract

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

Citing Articles

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.

Sahu S, Castro M, Muldoon J, Asija K, Wyman S, Krishnappa N Nat Protoc. 2025; .

PMID: 40032999 DOI: 10.1038/s41596-025-01154-8.

Non-Invasive Delivery of CRISPR/Cas9 Ribonucleoproteins (Cas9 RNPs) into Cells via Nanoparticles for Membrane Transport.

Tashima T Pharmaceutics. 2025; 17(2).

PMID: 40006568 PMC: 11859894. DOI: 10.3390/pharmaceutics17020201.

Widespread Gene Editing in the Brain via In Utero Delivery of mRNA Using Acid-Degradable Lipid Nanoparticles.

Gao K, Han H, Cranick M, Zhao S, Xu S, Yin B ACS Nano. 2024; 18(44):30293-30306.

PMID: 39445691 PMC: 11544762. DOI: 10.1021/acsnano.4c05169.

Gene therapy for CNS disorders: modalities, delivery and translational challenges.

Gao J, Gunasekar S, Xia Z, Shalin K, Jiang C, Chen H Nat Rev Neurosci. 2024; 25(8):553-572.

PMID: 38898231 DOI: 10.1038/s41583-024-00829-7.

Advances and challenges in modeling inherited peripheral neuropathies using iPSCs.

Van Lent J, Prior R, Perez Siles G, Cutrupi A, Kennerson M, Vangansewinkel T Exp Mol Med. 2024; 56(6):1348-1364.

PMID: 38825644 PMC: 11263568. DOI: 10.1038/s12276-024-01250-x.

References

Oura S, Noda T, Morimura N, Hitoshi S, Nishimasu H, Nagai Y . Precise CAG repeat contraction in a Huntington's Disease mouse model is enabled by gene editing with SpCas9-NG. Commun Biol. 2021; 4(1):771. PMC: 8222283. DOI: 10.1038/s42003-021-02304-w. View

Liu J, Gaj T, Wallen M, Barbas 3rd C . Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering. Mol Ther Nucleic Acids. 2015; 4:e232. PMC: 4354341. DOI: 10.1038/mtna.2015.6. View

Doudna J . The promise and challenge of therapeutic genome editing. Nature. 2020; 578(7794):229-236. PMC: 8992613. DOI: 10.1038/s41586-020-1978-5. View

Tsai S, Nguyen N, Malagon-Lopez J, Topkar V, Aryee M, Joung J . CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods. 2017; 14(6):607-614. PMC: 5924695. DOI: 10.1038/nmeth.4278. View

Hadaczek P, Kohutnicka M, Krauze M, Bringas J, Pivirotto P, Cunningham J . Convection-enhanced delivery of adeno-associated virus type 2 (AAV2) into the striatum and transport of AAV2 within monkey brain. Hum Gene Ther. 2006; 17(3):291-302. DOI: 10.1089/hum.2006.17.291. View

Gao G, Vandenberghe L, Alvira M, Lu Y, Calcedo R, Zhou X . Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol. 2004; 78(12):6381-8. PMC: 416542. DOI: 10.1128/JVI.78.12.6381-6388.2004. View

Lopez-Delisle L, Rabbani L, Wolff J, Bhardwaj V, Backofen R, Gruning B . pyGenomeTracks: reproducible plots for multivariate genomic datasets. Bioinformatics. 2020; 37(3):422-423. PMC: 8058774. DOI: 10.1093/bioinformatics/btaa692. View

Charlesworth C, Deshpande P, Dever D, Camarena J, Lemgart V, Cromer M . Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 2019; 25(2):249-254. PMC: 7199589. DOI: 10.1038/s41591-018-0326-x. View

Ferdosi S, Ewaisha R, Moghadam F, Krishna S, Park J, Ebrahimkhani M . Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat Commun. 2019; 10(1):1842. PMC: 6478683. DOI: 10.1038/s41467-019-09693-x. View

10.

Chambers S, Fasano C, Papapetrou E, Tomishima M, Sadelain M, Studer L . Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009; 27(3):275-80. PMC: 2756723. DOI: 10.1038/nbt.1529. View

11.

Li A, Tanner M, Lee C, Hurley A, De Giorgi M, Jarrett K . AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9. Mol Ther. 2020; 28(6):1432-1441. PMC: 7264438. DOI: 10.1016/j.ymthe.2020.04.017. View

12.

Maeder M, Stefanidakis M, Wilson C, Baral R, Barrera L, Bounoutas G . Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019; 25(2):229-233. DOI: 10.1038/s41591-018-0327-9. View

13.

Nelson C, Wu Y, Gemberling M, Oliver M, Waller M, Bohning J . Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat Med. 2019; 25(3):427-432. PMC: 6455975. DOI: 10.1038/s41591-019-0344-3. View

14.

Samaranch L, San Sebastian W, Kells A, Salegio E, Heller G, Bringas J . AAV9-mediated expression of a non-self protein in nonhuman primate central nervous system triggers widespread neuroinflammation driven by antigen-presenting cell transduction. Mol Ther. 2014; 22(2):329-337. PMC: 3918916. DOI: 10.1038/mt.2013.266. View

15.

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

16.

Ramirez F, Bhardwaj V, Arrigoni L, Lam K, Gruning B, Villaveces J . High-resolution TADs reveal DNA sequences underlying genome organization in flies. Nat Commun. 2018; 9(1):189. PMC: 5768762. DOI: 10.1038/s41467-017-02525-w. View

17.

Aschauer D, Kreuz S, Rumpel S . Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013; 8(9):e76310. PMC: 3785459. DOI: 10.1371/journal.pone.0076310. View

18.

Madisen L, Zwingman T, Sunkin S, Oh S, Zariwala H, Gu H . A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2009; 13(1):133-40. PMC: 2840225. DOI: 10.1038/nn.2467. View

19.

Sedlazeck F, Rescheneder P, Smolka M, Fang H, Nattestad M, von Haeseler A . Accurate detection of complex structural variations using single-molecule sequencing. Nat Methods. 2018; 15(6):461-468. PMC: 5990442. DOI: 10.1038/s41592-018-0001-7. View

20.

Park I, Zhao R, West J, Yabuuchi A, Huo H, Ince T . Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2007; 451(7175):141-6. DOI: 10.1038/nature06534. View