PMC

Multiple steps are involved in the mechanism of SORT LNP tissue targeting, including formation of unique protein coronas. (A) A proposed three-step endogenous targeting mechanism for tissue-specific mRNA delivery by SORT LNPs in which 1) PEG lipid desorption 2) enables distinct plasma proteins to bind SORT LNPs, 3) resulting in cellular internalization in the target tissues by receptor-mediated uptake. (B) Ex vivo bioluminescence of major organs excised from C57BL/6 mice IV injected with liver, spleen, and lung SORT LNPs incorporating either sheddable PEG lipids (C14-PEG2K) or less sheddable PEG lipids (C18-PEG2K) (0.1 mg FLuc mRNA/kg body weight, 6 h). Total luminescence produced by each organ is reduced when less sheddable PEG lipid is used, suggesting that PEG lipid desorption is a key process for efficacious mRNA delivery by SORT LNPs. (C) Quantification of total luminescence produced by functional protein translated from FLuc mRNA in target organs of C57BL/6 mice IV injected with liver, spleen, and lung SORT LNPs incorporating either C14- or C18-PEG2K (0.1 mg FLuc mRNA/kg body weight, 6 h). (D) ELISA quantification of serum hEPO in C57BL/6 mice treated with liver, spleen, or lung SORT LNPs encapsulating hEPO mRNA (0.1 mg hEPO mRNA/kg body weight, 6 h). Using a less-sheddable PEG reduces SORT LNP potency. (E) SDS–PAGE of the plasma proteins adsorbed to the surface of mDLNP, liver SORT, spleen SORT, and lung SORT LNPs. LNPs with different organ-targeting properties bind distinct plasma proteins. (F) The average abundance of proteins with distinct biological functions in the protein coronas of mDLNP and liver, spleen, and lung SORT LNPs. The choice of SORT molecule leads to large-scale differences in the functional ensemble of plasma proteins which bind the LNP. (G) Isoelectric point distribution for the most enriched proteins which constitute 80% of the protein corona of the LNPs. A SORT molecule’s headgroup structure influences the pI distribution of the protein corona. (H) The top five most abundant plasma proteins that bind different SORT LNPs (n = 3). The chemical structure of SORT molecule affects the number one plasma protein that is most highly enriched on the surface of SORT LNPs. Data are shown as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t test (*P < 0.05).

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.

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).

(A) Depiction of the cells’ passaging and sorting scheme, with the initial sorted pools characterized in at the top, followed by the re-sorted sub-pools analyzed here. The % GFP+ above Pool 1 or Pool 2 in (A) or (B) respectively represents the % GFP+ cells in that Pool prior to sorting. The percentages listed for the sorted cells below that indicate the proportion of the unsorted pool that were sorted into the indicated samples, after gating as described in . (B) Analysis of zip codes that sorted GFP+ in Pool 1. Y axis indicates the GFP+ proportions determined in the second sort (within beige shaded box; this was a re-sorting of the sub-pool that had sorted GFP+ in the first sort and had been passaged separately for > I week) and X axis is GFP+ proportions from Pool I first sort (eg: X axis) (C) Analysis of zip codes that sorted GFP- in Pool 1. Y axis indicates the GFP+ proportions determined in the second sort (within beige shaded box; this was a re-sorting of the sub-pool that had sorted GFP- in the first sort and had been passaged separately for > I week) and X axis is GFP+ proportions from Pool I first sort (eg: X axis) (D, E, F) Analysis performed as in , for zip codes in Pool 2. (G) Stability of GFP+ proportions in Pool 1 over time. GFP+ proportions determined in the first sort ( data) plotted against the reconstructed second sort, as assessed by comparing GFP+ proportions for each zip code derived from data in Pool 1 at the second sort (Y axis, data from panels B and C) vs the first sort ( X axis). Second sort GFP+ proportions were reconstructed by weighting the GFP+ and GFP- sub-pool values determined in panels A and B as described in Materials and Methods. (H) Stability of GFP+ proportions in Pool 2 over time, performed as described in panel G for Pool 1.