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. 2018 Dec 12;140(49):17095-17105.
doi: 10.1021/jacs.8b08976. Epub 2018 Nov 16.

Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution

Cory D Sago  1 , Melissa P Lokugamage  1 , Fatima Z Islam  1 , Brandon R Krupczak  1 , Manaka Sato  1 , James E Dahlman  1
Affiliations

Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution

Cory D Sago et al. J Am Chem Soc. .

Abstract

Bone marrow endothelial cells (BMECs) regulate their microenvironment, which includes hematopoietic stem cells. This makes BMECs an important target cell type for siRNA or gene editing (e.g., CRISPR) therapies. However, siRNA and sgRNA have not been delivered to BMECs using systemically administered nanoparticles. Given that in vitro nanoparticle screens have not identified nanoparticles with BMEC tropism, we developed a system to quantify how >100 different nanoparticles deliver siRNA in a single mouse. This is the first barcoding system capable of quantifying functional cytosolic siRNA delivery (where the siRNA drug is active), distinguishing it from in vivo screens that quantify biodistribution (where the siRNA drug went). Combining this approach with bioinformatics, we performed in vivo directed evolution, and identified BM1, a lipid nanoparticle (LNP) that delivers siRNA and sgRNA to BMECs. Interestingly, chemical analysis revealed BMEC tropism was not related to LNP size; tropism changed with the structure of poly(ethylene glycol), as well as the presence of cholesterol. These results suggest that significant changes to vascular targeting can be imparted to a LNP by making simple changes to its chemical composition, rather than using active targeting ligands. BM1 is the first nanoparticle to efficiently deliver siRNA and sgRNA to BMECs in vivo, demonstrating that this functional in vivo screen can identify nanoparticles with novel tropism in vivo. More generally, in vivo screening may help reveal the complex relationship between nanoparticle structure and tropism, thereby helping scientists understand how simple chemical changes control nanoparticle targeting.

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Conflict of interest statement

Competing interests. C.D.S., M.P.L., and J.E.D. have filed for intellectual property related to material within this publication.

Figures

Figure 1.
Figure 1.
Systemically delivering RNA to bone marrow endothelial cells (BMECs) is challenging. (A) BMECs release local signals that regulate pericytes, immune cells, and hematopoietic stem cells in the bone marrow ‘niche’. (B) The dose (mg / kg siRNA) required to silence target gene expression in different vascular beds in vivo. BMECs are targeted much less efficiently than other vascular beds. (C) ICAM-2 protein expression after mice were treated with siLuciferase or siICAM-2 carried by the nanoparticle 7C1; the ‘original 80: 20’ 7C1 formulation does not deliver siRNA to BMECs. (D) A methodology to improve LNP delivery to BMECs; this utilizes an iterative high throughput in vivo screening method.
Figure 2.
Figure 2.
Nanoparticles co-formulated with siRNA and a DNA barcode can be used to readout quantify how >100 different LNPs functionally deliver RNA into the cytoplasm of target cells in a single mouse. (A) Unlike previous biodistribution screens, which cannot distinguish between bound particles, particles stuck in endosomes, and particles that delivered RNA into the cytoplasm, our method identifies LNPs that functionally deliver siRNA. We do so by isolating cells that are ICAMLow and sequencing barcodes in those cells. (B,C) ICAM-2 protein expression in lung endothelial cells after mice were treated with 7C1 carrying a barcode and either siLuc or siICAM-2. ICAM-2 protein expression decreased in a dose-dependent manner. (D) siRNA-mediated silencing also led to a dose-dependent increase in ICAMLow lung endothelial cells.
Figure 3.
Figure 3.
The efficiency with which >100 chemically distinct LNPs delivered siRNA to cells was tested simultaneously in vivo. (A) LNPs from library 1 were made with 7C1 lipomer and cholesterol. (B) 5 different PEG types were used in library 1; including PEGs with 14, 16, and 18 carbon alkyl tails and molecular weights of 2000 and 3000. (C) 24 different formulation ratios were used for each of the 5 PEG types in library 1. (D) Diameter of 115 LNPs from library 1 that were pooled and injected. The diameter of the pooled library was similar to the diameter of the individual LNPs. (E) Normalized DNA delivery in BMECs for 115 LNPs and the 2 negative controls, which were naked. (F) Correlation between LNP diameter (nm) and normalized DNA delivery in BMECs for all 115 LNPs in Library 1. (G) Correlation between LNP diameter (nm) and normalized DNA delivery in the top and bottom 10% LNPs based on performance from library 1. (H) Diameter (nm) of top and bottom 10% LNPs. Taken together, the data in (F-H) suggest the relationship between siRNA delivery and LNP size (between 20 and 200 nm) is non-existent.
Figure 4.
Figure 4.
LNP delivery to BMEC changes with PEG structure. (A) Enrichment analysis suggests that (A) low and high PEG Mole% as well as (B) C16PEG2000 and C18PEG2000 can promote delivery to in BMECs in vivo. Enrichment is described in the Supplement. (C) Paired comparison of LNPs with identical formulation ratios suggest that C18PEG2000 outperforms C18PEG3000 promotes BMEC targeting in vivo (P<0.05, Paired 2-tail T-Test).
Figure 5.
Figure 5.
Analysis of LNP size and chemical traits from a second library further suggests BMEC targeting is influenced by LNP chemical composition. (A) LNPs from library 2 were made with 7C1 lipomer, cholesterol, and DSPC. (B) Two different PEG types were used in library 2 - C16PEG2000 and C18PEG2000. These structures were selected based on data from LNP library 1. (C) 20 different formulation ratios were used for each of the two PEG types in library 2. (D) ICAM-2 protein silencing in BMECs 3 days after mice were injected with the library of LNPs at a total dose of 1.5 mg / kg. Notably, ICAM-2 silencing was more potent in BMECs than library 1. (D) Normalized DNA delivery in BMECs for 31 LNPs and 2 naked barcodes; as expected the naked barcodes performed poorly. (E) Correlation between LNP diameter (nm) and normalized DNA delivery in BMECs for all 31 LNPs in Library 2. (F) Correlation between LNP diameter (nm) and normalized DNA delivery in the top and bottom 10% LNPs based on performance from library 2. (G) Diameter (nm) of top and bottom 10% LNPs. Taken together, (E-G) further suggest BMEC targeting is not influenced by LNP size between 20 and 200 nm. (H) Enrichment of LNPs containing PEG Mole % between 15–20% in BMECs in vivo. (I) Enrichment of LNPs containing C18PEG2000 in BMECs in vivo. (J) Enrichment of LNPs containing 80 mole % 7C1 in BMECs in vivo. (K) Enrichment of LNPs containing 0 mole % DSPC in BMECs in vivo. (L) Enrichment of LNPs containing 0.1 – 10 mole % cholesterol in BMECs in vivo.
Figure 6.
Figure 6.
Comparing BM1 and the original 80: 20 7C1 formulation reveal differences in nanoparticle behavior. (A) A table of the chemical properties enriched by the in vivo screens, the original 7C1 formulation, and BM1. Notably, BM1 has all the properties that were enriched in BMECs selected from LNP library 2. (B) Diameter, polydispersity index, and pKa for original 7C1 and BM1. There are no significant differences between the formulations. (C) In vitro uptake of 7C1 and BM1 in vitro quantified as Alexa647 MFI 1 hour after immortalized aortic endothelial cells were treated with fluorescent LNPs. The original formulation is (D) is inhibited by the endocytosis inhibitors genistein (caveolin) and chlorpromazine (clathrin), whereas BM1 uptake is only inhibited by chlorpromazine. (E) ICAM-2 protein silencing in BMECs 3 days after mice were treated with siLuc carried by original 7C1 or BM1. BM1 delivered siICAM-2 to BMECs much more efficiently than 7C1 (P<0.01, Unpaired 2-tail T-test) (F) Histogram of ICAM-2 protein expression in BMECs following the administration of PBS or BM1 carrying siLuc or siICAM-2 at 1 mg / kg. (G) ICAM-2 protein silencing mediated by a 1.0 mg / kg injection of 7C1 and BM1 in lung and heart endothelial cells. After evolving BM1 to target bone marrow, we did not observe any increased potency in lung or heart EC delivery. (H) The percentage of BMEC loci with targeted insertions or deletions (indels, i.e., mutations) after BM1 was formulated with another small RNA (sgICAM-2) and injected into Cas9 mice at a dose of 1 mg / kg.
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References

    1. Semple SC; Akinc A; Chen J; Sandhu AP; Mui BL; Cho CK; Sah DW; Stebbing D; Crosley EJ; Yaworski E; Hafez IM; Dorkin JR; Qin J; Lam K; Rajeev KG; Wong KF; Jeffs LB; Nechev L; Eisenhardt ML; Jayaraman M; Kazem M; Maier MA; Srinivasulu M; Weinstein MJ; Chen Q; Alvarez R; Barros SA; De S; Klimuk SK; Borland T; Kosovrasti V; Cantley WL; Tam YK; Manoharan M; Ciufolini MA; Tracy MA; de Fougerolles A; MacLachlan I; Cullis PR; Madden TD; Hope MJ, Rational design of cationic lipids for siRNA delivery. Nat Biotechnol 2010, 28 (2), 172–6. - PubMed
    1. Love KT; Mahon KP; Levins CG; Whitehead KA; Querbes W; Dorkin JR; Qin J; Cantley W; Qin LL; Racie T; Frank-Kamenetsky M; Yip KN; Alvarez R; Sah DW; de Fougerolles A; Fitzgerald K; Koteliansky V; Akinc A; Langer R; Anderson DG, Lipid-like materials for low-dose, in vivo gene silencing. Proceedings of the National Academy of Sciences of the United States of America 2010, 107 (5), 1864–9. - PMC - PubMed
    1. Dong Y; Love KT; Dorkin JR; Sirirungruang S; Zhang Y; Chen D; Bogorad RL; Yin H; Chen Y; Vegas AJ; Alabi CA; Sahay G; Olejnik KT; Wang W; Schroeder A; Lytton-Jean AK; Siegwart DJ; Akinc A; Barnes C; Barros SA; Carioto M; Fitzgerald K; Hettinger J; Kumar V; Novobrantseva TI; Qin J; Querbes W; Koteliansky V; Langer R; Anderson DG, Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates. Proceedings of the National Academy of Sciences of the United States of America 2014, 111 (11), 3955–60. - PMC - PubMed
    1. Dahlman JE; Barnes C; Khan OF; Thiriot A; Jhunjunwala S; Shaw TE; Xing Y; Sager HB; Sahay G; Speciner L; Bader A; Bogorad RL; Yin H; Racie T; Dong Y; Jiang S; Seedorf D; Dave A; Singh Sandhu K; Webber MJ; Novobrantseva T; Ruda VM; Lytton-JeanAbigail KR; Levins CG; Kalish B; Mudge DK; Perez M; Abezgauz L; Dutta P; Smith L; Charisse K; Kieran MW; Fitzgerald K; Nahrendorf M; Danino D; Tuder RM; von Andrian UH; Akinc A; Panigrahy D; Schroeder A; Koteliansky V; Langer R; Anderson DG, In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight. Nat Nano 2014, 9 (8), 648–655. - PMC - PubMed
    1. Novobrantseva TI; Borodovsky A; Wong J; Klebanov B; Zafari M; Yucius K; Querbes W; Ge P; Ruda VM; Milstein S; Speciner L; Duncan R; Barros S; Basha G; Cullis P; Akinc A; Donahoe JS; Narayanannair Jayaprakash K; Jayaraman M; Bogorad RL; Love K; Whitehead K; Levins C; Manoharan M; Swirski FK; Weissleder R; Langer R; Anderson DG; de Fougerolles A; Nahrendorf M; Koteliansky V, Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells. Mol Ther Nucleic Acids 2012, 1, e4. - PMC - PubMed

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