A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

Finn, Jonathan D.; Smith, Amy; Patel, Mihir; Shaw, Lucinda; Youniss, Madeleine R.; Heteren, Jane van; Dirstine, Tanner; Ciullo, Corey; Lescarbeau, Reynald; Seitzer, Jessica; Shah, R. Y.; Shah, Aalok; Ling, Dandan; Growe, Jacqueline; Pink, Melissa; Rohde, Ellen; Wood, Kristy M.; Salomon, William E.; Harrington, William F.; Dombrowski, Christian; Strapps, Walter; Chang, Yong Keun; Morrissey, David

doi:10.1016/j.celrep.2018.02.014

Cited by 632 publications

(489 citation statements)

References 32 publications

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“…There is great interest in protein and nucleic acid-based therapeutics for airway diseases, but delivery to respiratory epithelial cells remains exceedingly challenging. Several viral vectors 3,6,46 , non-viral liposomes 21,47,48 , and other nanoparticle formulations are in development to address delivery challenges 49,50 , but none have advanced to widespread clinical utility. While some viral vectors overcome delivery barriers 4,5,51 , they are complex biologics and genetic payloads may exceed the capacity of some vectors.…”

Section: Discussionmentioning

confidence: 99%

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

et al. 2019

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The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.

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Section: Discussionmentioning

confidence: 99%

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

et al. 2019

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“…By contrast, all previous systemically administered nanoparticle gene editing has occurred preferentially in hepatocytes. 46,48–50 This is the first report of sgRNA-mediated in vivo editing in hepatic endothelial cells.…”

Section: Resultsmentioning

confidence: 87%

Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery

et al. 2018

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Lipid nanoparticles (LNPs) are formulated using unmodified cholesterol. However, cholesterol is naturally esterified and oxidized in vivo, and these cholesterol variants are differentially trafficked in vivo via lipoproteins including LDL and VLDL. We hypothesized that incorporating the same cholesterol variants into LNPs - which can be structurally similar to LDL and VLDL – would alter nanoparticle targeting in vivo. To test this hypothesis, we quantified how >100 LNPs made with 6 cholesterol variants delivered DNA barcodes to 18 cell types in wildtype, LDL R−/−, and VLDLR−/− mice that were both age-matched and female. By analyzing ~2,000 in vivo drug delivery data points, we found that LNPs formulated with esterified cholesterol delivered nucleic acids more efficiently than LNPs formulated with regular or oxidized cholesterol when compared across all tested cell types in the mouse. We also identified an LNP containing cholesteryl oleate that efficiently delivered siRNA and sgRNA to liver endothelial cells in vivo. Delivery was as - or more - efficient than the same LNP made with unmodified cholesterol. Moreover, delivery to liver endothelial cells was 3X more efficient than delivery to hepatocytes, distinguishing this oleate LNP from hepatocyte-targeting LNPs. RNA delivery can be improved by rationally selecting cholesterol variants, allowing optimization of nanoparticle targeting.

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“…This strategy guarantees that these two components are delivered to the same individual cells, and has achieved greater editing efficiency [202]. An LNP-based delivery system that co-formulated Cas9 mRNA and gRNA into a single particle for simultaneous delivery resulted in a significant editing of the mouse transthyretin gene in the liver, with > 97% reduction in serum protein levels that persisted for at least 12 months [203]. …”

Section: Biomedical Applicationsmentioning

confidence: 99%

Biomedical applications of mRNA nanomedicine

et al. 2018

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As an attractive alternative to plasmid DNA, messenger RNA (mRNA) has recently emerged as a promising class of nucleic acid therapeutics for biomedical applications. Advances in addressing the inherent shortcomings of mRNA and in the development of nanoparticle-based delivery systems have prompted the development and clinical translation of mRNA-based medicines. In this review, we discuss the chemical modification strategies of mRNA to improve its stability, minimize immune responses, and enhance translational efficacy. We also highlight recent progress in nanoparticle-based mRNA delivery. Considerable attention is given to the increasingly widespread applications of mRNA nanomedicine in the biomedical fields of vaccination, protein-replacement therapy, gene editing, and cellular reprogramming and engineering.

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A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

Cited by 632 publications

References 32 publications

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery

Biomedical applications of mRNA nanomedicine

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