EXTRACELLULAR VESICLES FOR INHALATION

Abstract

Vesicles, including exosomes, having a coating of a hydrophilic, neutral polymer such as PEG have an increased ability to form a suspension or colloid compared to uncoated vesicles. This enables the coated vesicles to be used to form aerosol droplets such that a liquid formulation containing vesicles can be used in a nebulizer for inhaled administration thereof. Such coated vesicles are also able to pass through mucus and can deliver their cargo into lung cells. Exosomes from mesenchymal stem cells can deliver additional proteins, miRs, mRNAs and other nucleic acid sequences to lung cells providing a regenerative gene therapy for CF, COPD lung cancer and other lung diseases.

Claims

1. An aerosolizable composition comprising exosomes from mesenchymal stem cells (MSCs) having a surface coating of the hydrophilic polymer polyethylene glycol (PEG), the vesicles carrying a cargo comprising a microRNA (miR), an anti-miR, mRNA, a long non-coding RNA, a circular RNA, a small interfering RNA, a short hairpin RNA, a piwi-interacting RNA, a CRISPR RNA sequence, modifications of the foregoing or artificially designed nucleic acid sequences, protein, cytokine or lipid, wherein the PEG has a molecular weight of less than 5 kDa, the surface coating covers at least 65% of the exosome surface and substantially neutralises the surface charge of the exosomes.

2. A composition according to claim 1, wherein the hydrophilic polymer comprises DSPE-PEG.

3. A composition according to any preceding claim, wherein the surface coating polymer has a molecular weight of <3 kDa.

4. A composition according to any preceding claim, wherein the exosomes have a cargo comprising a microRNA (miR).

5. A composition according to claim 4, wherein the miR, is selected from miR-125b-5p, miR-125b-1-3p, miR-513a-5p and miR-17. The stem-loop (pre-microRNA) sequence hsa-mir-125b-1 may be overexpressed to yield EVs containing both miR-125b-1-3p and miR-125b-5p mature sequences.

6. A composition according to any preceding claim wherein the exosomes have a cargo comprising an mRNA or its translated protein.

7. A composition according to claim 6, wherein the mRNA is a modified CFTR mRNA.

8. A composition according to any preceding claim, comprising an aqueous suspension or colloid of the exosomes.

9. A composition according to any preceding claim, for use in therapy.

10. A composition for use according to claim 9, for use in therapy of lung disease.

11. A composition for use according to claim 9 or 10, for use in cancer therapy.

12. An exosome colloid or suspension for use in therapy, wherein the exosomes have a therapeutic cargo comprising a microRNA (miR), an anti-miR, mRNA, a long non-coding RNA, a circular RNA, a small interfering RNA, a short hairpin RNA, a piwi-interacting RNA, a CRISPR RNA sequence, modifications of the foregoing or artificially designed nucleic acid sequences, protein, cytokine or lipid, and a surface coating of the hydrophilic polymer polyethylene glycol (PEG), wherein the PEG has a molecular weight of less than 5 kDa, and the surface coating covers at least 65% of the exosome surface and substantially neutralises the surface charge of the exosomes.

13. An exosome colloid or suspension for use according to claim 10 or 12, wherein the therapy is for treatment of cystic fibrosis, COPD, lung cancer or another lung condition.

14. An exosome colloid or suspension for use according to claim 12 or claim 13, wherein the exosomes comprise a therapeutic cargo which comprises at least one exogenous mRNA (e.g. in vitro transcribed mRNA), protein, miR or antimiR.

15. An exosome colloid or suspension for use according to any one of claims 12-14, wherein the exosomes are derived from mesenchymal stem cells genetically modified to overexpress specific miRs, antimiRs, mRNAs or other nucleic acid and mature protein therapeutic cargoes.

16. An exosome colloid or suspension for use according to any one of claims 12-15, wherein the treatment includes aerosolization of the colloid or suspension.

17. A method of treating a patient having a lung disease, comprising: providing a composition according to any of claims 1 to 8; forming an aerosol of vesicles of the composition; and administering the aerosol to the patient.

18. A method according to claim 17, comprising forming the aerosol using a nebuliser.

19. A method according to claim 18, comprising forming the aerosol using a vibrating mesh nebuliser.

Description

EXAMPLES

[0092] To help understanding of the invention, a specific embodiment with a variant thereof will now be described by way of example and with reference of the accompanying drawings, in which:

[0093] FIGS. 1A and 1B are graphs of concentration v size of unPEGylated and PEGylated exosomes, in nanoparticle tracking analysis (NTA);

[0094] FIGS. 2A and 2B shows the zeta potential distribution of PEGylated and unPEGylated exosomes;

[0095] FIGS. 3A-3F are a series of transmission electron microscopy images of PEGylated and unPEGylated exosomes;

[0096] FIGS. 4A-4E is a series of flow cytometry graphs showing labelling of MSCs and exosomes with Far-Red dye;

[0097] FIGS. 5A-5C are confocal microscopy images of treated and untreated exosomes that have entered into cystic fibrosis bronchial epithelial cells having first penetrated the mucus layer in air-liquid-interface culture;

[0098] FIGS. 6a and 6b are nanoparticle tracking analysis readings for dPBS and 30 nm gold (Au) nanoparticles as controls;

[0099] FIGS. 7a and 7b are nanoparticle tracking analysis readings for Sample 1;

[0100] FIGS. 8a and 8b are nanoparticle tracking analysis readings for Sample 2;

[0101] FIG. 9 is a nanoparticle tracking analysis distribution for Sample 3;

[0102] FIG. 10 is nanoparticle tracking analysis distribution for Sample 4;

[0103] FIG. 11 is nanoparticle tracking analysis distribution for Sample 5;

[0104] FIGS. 12a and 12b are nanoparticle tracking analysis distributions for Sample 6;

[0105] FIG. 13 is nanoparticle tracking analysis distribution for Sample 7;

[0106] FIG. 14a is a plot showing zeta potential readings for PEGylated and non-PEGylated Sample 1;

[0107] FIG. 14b is a plot of zeta potential intensity distributions for tree separate measurement for PEGylated Sample 1;

[0108] FIG. 15a is a plot showing zeta potential readings for PEGylated and non-PEGylated Sample 2;

[0109] FIG. 15b is a plot of zeta potential intensity distributions for tree separate measurement for PEGylated Sample 2;

[0110] FIG. 16 is a plot showing mean zeta potential values for PEGylated and non-PEGylated Sample 4;

[0111] FIG. 17 is a plot showing mean zeta potential values for PEGylated and non-PEGylated Sample 5;

[0112] FIG. 18a is a plot showing zeta potential readings for PEGylated and non-PEGylated Sample 6;

[0113] FIG. 18b is a plot of zeta potential intensity distributions for tree separate measurement for PEGylated Sample 6;

[0114] FIG. 19 is a plot showing mean zeta potential values for PEGylated and non-PEGylated Sample 7;

[0115] FIG. 20 shows GFP Fluorescence for hMSC cells with EGFP-CFTR Transduction; and

[0116] FIG. 21 shows GFP fluorescence for hMSC cells with EGFP-Negative Control Transduction;

[0117] FIG. 22 shows the CFTR-EGFP positive MSCs taken on a fluorescence microscope following seeding the cells on slides, fixing and staining the nuclei with DAPI (blue) at ×10 magnification;

[0118] FIG. 23 shows the CFTR-EGFP positive MSCs taken on a fluorescence microscope following seeding the cells on slides at ×10 magnification;

[0119] FIG. 24 shows the CFTR-EGFP positive MSCs taken on a fluorescence microscope following seeding the cells on slides, fixing and staining the nuclei with DAPI (blue) at ×20 magnification; and

[0120] FIG. 25 shows the CFTR-EGFP positive MSCs taken on a fluorescence microscope following seeding the cells on slides at ×20 magnification.

Example 1

[0121] Aims of the example were to isolate exosomes from the human mesenchymal stem cells (hMSC) and to effectively modify their surface with low-molecular-weight lipid-modified polyethylene glycol (lipid-PEG) to improve exosome properties of aerosolization and mucus penetration.

Fluorescent Labelling of hMSCs

[0122] Frozen bone marrow MSCs (Donor #163) were thawed before they plated directly into 15 T-175 flasks (1×10.sup.6 cells for each flask) in EV-depleted complete culture media (CCM) with 10 ng/ml of human basic fibroblast growth factor (bFGF), passaged when they reached 80-85% confluency. Cells were pooled together before they were pelleted and resuspended in PBS, incubated in the dark with 2 μg/1×10.sup.6 cells Far-Red dye (excitation/emission ˜630/661 nm) (Cell Trace) for 20 min at the room temperature (this will label exosomes as Far-Red dye labels cytosolic proteins). After incubation, MSC culture media was added to stop the reaction and incubated further for another 5 min before pelleting the cells them and resuspending in EV-depleted CCM.

hMSC Culture for Exosome Isolation

[0123] Far-Red labelled hMSCs (Donor #163) were cultured in complete conditioned medium (CCM) and exosomes were isolated according to the previously published data (Thery et al., 2006). Briefly, labelled MSCs were directly seeded into 30 T-175 flasks (1×10.sup.6 cells for each flask) in CCM with 10 ng/ml of human basic fibroblast growth factor (bFGF). After 24 hours, media from all the flasks were discarded and replaced with fresh media. Within 3-4 days cells were reached 80% confluency, conditioned media was then collected from each flask and cells were counted from randomly selected 5 flasks. Conditioned media from all the flasks were pooled together before exosome isolation. For exosome isolation, the media was centrifuged at 400×g for 10 min and 2000×g for 30 min to remove cell debris and apoptotic bodies respectively. After each spin, the pellet was discarded and the supernatant was used. The supernatant was then filtered using a 220 nm vacuum filter for further purification and stored at 4° C. until ultracentrifugation (Sorvall Discovery 100SE, Hitachi). Finally, the supernatant was ultracentrifuged at 120000×g for 75 min to pellet exosomes. The pellet was resuspended in PBS to wash exosomes, spun again at the same high speed, the resulting pellet was resuspended in 100 μl PBS and stored at −80° C. until further experiments. All the centrifugation steps were performed at 4° C. and sterile tubes were used.

Post-Isolation Modification of Exosomes by DSPE-PEG

[0124] DSPE-PEG (2000) Amine (Avanti Polar Lipids, Inc) was purchased from Sigma Aldrich (Wicklow, Ireland). PEGylated exosomes were prepared based on previously published data on Pegylated liposomal preparation (Li and Huang et al., 2009). A final volume 300 μl of exosome suspension was prepared by mixing 50 μl of concentrated exosome with PBS and 37.8 μl of aqueous solution DSPE-PEG (10 mg/ml) (final concentration of 1.26 mg of PEG in mL of exosome). The sample was then incubated at 37° C. for 1 hr and vortexed at every 10 min to prevent clumping of PEG molecules or exosomes. After incubation, the sample was suspended in more PBS and finally ultracentrifuged at 120000×g for 75 min to wash off unbound PEG molecules. The exosome pellet was then resuspended in 50 μl PBS and stored at −80° C.

Nanoparticle Tracking Analysis (NTA)

[0125] Both PEGylated and unPEGylated exosome samples were analysed via Nanoparticle Tracking Analysis (NTA) (Malvern UK) using a NanoSight NS 500 system running NTA version 3.2 using optimized and validated protocols (Maguire et al., 2017, Hole et al., 2013, Gerlach et al., 2017). All the samples were analysed using a NanoSight NS 500 equipped with a 405 nm laser and 430 nm long pass filter. All samples were stored on dry ice prior to analysis. Dilutions for NTA were made up in DPBS buffer (Gibco) that was certified particle free by NTA immediately prior to measurement. Samples were vortexed briefly before loading to ensure adequate mixing and breaking up of weakly bound exosome clusters. Each sample was diluted manually in PBS to obtain an optimum particle concentration suitable for NTA (between 20 and 70 exosomes per field of view), with each dilution factor for being recorded in the automatically generated reports. A total of six×60-second videos were recorded for each exosome sample and the detection threshold during analysis was selected to ensure that only distinct nano-objects were analysed, to ensure that artefacts were removed.

Zeta Potential Measurement

[0126] The zeta potential of exosomes was measured using a Litesizer 500, a light-scattering instrument for particle analysis (Litesizer™ 500, Anton Paar Ltd, UK). The zeta potential was measured at slipping plane (15.sup.0) of the exosome vesicle and ensured the concentration of particles in each dilution was high enough to provide meaningful measurements by generating a mean detected light intensity of >20 kcounts/s.

Flowcytometric Analysis of Exosome Sample (with Bead Coupling)

[0127] For flow cytometric analysis, exosomes (2×10.sup.9 total particles) isolated from Far-Red labelled MSCs were incubated with 10 μl of aldehyde latex beads (0=4 μm) in PBS for 15 min in room temperature without rotation. The sample was then mixed with 950 μl of PBS and incubated overnight at 4° C. with rotation. The remaining binding sites of exosome-beads were then blocked by adding 1M glycine in PBS for 30 min at room temperature. The exosome-coated bead samples were then centrifuged at 1700 RCF for 5 min and the pellet was resuspended in 0.5% BSA in PBS solution. Samples were washed 3 times in a similar manner and resuspended in a final volume of 1 ml of 0.5% BSA in PBS. Exosome-coated bead samples were then either conjugated with a PE-conjugated mouse anti-human antibody to detect CD 9, CD 63, and CD 81 or left untreated as a control. Isotype control was also made by incubating exosome-coated beads with PE-conjugated mouse IgG1 κ, isotype 1 or 2. All the samples were then analysed using BD FACS CANTO and data analysed using FlowJo software.

Transmission Electron Microscopy (TEM)

[0128] Morphological examination of both PEGylated and unPEGylated exosomes were performed using an electron microscope (EM). Exosomes were fixed with 2% paraformaldehyde (PFA), washed thrice with PBS prior to being adsorbed to Formvar/Carbon 200 mesh gold EM grids. EM grids were then blocked with PBS/50 mM glycine and PBS/5% BSA before treating them with mouse-anti-human primary antibodies to either CD63 (cat #sc5275, Santa Cruz) or TSG-101 (cat #sc7964, Santa Cruz) (secondary only control on PBS/0.1% BSA) overnight and followed by 30 min in the goat-anti-mouse IgG-Gold secondary antibody (cat #G7652, Sigma). Grids were then fixed with 1% glutaraldehyde and incubated in 2% phosphotungstic acid in PBS to add contrast to the exosome membrane. Exosome grids were then washed in PBS and allowed to air dry before analysing them with TEM microscope.

Mucus Penetration and Cell Internalisation Experiments with Cystic Fibrosis Epithelial Cells Cultured at Air-Liquid-Interface.

[0129] Cystic fibrosis (CF) is a genetic disease in which impaired innate host defence results in repeated, severe airway infections. Airway epithelial cell cultures (AECCs) at air-liquid interface differentiate into cells typically encountered in the bronchial epithelium (ciliated epithelial and goblet cells) and produce thick, viscous dehydrated mucus at the apical surface which is exposed to air. The Air-Liquid-Interface model is thus an accurate recapitulation of the conditions that an aerosol delivered gene therapy will encounter in the actual CF lung. Surprisingly, we found that mucus penetration and cell internalisation was achieved by application of high concentrations of non-treated exosomes to the mucus surface of ALI cultures. 25 μl of treated exosomes and untreated exosomes were then applied to the apical mucus surface of the ALI cultures with the dose of treated exosomes being approximately half of the untreated exosomes. (2.65E+07 exosomes per microlitre of untreated control and 1.22E+07 exosomes per microlitre of treated test formulation). After 8 hours of incubation the samples were washed 3 times with PBS, fixed in 4% PFA and stained with phalloidin (tight junctions) and Hoechst (nuceli)

[0130] Confocal fluorescent microscopic analysis via Zeiss LSM 710 Confocal. Scanning for xyz image analysis of epithelial cells in treated and untreated samples.

Results

[0131] Exosomes isolated from the human bone marrow MSCs were characterized using different methods to quantify their size, to measure their surface charge and to identify them with the presence of some surface markers. Both PEGylated and unPEGylated exosomes were used for characterization (Except for flow cytometry). We isolated exosomes using differential ultracentrifugation as this method remains the most widely used standard method to isolate exosomes from different biological fluids or cell culture supernatants (Chia et al., 2017, Chia et al., 2016, Thery et al., 2006). The nanoparticle tracking analysis (NTA) of both unPEGylated and PEGylated exosomes revealed that their mean size was 143.2±3.5 nm and 165.6±6.0 nm respectively (FIGS. 1A&B). On the other hand, when PEG molecules alone suspended in PBS were analysed with NTA they failed to qualify the quality control criteria. This indicates that the number of particles showed in the PEGylated exosomes sample was not an artefact caused by PEG molecules. As NTA measures the particle hydrodynamic diameter of the particle, the measured diameter of both exosome samples obtained by TEM was smaller than the NTA analysis measured.

[0132] As indicated by the NTA analysis, the mean size of PEGylated exosomes was slightly higher than of unPEGylated exosomes. This size difference is attributed to incorporation of PEG molecules into the surface of the exosomes. Additionally, the zeta potential distribution was measured using a Litesizer 500. The surface charge was measured multiple times and the zeta potential of unPEGylated exosomes was about −16.5 mV and the PEGylated were about −2.6 mV (FIGS. 2A&B). The y axis shows the relative frequency which gives a probability of a particular charge recorded over a particular time period—in this case the exosome charge was recorded a 1000 frames per minute. This is a clear indication that exosomes have been effectively PEGylated and the presence of PEG molecules on the membrane of exosomes nearly neutralised the surface charge. Furthermore, as exosome samples required an additional ultracentrifugation step to remove excess PEG molecules, the actual particle count after the isolation of exosomes may be higher than the number obtained after the PEGylation procedure. TEM analysis of immunogold labelled exosomes samples confirmed the presence of CD63 and TSG-101 markers (FIG. 3) and most of the exosomes were under 100 nm size. FIGS. 3A-3F shows transmission electron microscopy (TEM) images of immunogold labelled (CD63 & TSG 101) exosomes (PEGylated and unPEGylated) (black line, 100 nm). Both FIGS. 3A and 3B represent control group secondary only exosomes, unPEGylated and PEGylated respectively; FIG. 3C represents PEGylated CD 63 exosomes; FIG. 3D represents unPEGylated CD 63 exosomes; FIG. 3E represents PEGylated TSG 101 exosomes; and FIG. 3F represent TSG 101 unPEGylated exosomes. However, the number of CD63 and TSG-101 labelled PEGylated exosomes were markedly less than unPEGylated exosomes but did not show any observable change in the morphology. This might be an indication of effective PEGylation of exosome surface which prevented antibodies from binding their respective antigen. Additionally, the labelling status of both MSCs and exosomes with Far-Red dye was confirmed by the flow cytometry analysis (FIG. 4). FIGS. 4 A-E show detection of Far-Red+MSCs and bead conjugated exosomes by flow cytometry. FIGS. 4A-4C show images of Far-Red+ exosome coated beads, and FIGS. 4D and 4E show Far-Red positive MSCs (gated population). FIG. 4A indicates the bead population (gated population) and FIGS. 4B & 4C show the gated population for Far-Red+ exosome-coated beads.

[0133] We thus effectively modified the surface of human bone marrow-derived exosomes by a simple incubation method with low-molecular-weight DSPE-PEG. We used DSPE-PEG (i.e. PEG with a cross-linked lipid) for this experiment to incorporate the lipid portion to the bi-layer of exosomes to anchor the polymer.

[0134] In this example, we adopted “post-insertion” methods, used to PEGylate pre-formed liposomes (Nag et al., 2013), to anchor the polymer to the exosome surface after isolating exosomes by ultracentrifugation. In this method, we mixed fluorescently labelled exosome suspension with a certain concentration DSPE-PEG to allow incorporation of PEG to the exosome membrane. In liposomal preparations, samples are usually incubated at a higher temperature to achieve faster and maximum insertion of PEG. However, higher temperature conditions are not compatible with exosomal preparations, so we performed PEGylation procedures at 37° C. The PEGylated and unPEGylated exosomes were characterized by different methods and our results indicate that exosome surface has been modified by PEG in the PEGylated samples as indicated by the increase in mean hydrodynamic diameter, nearly neutralised surface charge and decreased TSG 101 and CD 63 positive exosomes compared to unPEGylated exosomes.

[0135] Confocal fluorescent microscopic analysis of the samples clearly shows that the fluorescent Far Red stained PEGylated exosomes were able to internalise into the epithelial cells underlying the mucus layer at less than half the dose of the non-PEGylated control exosomes. This is a highly significant improvement and the improved efficiencies will result in a huge reduction in stem cell culture volumes required to produce exosome treatments. The localisation of the exosomes to the areas surrounding the cell nuclei is also apparent which is where the cell's protein translation machinery is located i.e. on ribosome lining the endoplasmic reticulum. The degree of cell uptake is notable considering that smaller sized viral vectors can become completely trapped in these mucus layers. The invention hence envisages that modified exosomes will be capable of modulating gene expression—for example the introduction of only approx. 100 microRNA molecules are required to provide detectable changes in a cell. Similarly, protein producing mRNA transcripts in a cell only average approx. 40 transcripts in the case of the CFTR protein, which is also easily obtainable with the exosome internalisation into target cells demonstrated by the invention.

[0136] Referring to FIG. 5, the mucus penetration and cell internalisation experiments demonstrate the ability of surface modulated extracellular vesicles, particularly exosomes, to pass through mucus and enter the apical cells. The nucleus of the cells are coloured purple and the cell membranes can be identified from the tight junctions, coloured green. It is clear that the red dots, namely the exosomes, have penetrated the cells and are clustered around the nuclei. Thus this ability of PEGylated exosomes has been demonstrated.

[0137] FIGS. 5A-5C are confocal microscopy images of well-differentiated Cystic Fibrosis bronchial epithelial cell cultures. 25 μl of exosomes were applied to the surface of the mucus layers overlying the apical cell surfaces prior to washing, fixing and staining of the samples. For FIG. 5A 3.05×10.sup.8 exosomes in 25 μl of PSB (the exosomes treated to be densely coated with DSPE-PEG2000 and a surface charge of −2.5 mV) were applied to surface of the mucus layer. For FIG. 5B 6.63×10.sup.8 exosomes in 25 μl of PSB (the exosomes being untreated exosomes, having no surface modification and a surface change of −19.5 mV) were applied to the surface of a mucus layer. As seen comparable mucus penetration and cell internalisation of treated exosomes was achieved with less than half of the applied exosomes. FIG. 5C is a labelled close up image showing exosomes in the apical compartments of epithelial cells and localised about the nuclei (endoplasmic reticulum and ribosomal protein translation machinery).

[0138] The surface engineered exosomes may be of beneficial therapeutic use when delivered without any genetic modification of their contents or genetic modification of the parent cells can optimize the exosomes contents for delivery into the targeted cells and the treatment of specific diseases. Further studies of the kinetics of mucus diffusion and cell internalization are planned as the speed of delivery is an important consideration that will influence the extent of intracellular delivery in vivo. The surface modified exosomes show accelerated uptake which is of significant benefit to avoid mucociliary clearance mechanisms and this may be demonstrated by comparing the permeation kinetics of equal ‘doses’ of surface modified and unmodified exosomes applied to the mucosal surface in a variety of concentrations with real time confocal microscopy video analysis.

[0139] The required doses of exosomes that need to be delivered by aerosol can also be approximately extrapolated from the in vitro mucus penetration experiments. A known number of exosomes were applied to a known surface area of mucus covered epithelial cells (60 mm.sup.2) and very significant epithelial cell uptake was demonstrated. Some key assumptions are; [0140] 1. The viscosity of a given concentration of exosome formulation and achievable aerosol output at this viscosity; [0141] 2. That surface engineered exosomes will penetrate relatively quickly through the mucus layer which is reasonable given that concentration will greatly influence this via concentration driven diffusion and relatively very dilute exosome formulations were employed in the pilot studies (0.00063928% v/v of surface modified exosomes).

[0142] The following table illustrates the exosome loading capacity of aerosol droplet of various sizes @ a 1% w/v concentration of exosomes:

TABLE-US-00001 # Exosomes @ X % loading (e.g. 6% est @2 Droplet Volume Volume Exosome Exosome Exosome cP, 12%, cP MMAD Droplet Droplet Droplet Diameter Volume volume est @ ~5.2) μm radius μm3 microlitres μm μm.sup.3 microlitres per droplet 10 5 523.6  5.236E−07 0.1 0.000524 5.24E−13 9992.37 7 3.5 179.59 1.7959E−07 0.1 0.000524 5.24E−13 3427.29 6 3 113.1  1.131E−07 0.1 0.000524 5.24E−13 2158.40 5 2.5 65.45  6.545E−08 0.1 0.000524 5.24E−13 1249.05 4.5 2.25 47.71  4.771E−08 0.1 0.000524 5.24E−13 910.50 4 2 33.51  3.351E−08 0.1 0.000524 5,24E−13 639.50 3 1.5 14.14  1.414E−08 0.1 0.000524 5,24E−13 269.85 2 1 4.19  4.19E−09 0.1 0.000524 5.24E−13 79.96 1 0.5 0.52   5.2E−10 0.1 0.000524 5.24E−13 9.92 0.1 0.05 0.000524  5.24E−13 0.1 0.000524 5.24E−13 0.01 X % = 1%

[0143] For example, in the case of Cystic Fibrosis, the targeted lung area is the total airway surface area from trachea to bronchioles which is approx. 2,471+/−320 cm.sup.2 in the adult human lung. This surface area could be treated via a deposited aerosol volume of approx. 70 microlitres of a 1% w/v exosome formulation.

[0144] The surface area of the distal lung is far greater with approx. 18 times more alveolar cells than bronchial epithelial cells and targeting this area is desirable for treating emphysema and idiopathic pulmonary fibrosis for example. The cellular uptake of exosomes is expected to be far higher in these regions of the lung due to the lack of mucus barrier.

[0145] In conclusion, we have shown that exosomes were effectively PEGylated using lipid-conjugated PEG by a simple incubation method and the resultant exosomes penetrated viscous CF mucus and were internalised into epithelial cells, demonstrating a pulmonary exosome-based gene therapy platform or simply as therapeutically useful exosomes for treating inflammatory lung diseases.

[0146] PEGylation also results in a sterically stabilised formulation of exosomes that has significantly reduced viscosity compared to non-PEGylated formulations. Generation of an aerosol containing such exosomes possessing a dense hydrophilic corona to shield from hydrophobic interaction induced agglomeration is hence facilitated, meaning that aerosols for delivery of exosomes at an acceptable and useful concentration are now facilitated and enabled at higher concentrations. The pegylated exosomes demonstrate improved mucus penetration, this property then facilitating their passage through lung mucus to lung tissue, further enhancing the efficacy of the pegylated exosomes in therapeutic applications against lung disease. Lastly, PEGylated monoclonal antibodies and nanoparticles may also display enhanced ‘stealth’ properties that help them to avoid uptake and clearance by phagocytes such as alveolar macrophages which are present in increased numbers in inflammatory lung diseases.

[0147] The invention thus provides coated vesicles e.g. exosomes that can be used in gene therapy for CF, COPD, Adenocarcinoma and other lung conditions.

Example 2

[0148] The aims of this example were to show the surface modification of exosomes can be effectively achieved and that genetically modified mesenchymal stem cell populations of bioengineered cells, over-expressing CFTR gene can be successfully established.

BT-20 and hMSC Cell Line Culture Techniques

[0149] BT-20 cells sourced as BT-20 human epithelial cells from ATCC® HTB-19™ were cultured in DMEM media supplemented with 10% FBS and 100 IU/ml of Penicillin/Streptomycin as complete condition media (CCM). Human Mesenchymal Stem Cells Donor No. 096 were used, the cells were cultured in Gibco's MEM Alpha Medium with 10% FBS and 100 IU/ml of Penicillin/Streptomycin and 1 ng/ml of human basic fibroblast growth factor (bFGF).

[0150] The feeding of the cells was done 3 days a week using aseptic technique, which was to remove spent media from culture flask with a pipette and add to waste bottle followed by replenishing of culture flask(s) with appropriate volume of fresh media (supplements included) to the ceiling of the culture flask so that the cell culture (monolayer) is not disturbed by the flow. The flask was then removed from the hood tilting the flask so its lid faced inward and placed in an incubator at 37° C. with 5% CO.sub.2. The cells were examined routinely with a light microscope for signs of contamination such as turbidity, cells in suspension, change in media colour, large clumps or particles etc.

[0151] For splitting of the cells every 7-10 days and before cells reach 80-90% confluence, the cells were washed with 3-5 ml of PBS ensuring that it was delivered to the opposite side of the flask so as not to disturb the cells, followed by 5 ml of warmed T/E (Appropriate concentration, 37° C.) The flask was then rocked on its axis gently for 30 seconds, ensuring that all cells were covered, and the excess T/E poured off into waste a bottle by a pipette (leaving approx. 1 ml in flask). The cells were incubated with remaining trace of T/E at 37° C. for 3-5 mins, as the cells detached from the floor of the flask. The detached cells were resuspended in 4-6 ml of complete medium (volume depends on cell number expected). The cell suspension was then pipetted up and down with 10 ml pipette to ensure homogeneity and remove to 15 ml falcon. The flasks were incubated at 37° C.

Preparation for EV Free Media

[0152] EV free media is a pre-requisite of EV Isolation protocols for purified yield of EVs from cells in culture. This is to eliminate contamination of EV isolates of the cultured cells from microvesicles outside of the experimental setup.

[0153] 130-140 mls of 20% FBS+Basal Medium (1:5) in an autoclaved sterile glass bottle was prepared and appropriate amount of the above was ultra-centrifuged at 110,000×g for 18 hours, at 4° C. After ultracentrifugation, the supernatant was aspirated carefully onto a fresh 50 ml falcon tube making sure not to disturb the pellet. The supernatant from 50 ml Falcons was filtered onto a fresh autoclaved glass container. To this was added an equal portion of Basal Media to bring to 1:1 proportion, making the total FBS concentration in the above solution to 10%. This yielded 10% EV free media to be used for EV isolation cell culture. The condition media was stored at 4° C. up to 4 weeks supplemented with 100 IU/ml of P/S (or other supplements) before using for EV Isolation protocol.

Cell Culture for EV Isolation and Preparation of EV Rich Condition Media

[0154] EVs were isolated according to the MISEV2018 guidelines and further refined protocols (Thery et al., 2006). BT-20 cells were brought to enough numbers 3-4 T-175 cm.sup.2 flasks to seed 12×T-175 cm.sup.2 T-flasks in EV free media (2×10.sup.6 cells/flask). The cells were trypsynized and resuspended in EV depleted media and seeded onto 12×T-175 cm.sup.2 T-Flasks in 10 mls of EV depleted media for 24 hours to allow the cells to adhere to the floor of the flask. The media was then replaced with equal amount of fresh EV free media (Condition Media). The flasks were left to incubate for 48 hours.

[0155] After 48 hours, conditioned media was then collected from each flask and pooled into 50 ml Falcons and cells in the flasks were trypsynized, a cell count was taken from total cell lysate (Appropriate number of cells were then either passaged/frozen/discarded). EV free media Culture Supernatant was poured from T-Flasks into 50 ml Falcons (4 flasks per 50 ml falcons). Then the 50 mls falcons containing supernatant were brought to centrifuge, to remove cell debris at 300×g for 10 mins, temperature-21° C. The supernatant was then aspirated using 25 ml pipettes, leaving the bottom 2-3 ml residue undisturbed, into fresh 50 ml Falcons respectively. The supernatant then centrifuged at 2000×g for 10 mins at a temperature of −21° C. After centrifugation, the supernatant was collected leaving dead cells pellet within bottom 2-3 ml residue. Then using a 0.2 μm pore size sterile filter and syringe, the top 25 ml of supernatant was aspirated and collected into fresh autoclaved ultra-centrifuge tubes. The filtered condition media was then ultra-centrifuged at 110,000×g for 70 mins at 4° C. in the Hitachi Micro Ultracentrifuge. After ultracentrifugation 1 ml of supernatant was taken from one of the tubes into a 1.5 ml tube and stored at −80° C. (Supernatant Analysis 1). The rest of the supernatant was discarded into waste container without scraping the pellet. The pellet containing EVs was re-suspended with 1 ml of PBS in each UCT and pooled together into a single UCT. This was ultracentrifuged at 110,000×g for 70 mins at 4° C. After ultracentrifugation, 1 ml of supernatant was taken into 1.5 ml Eppendorf from a UCT and stored at −80° C. (Supernatant Analysis 2). The rest of the supernatant was discarded into the waste container without scraping the pellet. 85 μls of sterile PBS was pipetted onto EV containing UCTs and the marked side was scraped off for about 5 mins with 100 μl pipette tip. After 5-8 mins of scraping, all PBS resuspension had been collected. All the residue including bubbles was collect onto a tapered tube for future PEGylation. This was pulse centrifuged for 10 seconds to get rid of bubbles and the supernatant was mixed in LFH with a pipette. 15-20 μls was pipetted into tube (NTA/Zeta) and 10 μls to protein analysis tube. All the isolated EV samples were stored at −80° Celsius.

[0156] All the steps were performed using aseptic techniques and all the centrifugation steps were performed at −4° C.

Post-Isolation Modification of EVs by DSPE-PEG 2000 (AMINE)

[0157] DSPE-PEG (2000) Amine (Avanti Polar Lipids, Inc) was purchased from Sigma Aldrich (Wicklow, Ireland). PEGylated exosomes were prepared based on previously published data on Pegylated liposomal preparation (Kooijmans et al., 2016). A final volume of 1:4 ratio of isolated EV suspension (50 μls) was prepared by mixing concentrated EV suspension with PBS in a plastic or glass tube for samples 1, 2 and 6 and samples 4, 5 and 7 respectively. To this an equal ratio (or desired percentage) of 1:1 (ug protein:ug protein) DSPE-PEG 2000 Amine (10 mg/ml) was added which was kept for incubation at 37° C. for 1 hr, by placing the EV samples in a temperature controlled tube holder on top of a swaying rocker at low speeds to prevent clumping of PEG molecules or exosomes. After incubation, the EV-PEG suspension was introduced into 16-20 mls of PBS in an Ultracentrifuge tube. Ultracentrifugation was performed at 110000×g for 70 mins to wash off unbound PEG molecules. The EV pellet was then re-suspended in 75 μl of PBS and stored in appropriately sized glass containers with lid, and from this aliquot 20 μls for Zeta analysis in separate containers. This was stored at −80° C. or −20° C. for sample 1, 2, 6 and samples 4, 5, 7 respectively.

Nanoparticle Tracking Analysis (NTA)

[0158] Both PEGylated and unPEGylated exosome samples were analysed via Nanoparticle Tracking Analysis (NTA) (Malvern UK) using a NanoSight NS500 system running NTA version 3.2 using optimised and validated protocols (Maguire et al., 2017, Hole et al., 2013, Gerlach et al., 2017). All the samples were analysed using a NanoSight NS 500 equipped with a 405 nm laser and 430 nm long pass filter. All samples were stored on wet ice prior to analysis. Dilutions for NTA were made up in D-PBS [—MgCl.sub.2, —CaCl.sub.2)] (Gibco) that was certified particle free by NTA immediately prior to measurement. Samples were pipetted before loading to ensure adequate mixing and the breaking up of weakly bound EV clusters. Each sample was diluted manually in D-PBS to obtain an optimum particle concentration suitable for NTA (between 10 and 100 particles per field of view), with each dilution factor being recorded in the automatically generated reports. A total of six×60 second videos were recorded for each EV sample and the detection threshold during analysis was selected by the operator, to ensure that only distinct nano-objects were analysed and to ensure that artefacts were removed. As NTA measures the particle hydrodynamic diameter, i.e., the size of the solvated particle that is approximated as being spherical, the measured diameters may be larger than those obtained by electron microscopy (EM) based techniques. In general, exosomes possess diameters in the 30-150 nm size range. All samples were stored on dry ice prior to analysis. Dilutions for NTA were made up in DPBS buffer (Gibco) that was certified particle free by NTA immediately prior to measurement. Samples were vortexed briefly before loading to ensure adequate mixing and breaking up of weakly bound exosome clusters. Each sample was diluted manually in PBS to obtain an optimum particle concentration suitable for NTA (between 20 and 70 exosomes per field of view), with each dilution factor for being recorded in the automatically generated reports. A total of six×60-second videos were recorded for each exosome sample and the detection threshold during analysis was selected to ensure that only distinct nano-objects were analysed, to ensure that artefacts were removed.

Zeta Potential Measurement

[0159] The zeta potential of EVs was measured using a Litesizer 500, a light-scattering instrument for particle analysis (Litesizer™ 500, Anton Paar Ltd, UK). The zeta potential was measured by diluting the EV samples PEGylated/Non-PEGylated with PBS in 1:100 or 1:16 (EV Sample-PBS) in an Anton Paar Calliope Core Omega Cuvette using Smoluchowski approximation with Debeye Factor of 1.5 with equilibration time of 1 minute and target temperature of 25° C. with water as dispersion medium selected in the software as there was no option for PBS as dispersion medium. The samples intended for Zeta analysis were thawed in ice box/container and brought to the zeta analysis lab, where the samples were stored at 4° C. and taken out as used for the analysis, the dilution was done in the original tube containing PEGylated or Non-PEGylated EVs, just before the sample was to be added to the cuvette. The cuvette was washed using a syringe first time with DI-water and then three times with sterile PBS and then the sample was added using a sterile syringe. The maximum and minimum load capacity for each cuvette was 350 μls and 300 μls respectively. The concentration of particles in each dilution was high enough to provide meaningful measurements by generating a peak intensity for most of the zeta potential distribution of the particles (outliers included in the results).

Lentiviral Transduction of hMSCs with EGFP-Gene Constructs

[0160] Lentiviral transductions were performed on hMSCs Donor #096. The introduction of EGFP-CFTR and EGFP-NEG control was done to express amplified CFTR protein in the EGFP-CFTR transduced cells for production of CFTR gene and mRNA encapsulated RNAs which are to be further characterized once a viable population is reached. The transductions were completed using spin protocol.

[0161] hMSCs were split as normal and resuspended in 5 ml of complete media. The resuspended MSCs were pelleted by centrifuging at 1000×g for 4 mins after which complete media supernatant was discarded. The cells were resuspended gently in 5 ml basal media (no FBS, no antibiotic) and a cell count was done.

[0162] 1×10.sup.6 (or approximate) cells were further diluted in approx. 5 ml of basal media in separate 50 ml falcon tubes for each EGFP Lentivirus, positive control and negative control (receives no lentivirus). To each Experimental and Positive Control MOI of 3-4 (approximately 18 μls) of lentivirus was added. The negative control received no lentivirus and all the 50 ml falcon tubes were centrifuged for 90 mins at 2000×g at 37° C. after centrifugation discarded the media and re-suspend cell pellet again in complete media. The cells were then seeded onto appropriately sized T-Flasks (T175 or T-75) depending on the number of cells transduced and incubated in completed media supplemented with bFGF (1 ng/ml). After 48 hr incubation, the media was changed to complete media with bFGF and supplemented with of 4 μg/ml puromycin to select the transduced cells for 7-14 days, after which the cells were changed to normal complete media without puromycin. The cells were split every 7-10 days after the transduction was done.

Results

[0163] For the initial experiments, BT-20 Human Breast Cancer cell line was used for their high proliferative capacity and ease of culture. The cells were brought to 12×T-175 cm.sup.2 T-Flasks. The EVs were isolated from the above using differential centrifugation followed by two ultracentifugation steps according to MISEV guidelines following most widely used and proven methods and then different methods were implemented for EV characterization including size distribution through Nanoparticle Tracking Analysis and Surface Charge Analysis through Zeta Potential studies using Litesizer™ 500 instrument. Both the PEGylated and non-PEGylated EVs were characterised using established protocols and techniques to the best-known practice.

[0164] EVs isolated from BT-20 cells were characterized using different methods to quantify their size and to measure their surface charge. We isolated exosomes using differential ultracentrifugation as this method remains the most widely used standard method to isolate exosomes from different biological fluids or cell culture supernatants (Chia et al., 2017, Thery et al., 2006). The nanoparticle tracking analysis (NTA) of both PEGylated and unPEGylated exosomes revealed that NTA Analysis is not a viable option for diluted fractions of PEGylated EVs. PEGylated EVs might be subject to surface modification due to interaction with plastic surfaces in Ultracentrifuge Tubes or due to degradation in plastic containers, for this we used glass containers for storing PEGylated EV fractions for sample 4, 5 and 7 and performed Zeta analysis for characterization of those samples instead of NTA analysis.

BT-20 Cell Culture Overview

[0165] All parameters relating to cell culture are briefly presented as below in Tables 1a and 1b, showing values of cell counts, protein concentration of EV-Isolates, DSPE PEG:Protein percentage for PEG treatment, Zeta potential and NTA readings for isolate EV Fractions. For NTA analysis readings, the values for Sample 1 are unreliable due to low particle counts per frame (i.e. <3 particles per frame). This also applies to the Mode NTA After PEG for samples 2 and 6. <10 particle counts per frame were also noted for the Mode NTA Before PEG for samples 3, 4, and 7. However, these results fulfil quality assessment criteria. All data is represented as cells/ml for cell count, μls for volume, mV for Zeta potential and nM for mode size distribution for NTA analysis results.

TABLE-US-00002 TABLE 1a Total Cells from 12 T-175 cm.sup.2 Lysate Flasks on Total Protein Sample Isolation Total Cell Dead Volume EV Viable Conc. No Date Count/ml Cells/ml (ml) harvest day Cells (μg/ml) 1 14-Mar 1.37E+06 5.10E+04 25 3.41E+07 3.29E+07 208.33 2 15-Mar 1.61E+06 7.10E+04 25 4.01E+07 3.84E+07 391.67 3 21-Mar 3.20E+06 2.70E+04 13 4.16E+07 4.13E+07 2268.75 4 28-Mar 2.51E+06 2.60E+04 18 4.54E+07 4.48E+07 408.33 5 29-Mar 2.39E+06 9.20E+04 19 4.53E+07 4.36E+07 187.50 6 4-Apr 2.15E+06 2.95E+05 22 4.74E+07 4.09E+07 263.46 7 5-Apr 2.23E+06 1.19E+05 22 4.90E+07 4.64E+07 125.00

TABLE-US-00003 TABLE 1b PEG volume DSPE for PEG Mode Prot. In 100% Volume NTA Mode μg in prot. PEG: used to Average before NTA Sample EV Conc. Protein PEGylate pre-PEG PEG after No. sample (μls) (%) (μls) ZP (mV) (nM) PEG 1 10.42 1.04 100% 1.04 −14.55 106.5 93 2 19.58 1.96  50% 0.98 −11.62 110.5 97 3 −0.42 156 4 9.38 1.63  50% 0.82 −12.93 120 5 16.33 0.94 100% 0.94 −10.48 96 6 13.17 1.32  75% 0.99 −0.63 112 117 7 6.25 0.63 150% 0.94 −15.85 93.5

NTA Analysis:

[0166] Tables 2a and 2b set out below show values of replicate readings for NTA analysis as: Mode particle size distribution (nm), Total particle concentration and Particle concentration between 30-150 nm range in NPs/mL for isolated EV-fractions from BT-20 Cells, cultured in 12XT-175 cm T-Flasks following EV-isolation protocol (MISEV). Data is represented as Mean and SEM. Sample 1 and all the PEGylated Samples provided unreliable data as <3 particle counts per frame were recorded, which does not meet NTA quality assessment criteria. Samples 2 and 3 provided <10 particle counts were frame, though their measurement passes the quality assessment criteria. Sample 7 also has a particle count of <10 per frame on SEM, but again passes the quality assessment criteria.

TABLE-US-00004 TABLE 2a Mode Particle Size (NM) Sample # Replicates Mean SEM 1 6 106.5 9 2 6 110.5 5 3 4 156 43.5 4 6 120 26 5 6 96 3.5 6 6 112 5 7 93.5 6 PEGylated Sample 1 6 93 11.5 PEGylated Sample 2 6 97 4.5 PEGylated Sample 3 6 117 23.5 100 nm NIST 6 106 1 30 nm AUNPs 22 34.5 0.5 D-PBS Control 28 100 11

TABLE-US-00005 TABLE 2b Total Particle Particle Concentration Concentration between 30- (NPs/mL) 150 nm (NPs/mL) Sample # Mean SEM Mean SEM 1 9.50E+08 1.84E+08 5.81E+08 1.39E+08 2 1.06E+11 6.67E+08 7.30E+10 1.75E+09 3 2.66E+09 1.83E+00 1.08E+09 4.08E+08 4 3.25E+09 1.88E+08 1.11E+09 1.43E+08 5 2.72E+10 1.96E+09 1.76E+10 9.96E+08 6 4.78E+10 2.57E+09 3.65E+10 2.10E+09 7 5.68E+09 5.78E+08 3.34E+09 3.14E+08 PEGylated 8.20E+08 1.63E+08 7.36E+08 1.64E+08 Sample 1 PEGylated 1.40E+09 2.04E+08 1.08E+09 1.58E+08 Sample 2 PEGylated 7.52E+08 1.01E+08 3.60E+08 1.15E+08 Sample 3 100 nm NIST 1.70E+10 1.12E+09 1.15E+10 5.59E+08 30 nm AUNPs 6.62E+10 1.44E+09 6.22E+10 1.64E+09 D-PBS Control 8.54E+05 8.54E+05 6.53E+06 6.72E+05
1. Calibration of the system before each day of analysis was verified using a 100 nm NIST polystyrene nanosphere size standard (100 nm NIST) and a 30 nm gold citrate nanoparticle (30 nm AuNPs).
2. All samples in the table below passed quality assessment criteria as discussed.
3. Samples #1, PEGylated Sample #1, PEGylated Sample #2 & PEGylated Sample #6 provided unreliable results as a consequence of low particle counts per frame (i.e. <3 particles per frame, which is classified as ‘particle-free’). For this reason, they did not pass quality assessment criteria.
4. Samples #3 & #4 provided a low particle count per frame (i.e. <10). Though, their measurements passed quality assessment criteria.
5. Samples #7 provided a SEM of their respective particle counts per frame reaching <10. Though, their measurements passed quality assessment criteria, this may be cause for minor consideration in their concentration measurement and should be reported.

Controls:

[0167] FIGS. 6a and 6b show nanotracking analysis (NTA) reading for dPBS and 30 nm Gold (Au) nanoparticles as controls for particle size (nm) at X-axis and particle concentration distributions (particles/ml) at Y-axis. dPBS shows less than 1 count per frame i.e. particle free solution as expected and AuNPs show a definite peak with mean size of 34±0.5 nm and pass the quality control test. Table 3 below provided the respective values in mean and SEM.

TABLE-US-00006 TABLE 3 Mean Particle Total Particle Conc. Particle Conc. between Sample Size (nm) (NPs/mL) 30-150 nm (NPs/mL) # Replicates Mean SEM MEAN SEM Mean SEM 30 nm 22 34.5 0.5 6.62E+10 1.44E+09 6.33E+10 1.64E+09 AuNPs D-PBS 28 100 11 8.90E+06 8.54E+05 6.42E+06 6.72E+05 Control

Sample 1

[0168] FIGS. 7a and 7b show nanoparticle tracking analysis distribution for sample-1. EV samples for particle size (nm) at X-axis and particle concentration distributions (particles/ml) at Y-axis. Both the non-PEGylated and PEGylated samples had less than 3 counts per frame and didn't pass the quality control test for NTA analysis i.e. particle free solution. The mean size distribution and particle concentration is set out in Table 4a below and the total cell count and particle distribution of the source cell lysate is set out in Table 4b. Data is presented in Mean SEM, particles/cell and particles/given volume respectively.

TABLE-US-00007 TABLE 4a Mean Particle Total Particle Conc. Particle Conc. between Size (nm) (NPs/mL) 30-150 nm (NPs/mL) Sample 1 Replicates Mean SEM MEAN SEM Mean SEM Non- 6 106.5 9 9.50E+08 1.84E+08 5.81E+08 1.39E+08 PEGylated PEGylated 6 93 11.5 8.20E+08 1.63E+08 7.36E+08 1.64E+08

TABLE-US-00008 TABLE 4b Total Total Particle 30-150 nm Total Particles Viable conc. Particles Particles/ (30-150 nm)/ Cells (NPs/85 μls) (NPs/85 μls) Cell Cell 3.37E+07 8.1E+07 4.9E+07 2 2

Sample 2

[0169] FIGS. 8a and 8b show nanoparticle tracking analysis (NTA) distribution for sample 2. EV samples for particle size (nm) at x-axis and particles concentration distributions (particles/ml) at y-axis. The non-PEGylated sample shows a definite peak at 110.5±5 nm and passed the quality test for NTA analysis although the PEGylated samples had less than 3 counts per frame and didn't pass the quality control test for NTA analysis i.e. particle free solution. The mean size distribution and particle concentration is shown in Table 5a and the total cell count and particle distribution of the source cell lysate is shown in Table 5b. Data is presented as mean, SEM, particles/cell and particles/volume of sample respectively.

TABLE-US-00009 TABLE 5a Mean Particle Total Particle Conc. Particle Conc. between Size (nm) (NPs/mL) 30-150 nm (NPs/mL) Sample 2 Replicates Mean SEM MEAN SEM Mean SEM Non- 6 110.5 5 1.06E+11 6.67E+08 7.30E+10 1.75E+09 PEGylated PEGylated 6 97 4.5 1.40E+09 2.04E+08 1.08E+09 1.58E+08

TABLE-US-00010 TABLE 5b Total Total Particle 30-150 nm Total Particles Viable conc. Particles Particles/ (30-150 nm)/ Cells (NPs/85 μls) (NPs/85 μls) Cell Cell 3.8E+07 9.0E+09 6.2E+09 235 162

Sample 3

[0170] FIG. 9 shows nanoparticle tracking analysis (NTA) distribution for Sample 3 EV samples for particle size (nm) at x-axis and particle concentration distributions (particles/ml) at y-axis. The non-PEGylated sample shown has a mean size distribution at 1.56±43.5 nm and although it has passed quality test for NTA analysis but had less than 10 counts per frame for NTA analysis. The mean size distribution and particle concentrations is shown in Table 6a below, and the total cell count and particle distribution of the source cell lysate is shown in Table 6b. Data is presented as mean, SEM, particle/cell and particles/volume of sample respectively.

TABLE-US-00011 TABLE 6a Mean Particle Total Particle Conc. Particle Conc. between Size (nm) (NPs/mL) 30-150 nm (NPs/mL) Sample 3 Replicates Mean SEM MEAN SEM Mean SEM Non- 4 156 43.6 2.66E+09 1.83E+08 1.08E+09 4/08E+08 PEGylated

TABLE-US-00012 TABLE 6b Total Total Particle 30-150 nm Total Particles Viable conc. Particles Particles/ (30-150 nm)/ Cells (NPs/85 μls) (NPs/85 μls) Cell Cell 4.1E+07 2.3E+08 9.2E+07 5 2

Sample 4

[0171] FIG. 10 shows nanoparticle tracking analysis (NTA) distribution for Sample 4. EV samples for particle size (nm) at x-axis and particle concentration distributions (particles/ml) at y-axis. The non-PEGylated sample shown has a mean size distribution at 120±26 nm and although it has passed quality test for NTA analysis but had less than 10 counts per frame for NTA analysis. The mean size distribution and particle concentration is shown in Table 7a below, and the total cell count and particle distribution of the source cell lysate is shown in Table 7b. Data is presented as mean, SEM, particle/cell and particles/volume of sample respectively.

TABLE-US-00013 TABLE 7a Mean Particle Total Particle Conc. Particle Conc. between Size (nm) (NPs/mL) 30-150 nm (NPs/mL) Sample 4 Replicates Mean SEM MEAN SEM Mean SEM Non- 6 120 26 3.25E+09 1.88E+08 1.11E+09 1.43E+08 PEGylated

TABLE-US-00014 TABLE 7b Total Total Particle 30-150 nm Total Particles Viable conc. Particles Particles/ (30-150 nm)/ Cells (NPs/85 μls) (NPs/85 μls) Cell Cell 4.5E+07 2.8E+08 9.4E+07 6 2

Sample 5

[0172] FIG. 11 shows nanoparticle tracking analysis (NTA) distribution for Sample 5. EV samples for particle size (nm) at x-axis and particle concentration distributions (particles/ml) at y-axis. The non-PEGylated sample shown has a mean size distribution at 96±3.5 nm and although it has passed quality test for NTA analysis but had less than 10 counts per frame for NTA analysis. The mean size distribution and particle concentration is shown in Table 8a below, and the total cell count and particle distribution of the source cell lysate is shown in Table 8b. Data is presented as mean, SEM, particle/cell and particles/volume of sample respectively.

TABLE-US-00015 TABLE 8a Mean Particle Total Particle Conc. Particle Conc. between Size (nm) (NPs/mL) 30-150 nm (NPs/mL) Sample 5 Replicates Mean SEM MEAN SEM Mean SEM Non- 6 96 3.5 2.72E+10 1.96E+09 1.76E+10 9.96E+08 PEGylated

TABLE-US-00016 TABLE 8b Total Total Particle 30-150 nm Total Particles Viable conc. Particles Particles/ (30-150 nm)/ Cells (NPs/85 μls) (NPs/85 μls) Cell Cell 4.4E+07 2.3E+09 1.5E+09 53 34

Sample 6

[0173] FIGS. 12a and 12b show nanoparticle tracking analysis (NTA) distribution for Sample 5. EV samples for particle size (nm) at x-axis and particle concentration distributions (particles/ml) at y-axis. The non-PEGylated sample shown has a definite peak at 112±5 nm and passed the quality test for NTA analysis although the PEGylated sample had less than 3 counts per frame and didn't pass the quality control test for NTA analysis i.e. particle free solution. The mean size distribution and particle concentration is shown in Table 9a below, and the total cell count and particle distribution of the source cell lysate is shown in Table 9b. Data is presented as mean, SEM, particle/cell and particles/volume of sample respectively.

TABLE-US-00017 TABLE 9a Mean Particle Total Particle Conc. Particle Conc. between Size (nm) (NPs/mL) 30-150 nm (NPs/mL) Sample 6 Replicates Mean SEM MEAN SEM Mean SEM Non- 6 112 5 4.78E+10 2.57E+09 3.65E+10 2.10E+09 PEGylated PEGylated 6 117 23.50 7.52E+08 1.01E+08 3.60E+08 1.15E+08

TABLE-US-00018 TABLE 9b Total Total Particle 30-150 nm Total Particles Viable conc. Particles Particles/ (30-150 nm)/ Cells (NPs/85 μls) (NPs/85 μls) Cell Cell 4.1E+07 4.1E+09 3.1E+09 99 76

Sample 7

[0174] FIG. 13 shows nanoparticle tracking analysis (NTA) distribution for Sample 7. EV samples for particle size (nm) at x-axis and particle concentration distributions (particles/ml) at y-axis. The non-PEGylated sample shown has a mean size distribution at 93.5±6 nm and has passed quality test for NTA analysis meeting the criteria for purified EV sample but has less than 10 counts per frame for SEM. The mean size distribution and particle concentration is shown in Table 10a below, and the total cell count and particle distribution of the source cell lysate is shown in Table 10b. Data is presented as mean, SEM, particle/cell and particles/volume of sample respectively.

TABLE-US-00019 TABLE 10a Mean Particle Total Particle Conc. Particle Conc. between Size (nm) (NPs/mL) 30-150 nm (NPs/mL) Sample 7 Replicates Mean SEM MEAN SEM Mean SEM Non- 6 96 3.5 2.72E+10 1.96E+09 1.76E+10 9.96E+08 PEGylated

TABLE-US-00020 TABLE 10b Total Total Particle 30-150 nm Total Particles Viable conc. Particles Particles/ (30-150 nm)/ Cells (NPs/85 μls) (NPs/85 μls) Cell Cell 4.4E+07 2.3E+09 1.5E+09 53 34

Zeta Analysis

Sample 1 (PEG 100%)

[0175] FIGS. 14a and 14b show mean zeta potential values for PEGylated and non-PEGylated EV isolate sample 1 (PEG 100%). FIG. 14a is a plot showing series measurement readings for PEGylated EV isolate sample with mean zeta potential and distribution peak (mode) value for each run (particle concentration; 1:100 in PBS). FIG. 14b show the zeta potential intensity distributions for three separate measurement reading for PEGylated EV isolate sample 1 (particle concentration: 1:100 in PBS) with relative frequency (%) on the y=axis and zeta potential (mV) on the x-axis. The different curves represent three consecutive measurements of the separate sample dilutions from same EV isolate. The zeta potentials were determined using the Smoluschowski approximation. The data is represented as Mean and SD. All data is in mV.

Sample 2 (PEG 50%)

[0176] FIGS. 15a and 15b are equivalent to FIGS. 14a and 14b with respect of sample 2.

Sample 4 (PEG 50%)

[0177] FIG. 16 show the mean zeta potential values for PEGylated and nonPEGylated EV isolate sample 4 (PEG 50%). Zeta potential intensity distributions for three separate measurement reading for PEGylate EV isolated sample 4 (particle concentration: 1:16 in PBS) with relative frequency (%) on the y-axis and zeta potential (mV) on the x-axis. The different curves represent three consecutive measurements of the same PEGylated EV isolate. Tables 11a and 11b below shows single run measurement readings for non-PEGylated EV isolate sample with mean zeta potential, followed by specific values same sample after PEGylation. SD and distribution peak (mode) values for each run (particle concentration 1:100 in PBS). The zeta potentials were determined using the Smoluschowski approximation. The data is represented as Mean and SD. All data is in mV.

TABLE-US-00021 TABLE 11a Pre-PEG Mean Zeta Potential Readings in a 3 series replicate Average Mode NTA 1000 readings run of pre-PEG Before PEG (mV) readings (nM) −12.93 N/A N/A −12.93 120

TABLE-US-00022 TABLE 11b Sample 4 PEGylated Reading 1 Reading 2 Reading 3 Mean Zeta Potential (mV) −18.00 −22.95 −19.13 Standard Deviation (mV) 0.80 0.96 0.93 Distribution Peak −15.11 −22.16 −23.25

Sample 5 (PEG 100%)

[0178] FIG. 17 show the mean zeta potential values for PEGylated and non-PEGylated EV isolate sample 5 (PEG 100%). Zeta potential intensity distributions for three separate measurement reading for PEGylate EV isolated sample 4 (particle concentration: 1:16 in PBS) with relative frequency (%) on the y-axis and zeta potential (mV) on the x-axis. The different curves represent three consecutive measurements of the same PEGylated EV isolate. Tables 12a and 12b below shows single run measurement readings for non-PEGylated EV isolate sample with mean zeta potential, followed by specific values same sample after PEGylation. SD and distribution peak (mode) values for each run (particle concentration 1:100 in PBS). The zeta potentials were determined using the Smoluschowski approximation. The data is represented as Mean and SD. All data is in mV.

TABLE-US-00023 TABLE 12a Pre-PEG Mean Zeta Potential Readings in a 3 series replicate Average Mode NTA 1000 readings run of pre-PEG Before PEG (mV) readings (nM) −7.74 −10.63 −13.08 −10.48 96

TABLE-US-00024 TABLE 12b Sample 4 PEGylated Reading 1 Reading 2 Reading 3 Mean Zeta Potential (mV) −18.77 −17/38 −17.41 Standard Deviation (mV) 0.86 0.83 0.85 Distribution Peak −11.96 −20.41 −16.72

Sample 6 (PEG 75%)

[0179] FIGS. 18a and 18b show mean zeta potential values for PEGylated and non-PEGylated EV isolate sample 6 (PEG 100%). FIG. 18a is a plot showing series measurement readings for PEGylated EV isolate sample with mean zeta potential and distribution peak (mode) value for each run (particle concentration; 1:100 in PBS). FIG. 18b show the zeta potential intensity distributions for three separate measurement reading for PEGylated EV isolate sample 6 (particle concentration: 1:100 in PBS) with relative frequency (%) on the y=axis and zeta potential (mV) on the x-axis. The different curves represent three consecutive measurements of the separate sample dilutions from same EV isolate. The zeta potentials were determined using the Smoluschowski approximation. The data is represented as Mean and SD. All data is in mV.

Sample 7 (PEG 150%)

[0180] FIG. 19 show the mean zeta potential values for PEGylated and non-PEGylated EV isolate sample 7 (PEG 100%). Zeta potential intensity distributions for three separate measurement reading for PEGylate EV isolated sample 7 (particle concentration: 1:16 in PBS) with relative frequency (%) on the y-axis and zeta potential (mV) on the x-axis. The different curves represent three consecutive measurements of the same PEGylated EV isolate. Tables 13a and 13b below shows single run measurement readings for non-PEGylated EV isolate sample with mean zeta potential, followed by specific values same sample after PEGylation. SD and distribution peak (mode) values for each run (particle concentration 1:100 in PBS). The zeta potentials were determined using the Smoluschowski approximation. The data is represented as Mean and SD. All data is in mV.

TABLE-US-00025 TABLE 13a Pre-PEG Mean Zeta Potential Readings in a 3 series replicate Average Mode NTA 1000 readings run of pre-PEG Before PEG (mV) readings (nM) −15.49 −18.70 −13.35 −15.85 93.50

TABLE-US-00026 TABLE 13b Sample 4 PEGylated Reading 1 Reading 2 Reading 3 Mean Zeta Potential (mV) −9.21 −15.71 −16.82 Standard Deviation (mV) 0.90 0.71 0.56 Distribution Peak 4.59 −13.63 −14.73

Transduction of MSCs

[0181] Transduction of hMSCs Donor #096 was completed using established protocols. Lentivirus particles were sourced from Genecopoeia Ltd.

[0182] We currently have 4 batches of Transduced hMSCs Cells listed below, some of the initial attempts with MOI of 1 to 2 to transduce the hMSCs were unsuccessful and are not included.

Lentivirus Titre Volumes

[0183]

TABLE-US-00027 TABLE 14 Lentivirus TU/ml TU/μl LPP-CS-NEG 1.63E+08 1.63E+05 LPP-CS-CFTR 1.82E+08 1.82E+05 * 1TU = 100 copies of viral genomic RNA, which combined are able to infect 1 cell.
Transduction Volumes for hMSCs Transduction

TABLE-US-00028 TABLE 15 LPP-CS-NEG LPP-CS-CFTR Volume used Volume used Transduction Current Stocks (μls) (μls) Dates EGFP-NEG EGFP-CFTR 18.00 18.00 22-May 1 × T175 T-Flask 1 × T75 T-Flask 12.50 12.50 30-May 2 × T75 T-Flask 1 × T25 T- Flask 18.00 18.00 07-Jun 2 × T175 T-Flask 2 × T175 T-Flask 18.00 18.00 14-Jun 1 × T75 + 1 × T25 1 × T75 T Flask + T-Flask 1 × T25 T-Flask

[0184] Fluorescence Images are shown in FIGS. 20 and 21.

Discussion

[0185] In this study, modification of EV fractions produced by cells were studied. As indicated by the NTA analysis in the previous study, the mean size of PEGylated exosomes was slightly higher than of PEGylated exosomes, although these experiments could not establish any specific co-relation between the hydrodynamic size difference of PEGylated vs Non-PEGylated EVs. This may be 1) because of the incorporation of PEG molecules onto the surface of the EVs modifying their behaviour in suspension; 2) leaching out of PEGylated EVs from the use of plastic tubes used for storage/incubation; 3) leaching out of EVs from Ultra Centrifuge Tubes during ultracentrifugation washing step after PEGylation is done; 4) precipitation of PEGylated EVs by the use of plastic/silicon pipettes. Additionally, the zeta potential measured using a Litesizer indicated the surface charge of unPEGylated EVs was in the range of −6.65 to −18.6 mV for series readings in which maximum 1000 runs was done by Anton Paar Software until a set algorithm for Phase quality control was reached or average of 1000 runs was considered automatically by the machine, whereas for PEGylated EVs in Series run similar to above for sample 1, 2 and 6 yields maximum mean zeta potential value of −0.40 mV to a minimum of −23.6 mV was observed, although consecutive zeta potential readings were observed to have a visible trend of significantly lower values than the initial readings, suggesting the modification of surface characteristics of PEGylated EVs due to applied voltage and PEGylation of EVs effectively bringing the surface charge of EVs towards a neutral value. This phenomenon may indeed help to explain that the surface modification of the exosomes is reversible which is a possible explanation for the observation that surface modification with low molecular weight PEG did not hinder cellular transmembrane uptake of the exosomes as we had expected to occur.

[0186] Another zeta test by replicates of same sample was performed in single run tests although the results were inconclusive, may be because of the storage capacity or PEG interactions with plastic. To further reduce possible interaction of plastic tubes with PEG particles during PEGylation incubation step by using glass vials although the ultracentrifugation step using the plastic ultracentrifuge tubes could not be skipped, nor could the use of plastic/silicon pipettes during transfer of DSPE-PEG or PEGylated EVs. In future, we will modify our experimental techniques accordingly, through the use of glassware vessels and instruments such as pipettes and modification of the EV purification technique post PEGylation by changing to SEC chromatography. The DSPE-PEG was also observed to be possibly degraded when some of these experiments were performed although we observed positive values at the modes in some of the sample indicating that effective PEGylation was possible using more PEG. This is a clear indication that some EVs were PEGylated, as PBS itself wouldn't cause the distribution peak to shift towards positive values and that the presence of PEG molecules on the membrane of exosomes nearly neutralised the surface charge. Furthermore, as exosome samples required an additional ultracentrifugation step to remove excess PEG molecules, the actual particle count after the isolation of exosomes may be higher than the number obtained after the PEGylation procedure.

[0187] The hMSCs were also transduced, although with initial setbacks a stable transduction was achievable at about MOI=4 or larger using spin protocol. Fluorescence Images produced from the transduced cells show GFP fluorescence in all transduced cells which is the result of the CFTR-EGFP fusion protein and the EGFP control lentivirus. This is shown in FIGS. 22-25 show GFP positive MSCs taken on a fluorescence microscope following seeding the cells on slides, fixing and staining the nuclei with DAPI (blue). This confirms the presence of cells, not just autofluoresence. Two magnifications are provided showing GFP expression in all cells.

[0188] In conclusion, it has been shown that EVs can be PEGylated to impart mucus penetrating abilities without impacting on intracellular trafficking of the EVs—further investigation of PEGylation using refined and specific tools is possible as suggested—and the EVs can be characterized—again this can be further explored for large scale production. Characterization of EVs and proteins from CFTR overexpressed cells for over expression of CFTR protein using RNA, gene and protein analysis will further show effective transduction and proof of concept. The functional transfer of CFTR expression mediated by these exosomes (in comparison to the Negative Controls) will be evaluated in Air Liquid Interface models of CF epithelial cells as this model recapitulates the conditions, inclusive of the mucus barrier, that an inhaled aerosol will encounter once the aerosol droplets deposit in the CF lung. These experiments are expected to show significant improvements over the state of the art viral vector and nanoparticle mediated gene transfer approaches i.e. greater transduction efficiency, functional transfer of CFTR protein and mRNA to virtually all cells in culture and demonstration of functionality of introduced actives in CF epithelial cells by assessment of chloride ion transport in Using chamber experiments of transduced cell cultures.

[0189] Also, scaling up of EV isolation using high cell density Bioreactor technologies and using more efficient and commercially scalable ways of EV isolation such as Tangential Flow Filtration and Size Exclusion Chromatography may raise the yield of EVs. The use of immortalised stem cells that are transduced with the CFTR overexpression lentivirus to create a master cell bank that is capable of reliably producing the overexpressed recombinant nucleic acids over extended passages is also highly beneficial for commercial scale production of EVs. Further optimization of the PEGylation protocol may neutralize charge of EVs separated from CFTR transduced mesenchymal stem cells without major loss, especially the storage and transfer of DSPE-PEG and PEGylated samples using organic solution safe apparatus.

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