Extended Data Fig. 3: Optimization of ABE RNP LNPs. | Nature Biomedical Engineering

Extended Data Fig. 3: Optimization of ABE RNP LNPs.

From: Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations

Extended Data Fig. 3

(a,b) Immunoprecipitation of ABE RNP, free or encapsulated in the LNP with SM102 ionizable lipid and 1.5% DMG-PEG 2000, using 1D4 resin or control B6-30 resin. Elution was done using 1 mg ml-1 1D4 peptide (E) or using Laemmli sample buffer with DTT (E*). 1D4 and B6-30 resins treated with Laemmli sample buffer alone served as additional controls. The arrow (at the right) indicates the band corresponding to ABE; hash marks correspond to bands from mouse 1D4 and B6-30 antibodies detected by equine anti-mouse secondary HRP-linked antibody. (c) Relative cytotoxicity of LNP with ionizable lipid DODMA, with or without SOPS, demonstrated using fluorescence-microscopic images from three transfection experiments at 20 nM RNP (4.5 µg ml-1). (d) Stability (upon storage at 4 °C or -80 °C) of ABE RNP LNP made with 50% ionizable lipid and 1.5% DMG-PEG 2000, as tested with rd12 reporter cells and 20 nM ABE RNP. Three replicates, mean ± s.d. (e,f) Particle size distribution and delivery efficiency of ABE RNP LNP made with various lipid:RNA weight ratios. Asterisks denote 2.5% DMG-PEG 2000; otherwise, the concentration was 1.5%. Plots shown in panels e and f represent data that were averaged from n = 3 replicates, mean ± s.d. (g) Encapsulation of ABE RNP into LNPs with ionizable lipid SM102 and 2.5% DMG-PEG 2000. (h) Size exclusion chromatograms of ABE8e RNP (black) and ABE8e RNP LNP with SM102 lipid and 2.5% DMG-PEG2000, 40:1 lipid:sgRNA ratio (red) resolved on a Sephacryl S-500 HR column (top) and anti-Cas9 blots of collected fractions (bottom). (i,j) ABE editing in the rd12 reporter cells, using ABE RNP LNP visualized via fluorescence microscopy (here, 20 nM ABE8e RNP) and quantified by flow cytometry. Scale bar: 100 µm. Three replicates. (k) ERG responses of rd12 mice treated with ABE8e LNPs made using ionizable lipid SM102 and 1.5 – 2.5% DMG-PEG 2000 (2.3 and 2.5 µM, 515 and 560 ng, respectively), ABE8e N108Q LNPs made with 2.5% DMG-PEG 2000 (2.3 µM, 515 ng), or ABE8e LNPs with non-targeting guide RNA (2.5 µM, 560 ng), 1 µl per eye. At least 8 eyes, mean ± s.d, Kruskal-Wallis test with Dunn’s multiple comparisons test, ***P < 0.001; ****P < 0.0001. Uncropped blots are available within Source data.

Source data

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