DOI: 10.5061/dryad.d51c5b0hd
title: Data from: A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina

description: CRISPR/Cas9-based gene-editing technologies offer promise for treating
 inherited retinal diseases (IRDs); however, safe and efficient ocular
 delivery of precision editors remains challenging. To address this
 challenge, we report a new class of Coomassie brilliant blue (CBB)-derived
 lipidoids that bind and deliver proteins. Subretinal injection of Cre
 complexed with these lipidoids into mT/mG mice leads to robust
 recombination in the retinal pigment epithelium and photoreceptors. We
 employ the CBB-lipidoid platform to deliver adenine base editor (ABE)
 ribonucleoproteins (RNP). Incorporating CBB lipidoids into liposomes
 improves delivery efficiency. CBB11 stands out for facilitating precise in
 vivo ABE-mediated gene editing. Delivery of liposome-CBB11-RNP complexes
 results in a 120-fold increase in base editing compared to RNP alone and
 restores the scotopic ERG b-wave response in the rd12 mouse model. These
 results demonstrate the potential of CBB-augmented, liposome-RNP systems
 for therapeutic gene editing in the eye, paving the way for single-dose
 precision medicines to treat IRDs.
description: # Data from: A combinatorial synthetic strategy for developing
 genome-editing protein-delivery agents targeting mouse retina ####
 Description of the data and file structure CRISPR/Cas9-based gene-editing
 technologies offer promise for treating inherited retinal diseases (IRDs);
 however, safe and efficient ocular delivery of precision editors remains
 challenging. To address this challenge, we report a new class of Coomassie
 brilliant blue (CBB)-derived lipidoids that bind and deliver proteins.
 Subretinal injection of Cre complexed with these lipidoids into mT/mG mice
 leads to robust recombination in the retinal pigment epithelium and
 photoreceptors. We employ the CBB-lipidoid platform to deliver adenine
 base editor (ABE) ribonucleoproteins (RNP). Incorporating CBB lipidoids
 into liposomes improves delivery efficiency. CBB11 stands out for
 facilitating precise *in vivo* ABE-mediated gene editing. Delivery of
 liposome-CBB11-RNP complexes results in a 120-fold increase in base
 editing compared to RNP alone and restores the scotopic ERG b-wave
 response in the rd12 mouse model. These results demonstrate the potential
 of CBB-augmented, liposome-RNP systems for therapeutic gene editing in the
 eye, paving the way for single-dose precision medicines to treat IRDs. The
 data is organized in compressed subdirectories whose name correspond to
 figures in the main directory (Figure_2b, Figure_3, Figure_S1, etc.).
 Abbreviations: * ABE - adenine base editor - CBB - Coomassie Brilliant
 Blue * CBBZ - Coomassie Brilliant Blue with alkyne moiety - DAPI -
 4',6-diamidino-2-phenylindole * DMSO - dimethylsulfoxide - EndoV -
 endonuclease V * ERG - electroretinography - GFP - green fluorescent
 protein * HEK - human embryonic kidney cells - IRDs - inherited retinal
 diseases * kDa - kilodaltons - m/z - mass to charge ratio * MW - molecular
 weight - OCT - optical coherence tomography * PAGE - polyacrylamide gel
 electrophoresis - PdI - polydispersity index * RFP - red fluorescent
 protein - RPE - retinal pigment epithelium * RNP - ribonucleoprotein - TAS
 - tris-acetate-sucrose buffer * TEM - transmission electron microscopy
 Source_data.xlsx: contains data used to draw graphs and the statistical
 analyses as reported by GraphPad. The contents are described below.
 Figure_2b.zip. CBB-mediated delivery of Cre recombinase *in vitro* into
 color-switching Cre reporter cells (CS cells). Fluorescence microscopic
 images of HEK color-switch (CS) cells treated with Cre complexed with CBB
 lipidoids. C, Cre without CBB. (c, d) Quantification of GFP-to-RFP
 conversion in CS cells treated with the complexes of Cre recombinase with
 CBB compounds (c) by flow cytometry or with CBBZ compounds (d) by
 fluorescence microscopy. [Cre]/[CBB(Z)] = 1:8; Cre delivery mediated by
 Lipofectamine 3000 (LF) as a positive control is also shown. C, Cre
 recombinase only. NE, no enzyme. Figure_3b.zip Cell-specific delivery of
 Cre recombinase mediated by CBB lipidoids in the mT/mG mouse retina.
 Representative two-photon microscopy images indicating the switch in
 expression from tdTomato to eGFP, one week after subretinal injection of
 Cre recombinase complexed with CBB lipidoids. The RPE layer is orientated
 toward the top. C, Cre recombinase only. The scale is provided in
 micrometers. Images are representative of at least 3 eyes. Figure_4.zip.
 Delivery of ABE-RNPs to *rd12*-reporter cells mediated by CBB lipidoids.
 Fluorescence microscopy images of rd12 reporter cells treated with ABE8e
 NG RNP-CBB complexes. A = ABE RNP only; LF = ABE RNP with Lipofectamine
 3000, CBB = ABE with specified CBB compound). (c,d) Quantitative
 assessment of ABE-RNP delivery into *rd12*-reporter cells, mediated by
 lipidoid complexes of ABE8e with CBB compounds (c) by flow cytometry; or
 CBBZ compounds (d) by fluorescence-microscopic imaging only.
 [ABE8e]/[CBB(Z)] = 1:8. A, ABE8e-RNP only. NE, no enzyme. (f - Figure_4f)
 Representative urea-PAGE-gel image of products of ABE deamination cleaved
 by EndoV. The gel was imaged with a fluorescein filter. N, no enzyme; P,
 positive control: 60 bp DNA with inosine in the middle; A, ABE-RNP in TAS;
 D, ABE-RNP in TAS with 10% DMSO. Numbers indicate molar excess of the CBB
 compound *versus* ABE. Left to right: N; P; A; D; CBB22: 4, 8, 16; CBB11:
 4, 8, 16; CBB14: 4, 8, 16. (g) Fluorescence quantification of substrate
 and products of deamination and EndoV cleavage. Two independent samples
 per data point were assayed in parallel. Deaminated (%) is the ratio of
 intensity of the 30 nt band to total intensity of 30 and 60 nt bands.
 Figure_5.zip. Liposome delivery of Cre- and ABE-RNPs to reporter cells.
 (b) The size distribution of the CBB11-alone and CBB11-doped liposomes
 (Lipo1), in TAS buffer. Average of 3 replicates. (c) Fluorescence
 microscopic quantification of the recombination activity triggered by
 Cre-recombinase delivery, mediated by Lipo1 or Lipo2. CBB14 and
 Lipofectamine 3000 served as positive controls. C, Cre recombinase only;
 NE, no enzyme. Data are presented as a mean of 2 replicates. (d) The zeta
 potentials of Lipo1 and Lipo2 in the absence or the presence of ABE-RNP.
 The ratio of ABE-RNP to CBB11 is 1:8. Means of two biological replicates.
 (e - Figure_5e) Representative images of cryo-TEM of Lipo1, ABE-RNP only
 and Lipo1 with ABE-RNP (f) Titration of *in vitro* delivery of ABE8e-RNPs
 mediated by CBB11, CBB14, and Lipo1 ~5 h after the RNP was complexed with
 the CBB compounds and liposomes. The ratio [CBB]/[RNP] = 8:1. The total
 lipid amounts of Lipo 2 and Lipofectamine 3000 were comparable to those
 for Lipo1. (g, h) Titration of *in vitro* delivery of ABE8e-RNPs mediated
 by Lipo3/Lipo4 (g); and Lipo5/Lipo6 (h), respectively, after allowing 15
 min for complex formation in each case. The ionizable lipid concentration
 was always 80-fold greater than the concentration of ABE-RNP. ABE-RNP in
 TAS buffer served as negative control. (i - Figure_5i)
 Fluorescence-microscopic images of *rd12*-reporter cells treated with
 liposome-RNP complex. The RNP concentration was 100 nM. Representative of
 2 biological replicates. Fluorescence microscopy images of rd12 reporter
 cells treated with ABE8e NG RNP-CBB liposome complexes. e = enhanced. CH1
 = green channel, GFP. CH2 = red channel, TxRed. Overlay = overlay of CH1
 and CH2.\ CBB11 = 10x_XY08\ CBB14 = 10x_XY14\ Lipo1 = 10x_XY20\ Lipo2 =
 10x_XY26\ LF = 10x_XY02\ NE = 10x_XY38. Figure 6. ABE-RNP delivery
 mediated by CBB-containing liposomes *in rd12* mice. Four-week-old *rd12*
 mice were treated *via* subretinal injection with 20 µM ABE8e-RNPs with
 indicated reagents, with 8-fold molar excess of CBB and 80-fold molar
 excess of SM-102, where applicable. (a) B-wave amplitudes of ERG responses
 of *rd12* mice recorded two weeks after treatments. (b) Quantification of
 the visual chromophore 11-*cis* retinal in the eyes from ABE-treated
 *rd12* mice obtained after a 24-h dark adaptation. *n* = 6 eyes. (c-f)
 Quantification of (c) total DNA editing, (d) total cDNA editing, (e)
 precise DNA editing and (f) precise cDNA editing in the retinal pigment
 epithelium isolated from the treated *rd12* mice. (g) Off-target adenine
 genome-editing in *rd12* mouse RPE after delivery of ABE-RNPs by Lipo1.
 *N* = 4 isolates picked randomly from samples analyzed in c-d.
 Supplementary Figure 2. Identification of oxidation sites on oxCBB11 with
 mass spectrometry. (a,d) Two isomers of oxCBB11 that eluted from the
 silica gel column were injected into the LC-MS and analyzed by
 selected-ion monitoring at m/z 543.5. (b, e) The MS1 spectrum of the peak
 that eluted at 4.65 min (b) and the MS1 spectrum of the peak at 4.93 min
 (e) were similar. (c, f) The MS2 spectrum (c) of the ion at m/z 543.02 in
 panel b suggested hydroxylation events on one of the polyunsaturated fatty
 acid moieties. The MS2 spectrum (f) of the ion at m/z 543.02 in panel e is
 consistent with hydroxylation events on the tertiary amine of CBB11.
 Supplementary Figure 4. Concentration dependence of the in vitro delivery
 of gene editors mediated by representative CBB compounds. (a)
 Quantification by fluorescence microscopic imaging of GFP-to-RFP
 conversion in CS cells. Cells were treated with Cre recombinase (0.5 μM)
 complexed with CBB compounds at increasing concentrations (1-8 μM). The
 cells emit green fluorescence at baseline and red fluorescence after
 editing. (b) Quantification by fluorescence microscopic imaging of the
 area ratio of eGFP to mCherry in rd12-reporter cells treated with ABE-RNPs
 (100 nM) complexed with CBB compounds at increasing concentrations
 (0.2-1.6 μM). Supplementary Figure 5. Relative viability of reporter cells
 treated with Cre or ABE-RNPs, along with CBB compounds. Color-switching
 cells treated with Cre recombinase and CBB compounds 1-22 (a), or
 rd12-reporter cells treated with ABE-RNPs and CBB compounds 1-22 (b), were
 assessed for viability by DAPI exclusion, measured with flow cytometry.
 LF, lipofectamine 3000; C, Cre recombinase only; A, ABE-RNP only; NE, no
 enzyme. Supplementary Figure 6: Cre is complexed by CBB compounds into
 large macromolecular complexes. The particle size distributions of Cre
 complexed with CBB11 and CBB14. CBB11-Cre formed homogenous particles (d =
 1443 nm, PdI = 0.182), whereas CBB14-Cre formed heterogenous particles
 (average d = 1514 nm, PdI = 0.33) with two species whose d were 167 and
 2129 nm. d = diameter; PdI = polydispersity index. Supplementary Figure 7
 - Figure_S7.pptx: In vivo two‐photon excitation microscopy demonstrating
 Cre recombinase delivery via CBB lipidoids or lipid-nanoparticle
 formulations in the intact mT/mG mouse eye. One week after subretinal
 injection of the Cre‐containing vector, tdTomato‐to‐eGFP expression
 switching was assessed in the mT/mG model. The left panels show the eGFP
 channel (green), and the right panels display the overlay of the tdTomato
 (red) and eGFP (green) channels. The RPE layer, oriented toward the top,
 is marked by open yellow triangles, and the photoreceptor layer is denoted
 by open blue triangles. Scale numbers correspond to micrometers. Images
 are representative of n = at least 3 eyes. Supplementary Figure 8 -
 Figure_S8.zip: Size-exclusion chromatography of CBB liposomes. (a)
 Coomassie fluorescence scan of a 96-well plate containing samples
 collected from PD10 desalting columns. Ve = elution volume. (b,c) A
 photograph (b) and a Coomassie fluorescence scan (c) of the PD10 desalting
 columns after the size-exclusion chromatography. Column Ve (ml) 1 2.00 2
 2.25 3 2.50 4 2.75 5 3.00 6 3.25 7 3.50 8 3.75 9 4.00 10 4.25 11 4.50 12
 4.75 Figure_S9.zip: Addition of CBB11 boosts the liposomal delivery of
 ABE8e-RNPs in rd12-reporter cells. Lipo3-6 correspond to formulations as
 in Fig 5. (a) The particle size distribution of Lipo3 (dark blue) was
 average diameter = 155 nm, with PDI = 0.19; and Lipo4 (orange) was average
 diameter = 155 nm, with PDI = 0.20. (b) The particle size distribution of
 Lipo5 (pale blue) was average diameter = 154 nm, with PDI = 0.17; and
 Lipo6 (yellow) was average diameter = 161 nm, with PDI = 0.15. (c) The
 zeta potential of Lipo3 (dark blue) was -2.64 mV, slightly higher than
 that of Lipo4 (orange), -4.11 mV. (d) The zeta potential of Lipo5 (pale
 blue) was -3.25 mV, slightly lower than that of Lipo6 (yellow), -2.25 mV.
 The measurements for a-d were done 3 replicates. (e) Representative
 fluorescence-microscopic images of rd12-reporter cells treated with
 liposome-RNP complex. The RNP concentration was 50 nM. Lipofectamine 3000
 (LF) served as positive control, and ABE8e-RNPs delivered without vehicles
 (A) served as negative control. The images are representative of 4
 biological replicates. CH1 = green channel, GFP. CH2 = red channel,
 TxRed.\ XY16 = Lipo3\ XY22 = Lipo4\ XY28 = Lipo5\ XY34 = Lipo6\ XY40 =
 Lipofectamine 3000\ XY45 = ABE RNP only. Figure_S10.zip: Representative
 cryo-TEM images of Lipo1. The round vesicles in the images represent lipid
 bilayers of Lipo1 surrounding aqueous cores. Scale bar = 100 nm.
 Figure_S11_RIMG3165.JPG: : Digestion of ABE8e-RNPs (without, or with
 Lipo1) with proteinase K. ABE8e-RNPs were incubated with proteinase K
 (protein:enzyme weight ratio = 100:1) at room temperature for 5, 10, 15,
 or 60 min. ABE8e-RNPs with Lipo1 in the absence of protease K served as a
 negative control.  Left to right: Protein molecular weight marker Bio-Rad
 Precision Plus Prestained; Lipo1 + Proteinase K - 5 min, 10 min, 60 min;
 Lipo1 - Proteinase K + 5 min, 15 min, 60 min; Lipo1 + Proteinase K + 5
 min, 10 min, 60 min; Protein molecular weight marker. From top to bottom,
 protein molecular weight marker bands represent molecular weight (MW) in
 kDa: 250, 150, 100, 75, 50, 37, 25, 20, 15. Molecular weight of ABE: 187.1
 kDa. Figure_S12.zip. (a) Fluorescence microscopic quantification of
 delivery of ABE8e RNP into rd12 reporter cells mediated by liposomes with
 CBB11 (Lipo1, Lipo7) and SM102 (Lipo1, Lipo2). Lipofectamine 3000 (LF) was
 used as a positive control, buffer (TAS) as a negative control. (b)
 Representative fluorescence microscopic images of rd12 reporter cells
 treated with ABE8e RNP complexed with liposomes. The images are
 representative of 4 replicates. Figure_S13.zip.. Treatment of TIGER-HEK
 cells with ABE-RNPs. (b, c) The conversion of non-fluorescent TIGER HEK
 cells to fluorescent after the treatment with ABE8e-NG-RNPs (b) or
 ABE8e-N108Q-NG-RNPs (c), complexed with CBB11, CBB14, Lipo1, or Lipo2.
 [CBB]/[RNP] ratio = 8:1. Results for Lipofectamine 3000 (as a positive
 control) were comparable to Lipo1. Cells treated with ABE-RNP without any
 vehicles (TAS) served as a negative control. Figure_S14.zip: Evaluation of
 the safety and efficacy of Lipo1. (a) Particle size distributions of fresh
 Lipo1, diameter 148 nm and PDI = 0.23; and stored Lipo1, size = 140 nm,
 PDI = 0.16. (b) Fluorescence microscopic quantification of the ratio of
 eGFP to mCherry for rd12 reporter cells, treated with the fresh Lipo1-RNPs
 (100 nM). RNPs with Lipofectamine 3000 (LF) served as positive control,
 and RNPs alone (A) as negative control; [CBB]/[RNP] ratio = 8:1. (c)
 Representative optical coherence tomography (OCT) images of female WT mice
 treated with 20 μM ABE-RNPs alone (R20), 160 μM CBB11 as Lipo1 (L160),
 with 10 μM RNPs and 80 μM CBB11 as Lipo1 (L80R10), or with 20 μM RNPs with
 160 μM CBB11 as Lipo1 (L160R20); control WT mice (WT N) were non-treated.
 The injection volume was 1 μl. N = 4 eyes per treatment. (d) ERG b-wave
 amplitudes of WT mice whose OCT scans are in panel c. (e) 11-cis-retinal
 levels in WT mice whose ERG responses are presented in d. (f) ERG b-wave
 amplitudes of rd12 mice treated with 0.5 μl of L80R10; 1 μl of L80R10; or
 1 μl of L160R20. Untreated WT mice (WT N) and rd12 mice (rd12 N) served as
 positive and negative controls, respectively. (g) Quantification of
 11-cis-retinal levels in rd12 mice whose ERG responses are presented in
 panel f. Figure_S15.zip. Flow cytometry gating strategies. Gating
 strategies used in flow cytometry quantification of (a) CS cells and (b)
 rd12 reporter cells. SSC-A: side scatter area; FSC-A: forward scatter
 area; SSC-H: side scatter, height; FSC-H: forward scatter height; FITC-A:
 green fluorescence intensity; PE-A: red fluorescence intensity; Pacific
 Blue-A: blue fluorescence intensity; P1, P2, P4: gates. Synthesis of CBB:
 Mass spectrometry traces of the CBB compounds. m/z = mass to charge ratio;
 Intensity = signal intensity. ## Sharing/Access information. Amplicon
 sequencing data are available in NCBI Sequencing Read Archive:
 PRJNA1240374 and PRJNA1358750. ## Code/Software. ImageJ was used for
 image-based estimation of conversion of fluorescent reporter cells.