Description of the data and file structure

The data is organized in compressed subdirectories whose names correspond to figures in the main directory (Figure_1, Figure_2, Figure_S1, etc.). Exception: Figure S3 is the uncropped, unadjusted image of the gel.

Figure_1.zip

Figure_1a: Anti-Cre immunoblot of Cre-eVLP before and after purification by sucrose cushion ultracentrifugation. Rafal H 2024-02-25 20h25m53s(Chemiluminescence).jpg = Chemiluminescence scan of the immunoblot. Rafal H 2024-02-25 20h25m53s(Cy3) = red channel scan of the immunoblot. Rafal H 2024-02-25 20h25m53s(Cy5).jpg = blue channel scan of the immunoblot.

Figure1b_VLP!_170713-2.jpg: A representative electron microscopic image of purified eVLPs.

Figure_1c,d: Quantification of mCherry delivery into HEK 293T cells by fluorescence microscopy. Brightfield = brightfield images of the cells. RFP = red fluorescence images of the cells. README.md = table of file names, time after application of the eVLP and red fluorescence intensity. 

Figure_1e: 

RIMG2419.JPG: SDS-PAGE gel images after staining with Coomassie Brilliant Blue of Cre-eVLPs produced in DMEM medium supplemented with 10% (or less) FBS, and purified by ultracentrifugation.

Rafal H 2024-10-09 16h43m59s = Immunoblot of the Cre-eVLPs with anti-Cre antibodies.

Rafal H 2024-10-09 16h55m15s = Immunoblot of the Cre-eVLPs with anti-VSV-G antibodies.

Chemiluminescence = signal from the antigen, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_1f.xlsx: Stable-isotope labeled quantification of Cre in eVLPs separated by ultracentrifugation. Gag designates a peptide specific for MLV Gag, Cre designates a peptide specific for Cre recombinase. Given values include molecular weight (MW) in g/mol, charges in each peptide, mass-to-charge ratio (m/z), molar concentrations of each peptide, signal intensities, and derived protein concentrations in samples.

Figure_1g.xlsx: Estimation by untargeted mass spectrometry of the relative abundance of classes of proteins in eVLPs. Labels are associated with FBS concentration in production culture. The detected proteins were assigned to VLP constituents, serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and Label Free Quantification (LFQ) of 0.01 were used as thresholds to judge the real presence of each protein in the samples.

Figure_1i: Delivery of functional Cre recombinase into HEK293-loxP-GFP-RFP cells via eVLPs purified by ultracentrifugation. The VLPs were standardized for Gag-Cre by Western blotting. CH1 = green channel, CH2 = red channel. README.md contains the assignment of the images to the appropriate concentrations of FBS in production cultures.

Figure_2.zip

Figure2_b: Coomassie-stained SDS-PAGE (RIMG2696.JPG) and anti-Cre immunoblot analyses (Rafal H 2025-03-08 00h28m21s) of Cre-eVLPs, purified by Capto Core 700. Chemiluminescence = signal from the antigen, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure2_c: Conversion of HEK color-switch cells by eVLPs, before and after CC700-purification. The eVLPs were standardized by immunoblot. README.md contains the assignment of images to data points.

Figure2_d: Heparin-Sepharose High Performance chromatography. Heparin.TXT = data as exported from Bio-Rad DuoFlow. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Fraction = corresponding fraction number. Heparin.tsv = Data used to plot the chromatogram.

Figure2_e: Anti-Cre Western-blot analyses of fractions collected from the purifications by heparin. Chemiluminescence = signal from Cre, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_2_f,j,n.xlsx: Abundance analysis of protein classes detected in eVLPs concentrated on centrifugal filters, before and after purification via heparin- Q-, or DEAE-chromatography. Samples: C = VLP purified by Capto Core 700, CH = VLP purified by Capto Core 700 and Heparin, CD = VLP purified by Capto Core 700 and DEAE, CQ = VLP purified by Capto Core 700 and Q. The detected proteins were assigned to VLP constituents (Cre, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure2_g: Conversion of HEK color-switch cells by eVLPs, before and after further purification via heparin chromatography. README.md contains the assignment of file names to the tested samples.

Figure2_h: Q-Sepharose High Performance chromatography. 121024 cre evlp cc700 q.TXT = chromatogram file as exported from the Bio-Rad DuoFlow chromatography system. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Fraction = corresponding fraction number. 121024 cre evlp cc700 q.WMF = plotted chromatogram.

Figure2_i: Anti-Cre Western-blot analyses of fractions collected from the purifications by Q. Chemiluminescence = signal from Cre, Cy3 = red channel, Cy5 = blue channel.

Figure2_k: Conversion of HEK color-switch cells by eVLPs, before and after further purification via Q chromatography. README.md contains the classification of images to data points.

Figure2_l: DEAE-Sepharose High Performance chromatography. 052824 cre evlp cc700ii deae 5 ml.txt contains numerical values as exported from Bio-Rad DuoFlow. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Fraction = corresponding fraction number. 052824 cre evlp cc700ii deae 5 ml.WMF = plotted chromatogram. 

Figure2_m: Anti-Cre Western-blot analyses of fractions collected from the purifications by DEAE. Rafal H 2024-05-30 11h37m35s(Chemiluminescence).jpg = signal from Cre. Cy3, Cy5 = red and blue fluorescence channels, respectively.

Figure2_o: Conversion of HEK color-switch cells by eVLPs, before and after further purification via DEAE chromatography. README.md contains the classification of images to data points.

Figure_3.zip: 

Figure_3a.xlsx: Stable isotope-labeled peptide quantification of Cre in Cre-eVLP preparations purified by ultracentrifugation alone. Gag designates a peptide specific for MLV Gag, Cre designates a peptide specific for Cre recombinase. Given values include molecular weight (MW) in g/mol, charges in each peptide, mass-to-charge ratio (m/z), molar concentrations of each peptide, signal intensities, and derived protein concentrations in samples. The labels correspond to the batch numbers of each eVLP sample.

Figure_3b.xlsx: Relative abundance of classes of proteins in eVLPs. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (Cre, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_3c: eVLP-preparation #47, resolved on a 4.7-ml Capto Core 700 column. 121024 cre evlp cc700 q.WMF = plotted chromatogram. 121024 cre evlp cc700 q.TXT = chromatogram. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_3d: Chromatogram of eVLP #47 collected from the CC700 column, concentrated, then applied and eluted from a HiPrep 16/60 Sephacryl S300HR size-exclusion-chromatography (SEC) column. 121124 cre evlp r cc q s300.WMF = plotted chromatogram. 121124 cre evlp r cc q s300.TXT = exported chromatogram. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_3e.xlsx: Classification of detected proteins in eVLP #47 before purification (“47”), after CC700 (“47C”), and after additional SEC chromatography (“47CS”). Labels are assigned to samples. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (Cre, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_3f.xlsx: Mass-spectrometric protein identification of eVLP preparation #19 before and after SEC.  Labels are assigned to samples. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (Cre, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_3h,i.pptx, Figure_3h_i.csv: Two-photon images of intact mT/mG mouse eyes 2 weeks after injection of Cre eVLP #19, before and after SEC purification. The table presents quantification results of the tdTomato-to-eGFP conversion in the RPE of Cre-eVLP-treated mT/mG mice.

Figure_4.zip: 

Figure_4a.txt: 
DEAE ion-exchange chromatogram of Cre-eVLPs prepurified on a 20-ml CC700 column, eluted with a steep 10 column-volume (CV) gradient (125–562 mM NaCl). Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_4b: Anti-Cre and anti-VSV-G immunoblots of fractions of Cre-eVLPs from experiment (A). Rafal H 2025-03-08 00h40m24s = anti-Cre, Rafal H 2025-03-08 20h26m32s = anti-VSVG. Chemiluminescence = signal from the antigen, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_4c.txt: DEAE ion-exchange chromatogram of Cre-eVLPs purified directly from the cell culture medium. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_4e,f.xlsx: Classification of proteins detected in eVLP preparations by untargeted mass spectrometry. Label-free quantification of proteins present in eVLP preparations purified by DEAE and ultracentrifugation. Labels are assigned to samples. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (Cre, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_4g: Fluorescence microscopic images of HEK color-switch cells, 48 h after application of Cre-eVLPs, purified via DEAE chromatography, and ultracentrifuged. The eVLPs were standardized for Gag-Cre content via quantitative immunoblot analysis. README.md assigns fluorescence micrographs to samples. CH1 = green channel, CH2 = red channel.

Figure_4h,i,j: Figure_4h.jpg and Figure 4i.jpg: Two-photon scans of eyes from mT/mG mice, 6 weeks after subretinal injection of Cre-eVLPs that were purified by DEAE chromatography and ultracentrifugation. Scales represent dimensions in μm. The data are representative of at least seven images. Figure_4h,i.xlsx: Stable-isotope labeled quantification of Cre in eVLPs separated by ultracentrifugation. Gag designates a peptide specific for MLV Gag, Cre designates a peptide specific for Cre recombinase. Given values include molecular weight (MW) in g/mol, charges in each peptide, mass-to-charge ratio (m/z), molar concentrations of each peptide, signal intensities, and derived protein concentrations in samples. Labels are assigned to samples. Figure_4j.xlsx: Estimation of the extent of color conversion in the RPE of the eyes of mT/mG mice treated with purified Cre eVLPs. 

Figure_5.zip:

Figure_5a: DEAE ion-exchange chromatogram of ABE-eVLPs, prepurified on a 20-ml CC700 column. 032125 abe vlp ccde.WMF = plotted chromatogram. 032125 abe vlp ccde.TXT = chromatography data. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_5b: Anti-Cas9 and anti-VSV-G immunoblots of fractions of ABE-eVLPs from experiment (A). Rafal H 2025-03-23 01h16m47s and Rafal H 2025-03-23 01h29m01s = anti-Cas9, Rafal H 2025-03-24 02h22m55s and Rafal H 2025-03-24 02h30m10s = anti-VSV-G. Chemiluminescence = signal from the antigen, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_5c: DEAE ion-exchange chromatogram of ABE-eVLPs, purified directly from the cell culture medium. 022825 abe evlp rde.WMF = plotted chromatogram, 022825 abe evlp rde.TXT = chromatogram data. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_5d: Anti-Cas9 and anti-VSV-G immunoblots of fractions of ABE-eVLPs from experiment (C). Rafal H 2025-03-18 03h56m27s = anti-Cas9, Rafal H 2025-03-04 00h21m53s = anti-VSV-G. Chemiluminescence = signal from the antigen, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_5e.xlsx; Figure_5g.xlsx: Mass spectrometric classification of proteins detected in purified ABE-eVLPs. Numbers denote peak numbers; S denotes concentration by ultracentrifugation. Labels are assigned to samples. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (Cas9, TadA, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_5f.xlsx; Figure_5h.xlsx: SIL peptide quantification of Cas9, TadA, and Gag in pooled fractions from each peak of DEAE eluate, concentrated by ultracentrifugation. Labels are assigned to samples. Gag designates peptide specific for MLV Gag, Cas9 designates peptide specific for Cas9, ABE designates peptide specific to TadA deaminase, PE2 designates peptide specific for reverse transcriptase. Given values include molecular weight (MW) in g/mol, charges in each peptide, mass-to-charge ratio (m/z), molar concentrations of each peptide, signal intensities, and derived protein concentrations in samples.

Figure_5j: Fluorescence microscopic images of rd12 color-switch reporter cells 48 h after application of purified ABE-eVLPs. README.md assigns images to samples.

Figure_6.zip:

Figure_6a.xlsx: Representative electroretinographic (ERG) traces recorded for rd12 mice treated with purified ABE eVLPs. WT = wild-type mouse, rd12 = non-treated rd12 mouse, CDS1, DS1, DS2 = rd12 mice treated with specific ABE eVLP.

Figure_6bc.xlsx: ERG a-wave and b-wave amplitudes recorded for treated rd12 mice, and statistical analysis as reported by GraphPad software.

Figure_6d: Example of the result of targeted amplicon sequencing analysis done by CRISPResso2. Amplicon sequencing reads were assigned to alleles of treated rd12 mouse with on-target, on-target and bystander, and bystander-only adenine deamination.

Figure_6e; Figure_6f: Targeted amplicon sequencing to assess editing efficiency in the Rpe65 rd12 locus in genomic DNA (D) and cDNA (E), isolated from the RPE of ABE-eVLP-treated rd12 mice. fastq.gz = compressed reads, .sh = bash commands to run CRISPResso analysis. README.md assigns reads to treatments of the mice.

Figure_7.zip

Figure_7a.txt: DEAE ion-exchange chromatogram of PE-eVLPs, prepurified on a 20-ml CC700 column. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_7b: Anti-VSV-G immunoblot of fractions of PE-eVLPs from experiment (A). Chemiluminescence = signal from VSV-G, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_7c.xlsx: Mass spectrometric classification of proteins detected in purified PE-eVLPs. Labels are assigned to samples. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (Cas9, MLV RT, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_7d; Figure_7e; Figure_7f.xlsx: DEAE ion-exchange chromatogram, anti-VSV-G immunoblot, and mass spectrometric classification of proteins analogous to (A–C) for PE-eVLPs, purified directly from the cell-culture medium. Figure 7d, 030225 pe3b rd12 evlp rde.WMF = plotted chromatogram, 030225 pe3b rd12 evlp rde.TXT = chromatography data: Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number. Figure_7e: Chemiluminescence = signal from VSV-G, Cy3 = red fluorescence, Cy5 = blue fluorescence. Figure 7f.xlsx, Labels are assigned to samples. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (Cas9, MLV RT, Gag, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_7g: Fluorescence microscopic images of rd12 color-switch reporter cells 48 h after application of purified PE-eVLPs. README.md assigns photographs to samples.

Figure_7h: Targeted amplicon sequencing to document editing efficiency in the Rpe65 rd12 locus in genomic DNA (gDNA) and cDNA isolated from the RPE of PE-eVLP-treated rd12 mice. fastq.gz = compressed reads, .sh = bash commands to run CRISPResso analysis. README.md assigns reads to treatments of the mice.

Figure_8.zip:

Figure_8a: Anti-Cas9 and anti-VSV-G immunoblots of fractions of PE-ENVLPEs+ collected from a 20-ml CC700 column. Rafal H 2025-03-21 13h13m06s = Cas9, Rafal H 2025-03-22 00h50m51s = Cas9 and VSV-G. Chemiluminescence = signal from antigens, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_8b.txt: DEAE ion-exchange chromatogram of PE-ENVLPEs+, prepurified on a 20-ml CC700 column. Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number.

Figure_8c: Anti-Cas9 and anti-VSV-G immunoblots of fractions of PE-ENVLPEs+ from experiment (B). Rafal H 2025-03-21 13h38m47s = Cas9, Rafal H 2025-03-22 01h02m04s = Cas9 + VSV-G. Chemiluminescence = signal from antigens, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_8d; Figure_8e: DEAE ion-exchange chromatogram and immunoblots analogous to (B, C) for PE-ENVLPEs+, purified directly from the cell-culture medium. Figure_8d.WMF = plotted chromatogram. Figure_8d.TXT = chromatography data, Time = time (sec), Quadtec1 = absorbance at 230 nm, QuadTec 2 = absorbance at 260 nm, QuadTec3 = absorbance at 280 nm, Gradient pump = Percentage of elution buffer in the system, Conductivity = conductivity of the solution (mS/cm), Volume = elution volume rounded to 0.1 ml, Fraction = corresponding fraction number. Figure_8e, Rafal H 2025-03-15 04h22m38s = Cas9, Rafal H 2025-03-15 23h34m16s = Cas9 + VSV-G. Chemiluminescence = signal from antigens, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_8f.xlsx: Mass spectrometric classification of proteins detected in purified PE-ENVLPEs+. Labels are assigned to samples. Columns in the Intensity ratio subtable designate the eVLP batch and replicate. The detected proteins were assigned to VLP constituents (PE2, Csy4, Gag, PCP, Pol, VSV-G), serum proteins, keratins, and host cell proteins ("Other") based on the descriptions from the Uniprot database. A score of 10 and a ratio of Label Free Quantification (LFQ) of each protein versus LFQ of Cre of 0.01 were used as thresholds to judge the presence of each protein in the samples.

Figure_8g: Fluorescence microscopic images of TIGER-reporter cells, 48 h after application of purified PE-ENVLPEs+. README.md assigns photographs to samples.

Figure_8h: Untitled-1.jpg: Two-photon fluorescence image of the posterior segment of the intact eye of a heterozygous TIGER-reporter mouse treated with PE-ENVLPEs+; scale is in μm. Figure_8h.xlsx: Labels are assigned to samples. Cas9 designates a peptide specific for Cas9, PE2 designates a peptide specific for reverse transcriptase. Given values include molecular weight (MW) in g/mol, mass-to-charge ratio (m/z), molar concentrations of each peptide, signal intensities, and derived protein concentrations in samples.

Figure_S1.zip: Figure_S1a: RIMG2221.jpg: Coomassie-stained SDS-PAGE of Cre-eVLPs, purified on 4.7 ml HiScreen Capto Core 400.  Rafal H 2024-01-24 22h04m36s: anti-Cre immunoblot analyses of Cre-eVLPs, purified on 4.7 ml HiScreen Capto Core 400. Chemiluminescence = signal from antigens, Cy3 = red fluorescence, Cy5 = blue fluorescence. Figure_S1b: RIMG2241.jpg: Coomassie-stained SDS-PAGE of Cre-eVLPs, purified on 4.7 ml HiScreen Capto Core 700.  Rafal H 2024-01-30 11h37m39s: anti-Cre immunoblot analyses of Cre-eVLPs, purified on 4.7 ml HiScreen Capto Core 700. Chemiluminescence = signal from antigens, Cy3 = red fluorescence, Cy5 = blue fluorescence.

Figure_S2.zip: Fluorescence microscopic images of HEK color-switch cells, 48 h after application of the eVLPs isolated by ultracentrifugation in independent experiments; eVLP #47, before and after sequential chromatographic purification; eVLP #19, before and after purification. README.md assigns photographs to samples.

Figure_S3.JPG: Purification of Cre-eVLPs assessed using silver staining. Cre-eVLP samples were subjected to DEAE chromatography with or without pre-purification on Capto Core 700 (CC700). The samples were analyzed by SDS-PAGE, and the gels were silver-stained. The samples were standardized for Gag-Cre by immunoblotting, and a quarter-volume of unpurified medium with eVLPs was loaded to avoid saturating the image and losing band resolution.

Figure_S4.zip: Analysis of ABE-eVLPs targeting Rpe65 rd12, purified by DEAE ion-exchange chromatography, with (a) or without (b) pre-chromatography on Capto Core 700. The samples were resolved using SDS-PAGE and developed by silver staining. Within each gel, the samples were standardized for VSV-G, and one-fourth volume of the medium (a) or one-fifteenth volume (b) containing eVLPs was loaded to avoid oversaturation of the signal.

Figure_S5.zip: Rafal H 2025-07-03 12h48m37s: Anti-RPE65 immunoblots of lysed eyecups, isolated from ABE-eVLP-treated rd12 mice. Rafal H 2025-07-03 19h03m22s: anti-β-actin immunoblots of lysed eyecups, isolated from ABE-eVLP-treated rd12 mice. Chemiluminescence = signal from antigens, Cy3 = red fluorescence, Cy5 = blue fluorescence. Figure_S5b.xlsx: Quantification of 11-cis-retinal in the eye homogenates from treated mice. Labels correspond to treatments.

Figure_S6.zip: (a, b) Analysis of PE-eVLPs targeting Rpe65 rd12. (c, d) Analysis of PE-ENVLPEs+ targeting the TIGER construct. The samples were resolved using SDS-PAGE and developed by silver staining. Within each gel, the samples were standardized for VSV-G, and one-fourth volume of the medium (a) or one-fifteenth volume (b,c,d) containing eVLPs was loaded to avoid saturation with the dye.

README.md (this file)

MS_fasta.zip contains a database of protein sequences used in mass spectrometric analysis in fasta format, downloaded from the Uniprot database.

Sharing/Access information

Raw mass spectrometry data are available: https://www.ebi.ac.uk/pride/archive/projects/PXD070195

Code/Software

Scripts used to analyze amplicon sequencing results with CRISPResso2 are attached in the respective figure subdirectories.