Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing

Abstract

CRISPR-Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues.

Fig. 1 |. Selective ORgan Targeting (SORT) allows lipid nanoparticles (LNPs) to be systematically and predictably engineered to accurately deliver mRNA into specific organs.

a, Addition of a supplemental component (termed a SORT molecule) to traditional LNPs systematically alters the in vivo delivery profile and mediates tissue specific delivery as a function of the percentage and biophysical property of the SORT molecule. This methodology successfully redirected multiple classes of nanoparticles. b, 5A2-SC8 SORT LNPs were formulated as indicated to make series of LNPs with 0% to 100% SORT lipid (fraction of total lipids). Here, inclusion of a permanently cationic lipid (DOTAP) systematically shifted luciferase protein expression from the liver to spleen to lung as a function of DOTAP percentage. c, Quantification data demonstrated that SORT molecule percentage is the most important factor for tissue-specific delivery. Data were shown as mean±s.e.m. (n=4 biologically independent animals). d, Relative luciferase expression in each organ demonstrated that fractional expression could be predictable tuned (0.1 mg/kg Luc mRNA, IV, 6h). Data are shown as mean±s.e.m. (n=4 biologically independent animals). e, Inclusion of an anionic SORT molecule enabled selective mRNA delivery to the spleen. Luciferase expression was observed only in spleen when introducing 18PA lipid into mDLNPs up to 40%. f, Ex vivo images of luminescence in major organs of DLin-MC3-DMA SORT LNPs and C12–200 SORT LNPs (0.1 Luc mRNA mg/kg, IV, 6h). g, Details of selected SORT molecule formulations.

Fig. 2 |. SORT relies on general biophysical properties and not exact chemical structures to deliver mRNAs encoding for therapeutically relevant proteins.

a, SORT molecules could be divided into specific groups with defined biophysical properties. Permanently cationic SORT lipids (DDAB, EPC, and DOTAP) all resulted in the same mRNA delivery profile. b, Anionic SORT lipids (14PA, 18BMP, 18PA) all resulted in the same mRNA delivery profile. c, Ionizable cationic SORT lipids with tertiary amino groups (DODAP, C12–200) enhanced liver delivery without luciferase expression in the lungs (0.1 mg/kg, 6h). d, Scheme for mRNA delivery of secreted proteins. e, High levels of hEPO expression persisted for > one week following administration of 0.3 mg/kg hEPO mRNA in 20% DODAP SORT LNPs. Data are presented as mean±s.e.m. (n=3 biologically independent animals). f, hEPO was quantified by ELISA in serum following IV administration of hEPO mRNA in lung-, spleen-, and liver-specific SORT LNPs, and MC3 LNPs. Data are presented as mean±s.e.m. (n=3 biologically independent animals). g, IL-10 was quantified by ELISA in serum following IV administration of mouse IL-10 mRNA in lung-, spleen-, and liver-specific SORT LNPs, and MC3 LNPs. mCherry mRNA SORT formulations and PBS were used as controls. Data are presented as mean±s.e.m. (n=3 biologically independent animals). A two-tailed unpaired t-test was used to determine the significance of the indicated comparisons of data from f and g. (*P < 0.05; **P<0.01; ***P < 0.001; ****P<0.0001).

Fig. 3 |. SORT LNPs enabled tissue-specific Td-Tomato activation by Cre mRNA delivery.

a, Schematic illustration shows that delivery of Cre mRNA activates Td-Tom expression in Td-Tom transgenic mice via Cre-mediated genetic deletion of the stop cassette. b, mDLNP and liver SORT LNPs (20% DODAP) induced Td-Tom fluorescence specifically in the liver and lung SORT LNPs (50% DOTAP) selectively edited the lung. Td-Tom fluorescence of main organs was detected 2 days following IV injection of Cre mRNA-loaded LNPs (n=3 biologically independent animals). c, Spleen SORT LNPs (30% 18-PA) induced gene editing in the spleen (note high liver background fluorescence in PBS injected mice). Data are presented as mean±s.e.m. (n=3 biologically independent animals). d, Confocal microscopy was employed to further verify effective tissue editing (n=3 biologically independent animals). Scale bars = 20 μm and 100 μm. e, FACS was used to quantify the percentage of TdTom⁺ cells within defined cell type populations of the liver, lung, and spleen (0.3 mg/kg, day 2). Data are presented as mean±s.e.m. (n=3 biologically independent animals).

Fig. 4 |. SORT LNPs mediated tissue-specific CRISPR/Cas gene editing of Td-Tom transgenic mice and C57/BL6 wild type mice by co-delivering Cas9 mRNA and sgRNA and by delivering Cas9 RNPs.

a, Schematic illustration shows that co-delivery of Cas9 mRNA (or Cas9 protein) and sgTom1 activates Td-Tom expression in Td-Tom transgenic mice. b, mDLNP and SORT LNPs (20% DODAP) induced Td-Tom fluorescence specifically in the liver and SORT LNPs (50% DOTAP) selectively edited the lung with 2.5 mg/kg dose (Cas9 mRNA/sgTom1, 4/1, wt/wt; measured day 10, n=3 biologically independent animals). c, tdTom expression was confirmed by confocal imaging of tissue sections (n=3 biologically independent animals). Scale bars = 20 μm and 100 μm. d, Cas9 mRNA and sgPTEN were co-delivered in SORT LNPs to selectively edit the liver, lung, and spleen of C57/BL6 mice with 2.5 mg/kg total mRNA (Cas9 mRNA/sgPTEN, 4/1, wt/wt; measured day 10, n = 3 biologically independent animals). The T7E1 assay indicated that tissue-specific PTEN editing was achieved. Editing was quantified by DNA sequencing and TIDE analysis. e, H&E sections and IHC further confirmed successful PTEN editing (n=3 biologically independent animals). Clear cytoplasm indicated lipid accumulation in H&E sections and PTEN loss in IHC images. Scale bar = 60 μm. f, Delivery of Cas9/sgTom1 ribonucleoprotein (RNP) complexes in 7% DOTAP or 55% DOTAP SORT LNPs induced Td-Tom fluorescence specifically in the liver and lungs, respectively (1.5 mg/kg sgTom1, day 7, n=3 biologically independent animals). tdTom expression was confirmed by confocal imaging of tissue sections. Scale bars = 20 μm and 100 μm. g, Liver- and lung-tropic SORT LNPs also delivered Cas9/sgPTEN RNPs to selectively edit the liver and lungs C57/BL6 mice (1.5 mg/kg sgPTEN; day 7, n = 3 biologically independent animals). The T7E1 and TIDE assays indicated that tissue-specific PTEN editing was achieved. h, C57BL/6 mice were IV injected three times (days 0, 2, 4) by co-delivery of Cas9 mRNA and modified sgPCSK9 with 20% DODAP SORT LNPs (n = 4 biologically independent animals). At day 9, ~60% Indel at PCSK9 locus of liver tissue was quantified by (i) T7E1 assay and (j) TIDE analysis, which resulted to ~100% PCSK9 protein reduction in (k) liver tissue (western blot) and (l) serum (ELISA), respectively. The results of h-l were given from 4 biologically independent animals. Data of j and l are presented as mean±s.e.m. A two-tailed unpaired t-test was used to determine the significance of the indicated comparisons of data from j and l. (*P < 0.05; **P<0.01; ***P < 0.001; ****P<0.0001).