DOI: 10.5061/dryad.c2fqz61q6
title: Scalable purification enables high-quality virus-like particles for therapeutic translation

description: Emerging molecular therapies introduce enzymatic activity into cells by
 delivering genes, transcripts, or proteins. Owing to their robust
 cell-entry capacity, virus-like particles (VLPs) represent a technology of
 choice in genome editing, where low doses of heterologous proteins and
 nucleic acids are essential. However, clinical translation of VLP vectors
 is hindered by inadequate purification methods. Current approaches,
 relying primarily on ultracentrifugation, suffer from inconsistent product
 quality and poor scalability. Here, we report the development of a broadly
 applicable purification strategy that improves the purity and therapeutic
 efficacy of genome-editing VLPs. Considering the characteristic properties
 of murine leukemia virus (MLV)-derived engineered VLPs (eVLPs) and
 HIV-derived engineered nucleocytosolic vehicles for loading of
 programmable editors (ENVLPEs+), we developed a workflow that involves
 single- and multi-modal chromatographic steps, effectively removing host
 cell proteins and cell-culture contaminants while improving VLP integrity
 and biological activity. Our purified VLPs displayed superior protein
 composition, consistency, and enhanced functional delivery compared to
 VLPs partially purified by conventional ultracentrifugation methods. Mass
 spectrometric analysis revealed a substantial decrease in contaminants,
 with VLP-specific proteins comprising &gt;90% of the final product. In
 vivo studies confirmed improved therapeutic outcomes when
 chromatographically purified VLPs were used. Our scalable purification
 platform addresses critical manufacturing bottlenecks and constitutes a
 starting point for further development of VLP therapeutics, enabling
 robust production of pure VLPs for diverse applications such as genome
 editing, vaccine development, and other uses that require intracellular
 protein delivery.
description: # Scalable purification enables high-quality virus-like particles for
 therapeutic translation ## 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](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.