RU2799315C2 - Extracellular vesicles for inhalation - Google Patents

Abstract

FIELD: pharmaceuticals; medicine.

SUBSTANCE: object 1 is an aerosolizable aqueous composition for delivering cargo to lung cells, containing exosomes from mesenchymal stem cells (MSCs) having a surface coating of a hydrophilic polyethylene glycol (PEG) polymer, and the exosomes carry the specified cargo containing microRNA (miR), anti-miR, mRNA, long non-coding RNA, cRNA, small interfering RNA, short hairpin RNA, piwi-interacting RNA, regular-spaced clustered short palindromic repeat (CRISPR) RNA sequence, modifications of the above or engineered nucleic acid sequences, protein, cytokine or lipid, where the PEG has a molecular weight of less than 5 kDa, the surface coating covers at least 65% of the surface of the exosomes. Object 2 is a method of treating a patient having a lung disease including providing a composition, forming an aerosol of exosomes of the composition, and administering the aerosol to the patient.

EFFECT: possibility of obtaining an aerosol containing exosomes which passes through the mucus layer with penetration into lung cells, for the treatment of lung diseases.

15 cl, 25 dwg, 16 tbl, 2 ex

Description

Introduction

The present invention relates to extracellular vesicles (EV, from the English. extracellular vesicles), in particular exosomes, compositions containing these vesicles, their uses and methods for their use and manufacture. The present invention also relates to the use of such vesicles in therapy and gene therapy, in particular for the treatment of diseases of the respiratory tract, such as cystic fibrosis (CF - cystic fibrosis), COPD (Chronic Obstructive Pulmonary Disease) and lung cancers.

Prior Art

Extracellular vesicles are nanoscale membrane vesicles encapsulated by a lipid bilayer and actively secreted by most prokaryotic and eukaryotic cells. Extracellular vesicles include exosomes, microvesicles, and membranous vesicles.

Vesicles are generally formed by exocytosis from living cells. Exosomes differ from many other extracellular vesicles in that they originate from endocytic compartments in cells and are expelled from the cell by exocytosis when the multivesicular bodies fuse with the plasma membrane.

The formation of multivesicular bodies is a multi-step process that begins with the formation of early endosomes. These early endosomes mature into late endosomes, and during this process several cargoes, either during degradation in the lysosomal compartment or being ordered into small vesicles to be secreted, are selectively enclosed within the endosomes by invagination of their membrane and gradually form many intraluminal vesicles. These endosomes with many intraluminal vesicles are currently referred to as late endosomes and then mature to form MBTs (multivesicular bodies) that eventually fuse with the plasma membrane to release these vesicles as exosomes.

Extracellular vesicles typically range in size up to about 1000 nm, while exosomes typically range in size from 30-150 nm. In addition to their membrane "signature" molecules, vesicles, including exosomes, naturally have an intravesicular cargo that can include a variety of proteins, RNA, and microRNAs in aqueous solution/suspension.

Vesicles, in particular exosomes, are involved in cellular communication and intercellular signal transduction. As a result, exosomes and other vesicles have recently begun to be used in experimental disease models to deliver drugs or desirable molecules. Due to their natural origin, exosomes retain a distinct advantage over liposome-based drug delivery as an efficient and reliable means of delivering various nucleic acid, macromolecular, and drug-based therapeutics.

The ability of vesicles to carry proteins, RNA, and microRNAs to the cytoplasmic nucleus makes them convenient vesicles for drug delivery. In addition, the presence of a lipid bilayer provides structural integrity, protects nucleic acids from degradation, and enables exosomes to withstand shear stress.

Exosomes are known to be able to easily penetrate cell membranes and also evade the immune system, due in part to their size. Because exosomes are naturally occurring nanovesicles, they do not have the cytotoxic effects that drug carriers based on liposomes or other nanoparticles might have, and have a better blood half-life and better penetration across various biological barriers.

Cystic fibrosis (CF) is a genetic disorder in which a patient inherits a mutation in both copies of the cystic fibrosis transmembrane conductance regulator (CFTR) protein gene. CFTR is involved in the production of various secretion products, in particular mucus, digestive juices, and sweat. While such secretions are usually thin, as a result of a mutation in patients with cystic fibrosis, the secretions are thick. In the lungs, these thick secretions result in significant sputum production, reduced clearance of this mucus, blockage of the airways due to mucus accumulation, persistent coughing, frequent infections in the lungs, and difficulty breathing.

While there are currently no known cures for CF, gene therapy is being explored as a possible potent cure. Various approaches have been tried in the case of gene therapy, including liposomes and viral vectors. However, such treatments have been found to be ineffective, mainly due to vector inefficiency and safety concerns. Viral vectors can elicit an unwanted immune response that prevents reintroduction and can also induce potentially carcinogenic insertional mutagenesis by integrating the viral DNA into the host genome.

Because CF causes significant problems in the lungs, efficient delivery to the lungs by gene therapy is considered to be a requirement for effective therapy.

Vesicles, including exosomes, appear to be possible delivery vehicles to the lung for gene therapy and other types of therapies in general, such as regenerative medicine, protein, antibody or small molecule based therapies. However, there are at least two significant difficulties. First, the mucus in the lung provides a significant barrier to any gene transfer agent reaching the lung cells. The mucus is a sticky, viscoelastic gel that protects against pathogens, toxins, and waste products at multiple entry points in the body, including the eyes, nose, lungs, gastrointestinal tract, and female reproductive tract. Many nanoparticles are highly mucoadhesive and are effectively entrapped in the rapidly releasing peripheral mucus layer, which greatly limits their spread across the mucosa as well as penetration into the underlying tissue. Clinical trials testing viral genetic vectors, including adenovirus (AdV) and adeno-associated virus (AAV), serotype 2, and non-viral liposome vectors for inhaled gene therapy, have failed to provide clinically meaningful benefit due to inefficient gene transfer to the respiratory epithelium, generation of therapy-inactivating host immune responses, and, in the case of liposomal gene therapy, delivery of sufficient doses has been another recognized problem. Various studies have shown that mucus-mediated entrapment makes it difficult for these vectors to reach the underlying epithelium and thus prevents successful gene transfer. The retention time of these trapped particles is limited by the rate of renewal of the peripheral mucus layer, which, depending on the organ, ranges from seconds to several hours. To ensure efficient delivery of particles, including pharmaceuticals, across mucosal surfaces, such particles must be capable of rapidly diffusing across the mucosal barrier, avoiding mucus adhesion and avoiding protection due to mucocyl pair clearance. In addition, immediately after crossing the mucosal barrier, it is also necessary to cross the barrier in the form of a cell membrane. It is also intended to provide a barrier preventing access to the cell, in particular by viruses and foreign particles.

Viral vectors can also be neutralized by existing immune responses or acquired immune responses upon repeated administration. The use of stem cell or EV exosomes that are immunologically privileged may circumvent this problem. The PEG (polyethylene glycol) coating can further enhance the stealth properties of stem cell exosomes by effectively shielding surface antigens.

In addition, aerosol delivery of therapeutically relevant doses of vesicles can be problematic with many standard nebulizers. Vesicles in an aqueous nanosuspension or colloid can aggregate or form agglomerates. Forces acting between particles, especially in concentrated nanofluids, lead to increased viscosity, which, in turn, leads to very significant difficulties in aerosolization. In addition, aggregation or agglomeration results in vesicles that do not form a true suspension or colloid, since the vesicles are not distributed throughout the liquid, but accumulate or adhere to the walls of the vessel in which the nanosuspension or colloid is held.

Efficient delivery of cargo, in particular nucleotide sequences and proteins, to lung respiratory epithelial cells remains a common problem for all approaches.

In Kooijmans et, "PEGylated and targeting extracellular vesicles display enhanced cell specificity and circulation time", Journal of Controlled Release, vo. 224, 7 January 2016, pp. 77-85 describe decorating extracellular vesicles with polyethylene glycol-conjugated targeting ligands to improve the specificity of the vesicles and prolong their residence time in the circulatory system before clearance. This article describes the production of extracellular vesicles from mouse neuroblastoma cells and does not address the problems of mucus penetration or cell membrane penetration. In fact, it has been pointed out in this article that PEGylation adversely affects cellular uptake.

In Myung Soo Kim, "Engineered macrophage-derived exosomes for targeting paclitaxel delivery to pulmonary metastases: in vitro and in vivo evaluations*", Nanomedecine: Nanotechnology, Biology and Medicine, vol. 14, no. 1,2 October 2017, pp. 195-204 also describes exosomes decorated with PEG conjugated to a cell/receptor specific ligand. Again, this document does not address any problem associated with the delivery of vesicles to the lung and does not mention crossing the mucosal barrier or cell membrane. In addition, this document describes the cargo - paclitaxel.

US9901600 on behalf of Alexander Mitsialis describes the use of exosomes for the treatment and/or prevention of lung diseases. Exosomes may include proteins or nucleic acids as cargo. These exosomes are uncoated and do not solve mucosal or cell membrane barrier problems. While this document suggests administration by injection or inhalation, there is no information on how aerosolization of exosomes can be achieved as required for inhalation.

It is desirable to provide a means of delivering gene therapy to lung cells using an inhaled delivery system. It is also desirable to provide a delivery vehicle that can pass through the mucus layer and permeate cell membranes, additionally the delivery vehicle should be carried in compositions that can be aerosolized.

The purpose of the present invention is to provide a vesicle, in particular an exosome, which can be aerosolized and pass through the mucus layer to enter lung cells. Another object of the invention is to provide vesicles, in particular exosomes, for use in therapy, specifically for the treatment of diseases of the lung.

Brief summary of the invention

According to the invention, an aerosolizable composition is provided containing extracellular vesicles from mesenchymal stem cells (MSCs) having a surface coating of a hydrophilic polymer polyethylene glycol (PEG), and these vesicles carry a cargo containing one or more of microRNA (miR), anti-MIR, mRNA, long non-coding RNA, circular RNA, small interfering RNA, short hairpin RNA, piwi-interacting RNA, clustered regularly interspaced short palindromic repeats (CRISPR) RNA sequences, modifications of the above, or artificially constructed sequences nucleic acids, protein, cytokine or lipid.

Such compositions suitably contain surface-coated vesicles in the form of a colloid or suspension. Coating with a hydrophilic polymer typically results in vesicles that can form a suspension or colloid in an aqueous composition and can penetrate mucus and enter cells to deliver a therapeutic payload.

The invention also provides the vesicles themselves and such a composition containing the vesicles, for example in the form of a colloid or suspension for use in therapy, where the vesicles have a therapeutic payload and a surface coating of a hydrophilic polymer.

Such a payload may contain a microRNA (miR), a microRNA modulating nucleic acid sequence such as an anti-miR/anthomir, or other non-coding nucleic acid sequences that can alter gene expression in diseased target cells (e.g., long non-coding RNA or circular RNAs that can act as miRNA sponges, thus exerting anti-miR actions, small interfering RNA or short hairpin RNA, for specific gene knockdown, piwi-interacting RNA for silencing genes or transposons involved in disease, etc. ), mRNA, lipids, proteins, cytokines or small molecules for delivery to target cells. All of the nucleic acids can be changed or the sequence can be designed for modified effects. Such shipments may contain one or more or two or more or all of the following: microRNA (miR), mRNA, anti-miR, other naturally occurring or artificially derived nucleic acid sequences, a lipid, a protein, a cytokine, and a small molecule therapeutic agent. Such cargoes can be introduced, for example, into exosomes during biogenesis by their parent cells (for example, via microRNA overexpression through genetic modification of the producing cell type), or the therapeutic molecule can be introduced into the exosome after it has been isolated from the culture medium (for example, using electro via radio, heat shock, transfer solutions/reagents, or other means of introducing a small molecule, protein, or nucleic acid sequence such as an anti-miRNA, mRNA, or microRNA). When vesicles, such as exosomes, are introduced into the cytoplasm of a lung target cell, they can provide treatment for cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), lung cancers, idiopathic pulmonary fibrosis, obstructive pulmonary disease, asthma, pulmonary hypertension, bronchopulmonary dysplasia and other pathological conditions and diseases of the lung.

The invention also provides a composition as described above for use in therapy.

The invention also provides a method for treating a patient having a lung disease, comprising:

providing the composition as described above;

aerosol formation of vesicles of the composition; And

administration of this aerosol to the patient.

Details of the Invention

Providing a hydrophilic polymer surface coating on the vesicles reduces their tendency to aggregate or agglomerate, resulting in a colloid or suspension of coated vesicles that can be aerosolized and also have the ability to pass through mucus to enter cells and, if present, deposit their additional therapeutic cargo that has been incorporated into the EV, either as a result of genetic modification of EV-producing cells, or otherwise incorporated exogenous cargo.

Preferably, the vesicles are exosomes, as used, for example, in the examples below. Alternatively, the vesicles may be microvesicles, membrane vesicles, or membrane particles.

Preferably, the vesicles, in particular exosomes, are derived from animal cells, more preferably from human cells. The cells may be somatic cells or stem cells.

In preferred embodiments of the invention, the exosomes are derived from mesenchymal stem cells, primarily human MCKs (hMCKs). Such vesicles exhibit inherent therapeutic properties, and delivery of these vesicles to target cells has been shown to modulate fibrosis, inflammation, tissue regeneration, and pro-survival activities. More preferably, vesicles, in particular exosomes, are derived from hTERT (human Telomerase reverse transcriptase) immortalized mesenchymal stem cells derived from adipose tissue or bone marrow. MSCs can be genetically modified to overexpress specific miRs; there are commercially available miRNA and anti-miR overexpressing constructs (eg, XMIRXpress lentivectors from Systems Biosciences, which can transform cells to overexpress preferred miR/antiR while the X motif directs high concentrations of miRNA to exosomes). MSCs can package miR data into exosomes or other vesicles, which can be harvested and purified using standard application techniques as described. Immortalized MSCs are an ideal source - a cell bank with a tremendous capacity for stable overexpression of cargoes as bioengineered nucleic acids and a distribution that is ideal for the production of vesicle-based therapeutics, in particular exosomes, at an industrial level.

Vesicles, in particular exosomes, can be used to treat many disease states. Hydrophilic polymer-coated vesicles can be aerosolized and, when deposited in appropriate areas of the lung, are also better able to pass through mucus and surprisingly, despite surface modification, they show continued ability to rapidly cross the cell membrane. In this regard, the vesicles of the invention are particularly suitable to be adapted for use in the treatment of pathological conditions of the lung such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), lung cancer, asthma, pulmonary hypertension, acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis, and other conditions affecting the lungs, but not limited to.

Vulnerable organs, particularly the respiratory tract, secrete viscoelastic mucus to capture and expel foreign pathogen and ultrafine particles. As a result, mucus provides a significant barrier to entry of therapeutic agents delivered to the lungs. While small molecule drugs can usually penetrate the mucosal barrier, which makes aerosol delivery of such therapeutics a successful delivery method, the mucosal barrier has proven to be very problematic for the delivery of biological molecules such as gene transfer agents or vectors for application. in gene therapy.

Vesicles, in particular exosomes, according to the invention are coated with a hydrophilic polymer. Advantageously, these coated vesicles have been found to have improved aerosol generating properties. Separately, coated vesicles have been found to more easily penetrate lung mucus, facilitating access to lung tissue in the treatment of lung disease or other therapeutic applications in which active agents are delivered to or through lung tissue.

Coated vesicles, in particular exosomes, of the invention have been found to form suspensions and/or colloids that have a reduced viscosity compared to uncoated exosomes, this effect is believed to result from steric stabilization of the exosomes in the nanosuspension/colloid. The formation of a suspension having dispersed particles or a colloid facilitates the formation of aerosols with aerosol particles in the size range suitable for delivery to the lungs and containing useful concentrations of vesicles. These compositions are useful for delivering directly to vesicles, such as exosomes, and are also useful for delivering vesicles containing additional components for therapeutic use by delivery to, or by being included in, or on the vesicles. Such exosomes may display additional cell-specific targeting moieties, either attached (eg, via click chemistry) or moieties that have been expressed as a result of genetic engineering. Such targeting ligands can enhance uptake of exosomes in specific cell types (eg, fibroblasts, immune cells, endothelial or epithelial cells).

The hydrophilic polymers used in the invention thus result in vesicles that exhibit reduced aggregation or agglomeration in solution forming a colloid and a suspension. Suitably, the polymers are essentially non-ionic and essentially uncharged. One result is that coated exosomes of the invention typically have a surface charge that is reduced compared to uncoated exosomes and tends to have a substantially neutral net surface charge.

A surface coating of a hydrophilic polymer, polyethylene glycol (PEG), indicates that the PEG is on the outer surface of the extracellular vesicle, such that its functionality is available. This allows the properties of the PEG to influence the properties of the extracellular vesicle, such as its hydrophilic properties. In view of this, the PEG is not an internal part of the other coating and does not have any significant coating on the PEG. The PEG coating is the outermost coating on extracellular vesicles. Additional ligands or other elements are not conjugated at the distal ends of the PEG. PEG forms essentially the only coating on extracellular vesicles.

The low molecular weight PEG preferably has a molecular weight of less than 3 kDa and preferably provides an EV with a dense surface coverage of 65% or more, which is sufficient to substantially neutralize the surface charge of the EV (namely, -8 mV to 0 mV).

Preferably, the surface coating will cover at least 65%, more preferably at least 70% and most preferably at least 75% of the surface of the vesicles to provide sufficient steric stabilization and surface charge neutralization while minimizing exosome agglomeration and aggregation. In the specific examples illustrated below, a surface coverage of approximately 70% or more was observed in coated exosomes that aerosolized and passed through the mucus.

The surface coating polymer typically has a low molecular weight, preferably less than 5 kDa, more preferably less than 4 kDa, more preferably less than 3 kDa, and most preferably 2 kDa to 3 kDa. In the specific examples illustrated below, the coating was formed by attaching approximately 2 kDa polymer chains to the surface of the vesicles. As a result, these chains do not provide steric distortion or mismatch with mucin chains that may occur in the mucosal layer of the lungs. The polymers are suitably included at a concentration that is sufficient to substantially neutralize the surface charge of the vesicles and prevent agglomeration or aggregation; such a dense coating will ensure that the conformation of the hydrophilic chains is in the optimum orientation/brush conformation. The required polymer concentration depends on many variables such as the chain length of the selected polymer, the efficiency of the polymer binding to the surface of the vesicles, and the molar concentration and particle size distribution of the isolated vesicle sample. For example, a polymer incubated with 100 nm exosomes can be used at a concentration of about 8 mole % or more to provide dense coverage and suitable post-modification of vesicle characteristics. However, care must be taken to avoid excessively high polymer concentrations, which can lead to damage to the vesicular membrane due to detergent-mediated disruptive actions at higher concentrations.

The vesicles of the invention typically have a size in the range of 20-1000 nm, more suitably in the range up to 300 nm. Exosomes typically have a size in the range of 30-150 nm, and microvesicles typically have a size in the range of 100-1000 nm. Generally, the surface coated exosomes of the invention are in the size range up to 200 nm, preferably 20-200 nm and most preferably 30-180 nm. Dimensions can be measured using dynamic light scattering techniques (Degiorgio, V., et al., 1979) or nanoparticle tracking analysis (NTA, used in the examples below).

As stated, preferably the vesicles are derived from a mesenchymal stem cell and therefore have the great advantage of having inherent regenerative and therapeutic properties. Studies have shown that unmodified MSC exosomes and MSCs themselves demonstrate anti-apoptotic, pro-angiogenic and anti-inflammatory effects due to many factors (for example, microRNA, mRNA, IncPHK, KGF (keratinocyte growth factor), HGF (hepatocyte growth factor, hepatocyte growth factor), VEGF (from the English vascular endothelial growth factor), IGF-1 (from the English insulin-like growth factor), TIMP3 (from the English tissue inhibitor of metalloproteinase - a tissue inhibitor of metalloproteinases)) and through a number of mechanisms (for example, as a result of polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, modulation of antigen presentation, modulation of cytokine release, increased glycolysis through ATP-forming enzymatic transfer and activation of survival kinases ( for example, ERK (from the English extracellular signal-regulated kinase - a kinase regulated by extracellular signals) and ACT ("AK" from the English. AKR mouse strain - a line of mice AKR, "T" from the English. thymoma - thymoma) through CD73), and by reducing complement activation through CD59).

These benefits can be enhanced by adding relevant cargo to the vesicle, such as exosomes. In fact, whatever the source of the vesicle, the cargo can provide a beneficial therapeutic effect.

Vesicles, in particular exosomes, can also carry cargo to be delivered to target cells. Preferably, the cargo is a microRNA (miR) or an anti-miRNA (anti-miR). Alternatively, the payload may be an mRNA or other endogenous or exogenous nucleic acid construct, an engineered sequence, a chemically modified or synthetic version of the foregoing, a protein, or a pharmaceutical small molecule. Nucleic acid can be of engineered sequence or modified base pairs, and be loaded into exosomes through a variety of mechanisms.

Vesicles are also useful for in vivo pulmonary delivery of CRISPR-CAS9 or other gene editing techniques, since intracellular delivery to cells located below the mucosal barrier is a problem that must be solved for successful gene editing in the case of respiratory diseases.

In some embodiments of the invention, the hTERT mRNA and the catalytic subunit of the hTERT enzyme can be incorporated into exosomes as a result of lentiviral hTERT immortalization of EV-producing stem cells, these active moieties are useful in combating cell aging in diseased target cells (e.g., alveolar progenitor cells). epithelium AEC2), which is a hallmark of COPD, IPF (idiopathic pulmonary fibrosis) and increasingly related to other lung diseases. Similarly, the other major component of the telomerase enzyme, TERC (hTR), can be incorporated into EV as an RNA molecule. These cargoes will act in target cells to enhance the potential of EV stem cells in regenerative medicine. It is important that these effects will be transient and reversible in nature as they will act in the cytoplasm and will not integrate into the genome of the target cells, which is an important feature of the regulation since no lasting effects will be observable in the target cells or indeed any daughter cells formed by target cells.

MicroRNAs (miRs) are small non-coding RNA molecules, typically on the order of 20-25 nucleotides, found in plants, animals, and some viruses that primarily function in RNA silencing and post-transcriptional regulation of gene expression. miRs function by base pairing with complementary sequences within mRNA molecules and are key regulators of gene expression. The load may also be anti-miR or antagonisms (antago-miR). The load can be tailored to suit the condition being treated or to modulate gene expression in a particular target cell type. Typically, conditions or disease states result in upregulation of some miRs and downregulation of other miRs. When the miR is down-regulated, replacement treatment for that miR may be effective, while when the miR is up-regulated, it may be effective to provide an anti-miR or an antagonist.

EV cargoes can be other non-coding nucleic acid sequences that can alter gene expression in diseased target cells. For example, long non-coding RNAs or circular RNAs (endogenous or artificially engineered) that can act as "sponges" or "mops" of microRNAs, thus exerting anti-miR activities; small interfering RNAs, or short hairpin RNAs, to knock down a specific gene; and piwi interacting RNAs for silencing a gene or transposon relevant to a disease.

When the payload is miR and the treatment is for COPD, preferably the payload is selected from one or more of: miR-125b-5p, miR-125b-1-3p, miR-513a-5p, miR-34c, miR-452, miR -146a, Let-7c, miR-576-3p, miR-513a-3p, miR-923, miR-937, miR-422a, miR-25, miR-99b, miR-24 and miR-187. More preferably miR is selected from miR-125b-5p, miR-125b-1-3p and miR-513a-5p, which represent novel genetic targets for disease management. The selected microRNAs, hsa-miR-125b-5p, hsa-miR-125b-1-3p and hsa-miR-513a-5p, are key regulators in COPD targeting important genes involved in COPD pathogenesis. These miRs are underexpressed in COPD smokers compared to non-COPD smokers and are known to target and downregulate several important mRNAs through microRNA-Target-Interactions (MTIs). that are involved in the pathogenesis of the disease.

Specifically, miRNA 125b-5p is a suitable cargo and has been shown to target and down-regulate the following important COPD-associated genes:

[1] ADAMTS4 (Aggrecanase-1) in human osteoarthritic chondrocyte studies. ADAMTS4 is a proteolytic enzyme involved in the disassembly of the extracellular matrix and is the most overexpressed mRNA (8.91-fold increase) in COPD smokers compared to non-COPD smokers. In addition, ADAMTS4 has been shown to be anti-angiogenic through VEGF binding, downregulation of ADAMTS4 may thus aid in the regeneration of alveolar compartment angiogenesis; [2] SFRP5, a WNT antagonist, whose levels were found to be significantly higher in COPD compared to healthy controls;

[3] DKK3, a notch antagonist whose downregulation may aid alveolar regeneration;

[4] the expression level of EGFR (Epidermal Growth Factor Receptor) in bronchial epithelial cells is significantly higher in smokers compared to healthy controls, and also higher in smokers with COPD than in smokers without COPD. EGFR plays an important role in the regulation of mucus production and goblet cell hyperplasia in the respiratory epithelium;

[5] Receptor for IL-6 (from the English. interleukin - interleukin) (IL6R), a well-known key pathway of inflammation in COPD;

[6] MMP13, a member of the MMP (matrix metalloproteinase) collagenase subfamily, has been shown to be upregulated in COPD alveolar macrophages and type II alveolar cells. MMP13 is involved as the main mediator of disease pathogenesis in response to cigarette smoke and exposure to the PR8 influenza virus in an animal model of COPD exacerbations;

[7] ANGPT2, an antagonist of ANGPT1 (angiopoietin). ANGPTL1 has reduced levels in advanced COPD compared to stable phase/normal levels (associated with vascular regression, levels highly correlated with loss of lung function);

[8] APC (from the English. adenomatous polyposis coli - adenomatous intestinal polyposis), a regulator of the WNT signaling pathway, inhibitory, since APC is a component of the beta-catenin destruction complex;

[9] MAPK14 (P38 inhibition is promising in COPD clinical trials); And

[10] Various other targets that are strongly associated with COPD: SGPL1, BMPR1B, BTG2, CEBRA, Fas, FGFR2, FZD6, GLI1, HK2, HMGA1, HMGA2, IGF1R, IGF2, MMP2, MMP26, MUC1, SMAD4, STAT3, TNF , TNFAIP3, TP53, and BCL2 are pro- and anti-apoptotic mediators, respectively, while BAK1, a BC1_2 antagonist/killer 1, is also a target, TP53INP1, VDR, ERBB2, and ERBB3.

In addition, upregulation of miR-125b-5p was shown to attenuate LPS-induced acute lung injury and inflammation in mice.

MiR-125b-1-3p is a suitable payload and has been shown to target COPD-relevant genes:

[1] Increased expression of TACSTD2 (aka TROP2) in airway basal cells potentially promotes airway remodeling in COPD and airway remodeling through increased levels of basal cell hyperplasia;

[2] SGPL1, sphingosine-1-phosphate (S1P) degrading enzyme with a 4.5-fold increase in expression level in alveolar macrophages in COPD compared with controls. Promotes defects in alveolar macrophages in the phagocytosis of apoptotic cells (efferocytosis) in COPD;

[3] FRZB, a WNT antagonist whose downregulation is expected to show a regenerative effect;

[4] TP53, overexpression of pro-apoptotic P53 in COPD type II alveolar cells. Thus downregulation is expected to have a pro-survival effect, it should be noted that 125b-5p also targets P53;

[5] S1PR1 (cell adhesion, immunomodulation);

[6] BIK, accelerates programmed cell death;

[7] MTFP1;

[8] MAP2K7 involved in environmental/cellular responses;

[9] BGLAP and [10] ITGA9.

MiR-513a-5p is also a suitable vesicle cargo of the invention due to its relevant effects on COPD gene expression:

[1] CBL-miR-513a-5p has been shown to target CBL, negatively affecting the phosphoinositide 3-kinase (PI3K)-AKT and nuclear factor-kB (NF-kB) pathways through their downstream genes, spleen tyrosine kinase ( SYK) and epidermal growth factor receptor (EGFR). Elevated levels of 513a-5p thus reduced the secretion of cytokine factors - interleukin (IL)-10, γ-interferon (γ-IFN), tumor necrosis factor-α (TNF-α - from the English tumor necrosis factor alpha) and IL -12;

[2] CB274 (programmed death ligand 1) PD-L1 protein is expressed more frequently in adenocarcinomas of the lung associated with emphysematous bullae (EB) than in lung adenocarcinomas without EB. PD-L1 - The immune checkpoint is not fully understood but is deregulated in COPD. The PD-L1 protein is induced in various non-lymphoid tissue cells, including epithelial, endothelial, and smooth muscle cells, in response to inflammatory cytokines. The well-known comorbidities of COPD and lung cancer make CD274 a target of particular interest;

[3] miR-513a-5p has many additional and interesting virtual predicted targets for investigation (TargetScan 7.2): AGER (encodes RAGE, upregulated in COPD, RAGE KO mice show reduced levels of inflammatory responses, and upregulation of RAGE causes increased levels of apoptosis of alveolar cells); ADAM9 and INHBA (aka Activin A, TGFB superfamily, elevated in COPD and associated with airflow limitation).

In addition, the miRs identified herein as being suitable for this invention have demonstrated functional MTI (weak) for a much larger number of COPD-associated genes.

Lung cancer is a highly significant and fatal comorbidity of COPD. The increased risk of lung cancer shown by individuals with COPD is independent of smoking history. In the examples below, the inventors aim to reduce the risk of lung cancer by modulating miR-21-5p in affected COPD and surrounding lung tissues, such a decrease in miR-21 levels is believed to be able to reduce the risk of developing lung cancer in patients with COPD and will be achieved by genetically modifying stem cells to overexpress an anti-miR to miR-21 sequence (hsa-miR-21-5p) that will be loaded into the EV. The miR-21-5p inhibitor is either a short RNA with a reverse complementary miRNA sequence, or it can take the form of a circular RNA sequence with multiple miR-21-5p binding sites and use of intron terminal sequences to drive the formation of the circular molecule. Alternatively, a naturally occurring or modified long non-coding RNA sequence can be used to achieve a reduction in miR-21 levels.

When the cargo is miR and the treatment is for CF, preferably the cargo contains miR-17 to reduce hyperinflammation in the lung in CF. CF is a multisystem disease, but the vast majority of complications and deaths are due to lung-related disease. Administration of anti-inflammatory MSC exosomes of the invention with miRNA cargoes is suitable for a significant improvement in lung function. In addition, exosomes delivered to the lungs are expected to have a degree of systemic uptake and systemic action due to the large surface area and pulmonary vasculature.

The shipment may additionally or alternatively contain mRNA. When the treatment is for CF, the cargo may be a modified CFTR mRNA. For example, modified CFTR mRNA may include mutated or deleted miR binding sites for miR-101, miR-223, miR-494, and/or miR-509-3p as they are upregulated in CF and downregulate CFTR expression. Alternatively, the introduced CFTR mRNA may include synthetic 3' and 5' UTRs (untranslated region) that are designed to be freely expressed in most cell types and will also lack upregulated miR binding sites. regulation in this disease. Recent studies have shown that blockade of miR target sites or site-directed mutagenesis at these sites resulted in increased levels of CFTR expression. In addition, the modified CFTR mRNA is resistant to in vivo miRNA-mediated intracellular degradation and therefore has a longer half-life for sustained in vivo expression of CFTR. In addition, enhanced efficacy compared to target site blockers can be achieved because the functional CFTR protein will be translated from the wild-type protein coding sequence, as opposed to saving the expression of endogenous CFTR mutant mRNA, which will cause cellular stress due to reactions of unfolded proteins. .

When exosomes are used in the treatment of lung cancer, in particular adenocarcinoma, the cargo may be one or more of miR-126-3p, miR-218-5p, miR-486-5p, miR-145-5p, miR-338-3p, miR-195-5p, miR-143-3p, miR-139-5p, miR-126-5p, miR-144-3p, miR-34c-5p, miR-30a-3p, let-7c-5p, miR- 451a, miR-1-3p and miR-133a-3p. Additionally or alternatively, the payload may be one or more of anti-miR miR-21-5p, miR-210-3p, miR-182-5p, miR-183-5p, miR-135b-5p, miR-9 -3p, miR-96-5p, miR-205-5p, miR-31-5p, miR-708-5p, miR-196b-5p, miR-375, miR-345-5p, miR-200a-3p and miR -130b-3p.

Other suitable payloads may include microRNAs or anti-miRs chosen for the treatment of other lung diseases, such as idiopathic pulmonary fibrosis.

The compositions are suitably provided as suspensions or colloids for delivery to the lungs by aerosolization using nebulizers. Preferably the suspensions or colloids are water-based. The compositions may also include, but are not limited to, other pharmaceutically acceptable excipients such as salts, surfactants, stabilizers, preservatives, cryoprotectants, buffers, and pH adjusters.

The compositions of the invention are designed to form aerosols containing droplets that can be delivered to the patient's lung. Suitable sizes and aerosol generating devices are known in the art, and aerosols typically contain droplets of 10 microns or less. Aerosolized droplets larger than 5 µm are generally considered too large for respiratory delivery under normal breathing conditions. For reaching and adhering to bronchial and alveolar cells, aerosol droplets are very suitable in the range of 1-5 μm, namely the aerosol includes droplets in this range. Those familiar with the field will appreciate that droplets larger than 5 µm in diameter can be deposited in desirable areas of the lung when appropriate limits are placed on inspiratory flow rates and aerosol rates. Larger droplets, being spherical, carry a much larger volume than smaller droplets, for example, a 10 µm diameter droplet has a thousand times the volume (and hence dose loading capacity) than a 1 µm diameter droplet. The targeting of aerosol deposition in the desired region of the lung can be effectively controlled by varying the aerosol delivery device and formulation parameters, suitably influencing the aerosol droplet size and inhalation flow rate. Aerosol droplet size can be measured, for example, by means of mass median aerodynamic diameter (MMAD) values, which are defined as the aerosol diameter at which 50% of particles are larger by mass and 50% are smaller.

In order to achieve successful aerosolization, it is generally preferred that the viscosity of the colloid or suspension is not too high; these options, however, also depend on the type and model of the device. Preferably, the viscosity should be less than 6 centipoise, such as less than 4 centipoise, more preferably less than 3 centipoise, and most preferably less than 2 centipoise. When the colloid or suspension is too viscous, the aerosolization method may result in foaming and splashing and thus significantly reduce aerosolization, or if aerosolization is possible, the resulting droplets may be too small to be usefully deposited in relation to the intended lung area. Likewise, when the particles in suspension or colloid tend to aggregate or agglomerate, then the aggregated particles are not aerosolized, but may, on the contrary, remain in the colloid or suspension of increasing concentration in the nebulizer reservoir. This, then, results in an increase in the viscosity of the suspension or colloid and an inability to aerosolize or a decrease in delivered dose. Free vesicles, including exosomes, have a pronounced tendency to aggregate or agglomerate, forming a viscous colloid or suspension, and thus cannot be effectively aerosolized. However, it has been found that the vesicles of the invention coated with a hydrophilic polymer show a significantly reduced tendency to aggregate and agglomerate. As a result, a colloid or suspension of suitable viscosity can be formed and the coated vesicles provided herein can be successfully aerosolized.

The size of the formed aerosol droplets is usually inversely proportional to the viscosity of the sprayed composition. The viscosity of nanoscale colloidal systems, such as exosome compositions, increases dramatically as the volume fraction of exosomes increases. As mentioned earlier, the steric stabilization of the vesicles of the invention can significantly attenuate this effect. This effect can be used to promote aerosol deposition of generated aerosols in the target region of the lung, where smaller droplets (at a constant rate and inspiratory flow rate) will be deposited in the small airways and peripheral/distal regions of the lung, and larger droplets will have more central pattern of deposition. For example, in the case of cystic fibrosis, a central aerosol deposition pattern is desirable, targeting areas of the bronchi and large airways where gas exchange does not occur. In contrast, in the case of emphysema, the distal alveolar regions and small airways are the main areas of interest, and smaller droplet size distributions are preferred.

Aerosolization can be carried out using a nebulizer. Nebulizers use oxygen, compressed air or gases and/or nozzles or ultrasound power to break up solutions, suspensions and colloids into small droplets, forming an aerosol that can be inhaled. Nebulizers have previously been designed to aerosolize small molecules, and all commercially available types are successful in doing so when the drug is in solution. However, the type of nebulizer used is more important in the case of large biological agents that can be damaged by nebulizers that transfer too much energy to the solution, suspension, or colloid and generate enough hydrodynamic stress to subject the biological molecules to shear, resulting in loss of activity. For example, ultrasonic nebulizers are absolutely unsuitable for delivering nanosuspensions, and compressor nebulizers cause continuous recirculation of the aerosol, resulting in repeated shear stresses that can damage biological molecules, such nebulizers also preferably aerosolize an aqueous carrier, resulting in concentration of the composition in the reservoir. The "dead volume" of a composition that is not aerosolized but remains in the reservoir is typically 1 ml in compressor nebulizers, and this is completely inapplicable and commercially impractical from a cost perspective relative to advanced therapies such as exosome formulations.

Of the officially approved nebulizer types, vibrating mesh nebulizers are particularly suitable for vesicle delivery because:

1. drops only change phase from liquid to air once when droplets are generated (compared to "reuse" and repeated shear stresses in compressor nebulizers);

2. ultrasonic nebulizers are not suitable for delivering nanosuspensions;

3. vibrating mesh nebulizers can be adapted to efficiently deliver small volume formulations with minimal residual dead volumes; And

4. Compressor nebulizers may preferably aerosolize the solvent and concentrate the remaining composition in a reservoir.

Surface acoustic wave (SAW) nebulizers, which generate surface acoustic waves on piezoelectric chips, have been shown to aerosolize suspensions of fragile nucleic acid molecules such as plasmid DNA vaccines and stem cells. The high frequency, low power SAW sputtering mechanism is useful in order to avoid minimal damage to biological molecules, including nucleic acids. SAW nebulizers are more successful at aerosolizing more viscous solutions and suspensions than many other types of nebulizer, making them ideal for aerosolizing suspensions and colloids formed from an aqueous mixture of exosomes, in particular exosomes coated with a hydrophilic polymer. The viscosity of the liquid determines, to some extent, the size of the droplets produced by the SAW nebulizer. However, these types of nebulizers have not yet been approved by any regulatory authority for use in clinical trials.

Examples

In order to aid the understanding of the invention, a particular embodiment with a variant thereof will now be described by way of example and with reference to the accompanying drawings, in which:

Fig. 1A and 1B are concentration versus size plots of non-PEGylated and PEGylated exosomes in nanoparticle trajectory analysis (NTA);

On FIG. 2A and 2B show the zeta potential distribution of PEGylated and non-PEGylated exosomes;

Fig. 3A-3F are a series of transmission electron microscopy images of PEGylated and non-PEGylated exosomes;

Fig. 4A-3F are a series of flow cytometry plots showing labeling of MSCs and exosomes with far red dye;

Fig. 5A-5C are confocal microscopy images of treated and untreated exosomes that entered bronchial epithelial cells in cystic fibrosis by first penetrating the mucosal layer in air-liquid culture;

Fig. 6a and 6b are nanoparticle trajectory analysis readings for dPBS (Dulbecco's Phosphate Buffered Saline) and 30 nm gold nanoparticles (Au) as controls;

Fig. 7a and 7b are nanoparticle trajectory analysis readings for Sample 1;

Fig. 8a and 8b are nanoparticle trajectory analysis readings for Sample 2;

Fig. 9 is the distribution according to the nanoparticle trajectory analysis for Sample 3;

Fig. 10 is the distribution according to the nanoparticle trajectory analysis for Sample 4;

11 is a distribution according to the nanoparticle trajectory analysis for Sample 5;

Fig. 12a and 12b are the distributions according to the nanoparticle trajectory analysis for Sample 6;

Fig. 13 is the distribution according to the nanoparticle trajectory analysis for Sample 7;

Fig. 14a is a graph showing zeta potential measurement readings for PEGylated and non-PEGylated Sample 1;

Fig. 14b is a plot of zeta potential intensity distributions for three separate measurements for PEGylated Sample 1;

Fig. 15a is a graph showing zeta potential measurement readings for PEGylated and non-PEGylated Sample 2;

Fig. 15b is a graph showing zeta potential intensity distributions for three separate measurements of PEGylated Sample 2;

Fig. 16 is a graph showing the mean zeta potential values for PEGylated and non-PEGylated Sample 4;

Fig. 17 is a graph showing the mean zeta potential values for PEGylated and non-PEGylated Sample 5;

Fig. 18a is a graph showing zeta potential measurement readings for PEGylated and non-PEGylated Sample 6;

Fig. 18b is a plot of zeta potential intensity distributions for three separate measurements for PEGylated Sample 6;

Fig. 19 is a graph showing the mean zeta potential values for PEGylated and non-PEGylated Sample 7;

On FIG. 20 shows the fluorescence of GFP (from the English. green fluorescent protein - green fluorescent protein) for IMSC cells transduced with EGFP (from the English. enhanced green fluorescent protein - enhanced green fluorescent protein) -CFTR; And

On FIG. 21 shows GFP fluorescence for hMCK cells transduced with EGFP-negative control;

On FIG. 22 shows CFTR-EGFP-positive MSCs photographed on a fluorescent microscope after seeding cells on glass slides, fixing and staining nuclei with DAPI (blue) at x10 magnification;

On FIG. 23 shows CFTR-EGFP-positive MSCs photographed on a fluorescent microscope after seeding the cells on glass slides at x10 magnification;

On FIG. 24 shows CFTR-EGFP-positive MSCs photographed on a fluorescence microscope after seeding cells on glass slides, fixing and staining nuclei with DAPI (blue) at x20 magnification; And

On FIG. 25 shows CFTR-EGFP-positive MSCs photographed with a fluorescence microscope after seeding the cells on glass slides at x20 magnification.

Example 1

The objectives of this example were to isolate exosomes from human mesenchymal stem cells (hMCK) and efficiently surface-modify them with low molecular weight lipid-modified poly and ethylene glycol (lipid-PEG) to improve the aerosolization and mucus penetration properties of the exosomes.

Fluorescent labeling of hMCK

Frozen bone marrow MSCs (Donor #163) were thawed before they were placed directly into 15 T-175 flasks (1×10 ⁶ cells for each flask) in complete culture media (CCM), depleted EVs, with 10 ng/ml of basic human fibroblast growth factor (bFGF), were subcultured when they reached 80-85% confluency. Cells were pooled together before being pelleted and resuspended and in PBS, incubated in the dark with 2 µg of 1×10 ⁶ far red cells (excitation/emission approx. 630/661 nm) (Cell Trace) for 20 min at room temperature (this will label exosomes as the far red dye labels cytoplasmic proteins). After incubation, MSC culture media were added to stop the reaction and were further incubated for another 5 min before cell sedimentation and resuspension in EV-depleted CCM.

hMCK culture for exosome isolation

Far red labeled hMCK (Donor #163) were cultured in complete conditioned medium (CCM), and exosomes were isolated according to previously published data (Thery et al., 2006). Briefly, labeled MSCs were directly seeded into 30 T-175 flasks (1 x 10 ⁶ cells for each flask) in CCM with 10 ng/ml basic human fibroblast growth factor (bFGF). After 24 hours, media from all flasks were discarded and replaced with fresh media. Within 3-4 days cells reached 80% confluence, then conditioned media were collected from each flask and cells were counted from 5 randomly selected flasks. Conditioned media from all flasks were pooled together before exosome isolation. To isolate exosomes, media were centrifuged at 400 x g for 10 min and 2000 x g for 30 min to remove cell debris and apoptotic bodies, respectively. After each rotation, 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 ultracentrifuged (Sorvall Discovery 100SE, Hitachi). Finally, the supernatant was subjected to ultracentrifugation at 120,000 xg for 75 min to pellet the exosomes. The pellet was resuspended in PBS to wash the exosomes, swirled again at the same high speed, the resulting pellet was resuspended in 100 µl of PBS and stored at -80°C until further experiments. All centrifugation steps were performed at 4° C. and sterile tubes were used.

Modification of exosomes after isolation with distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG)

Amine DSPE-PEG (2000) (Avanti Polar Lipids, Inc) was purchased from Sigma Aldrich (Wicklow, Ireland). PEGylated exosomes were prepared based on previously published data on the preparation of PEGylated liposomes (Li and Huang et al., 2009). A final volume of 300 μl exosome suspension was prepared by mixing 50 μl concentrated exosome with PBS and 37.8 μl aqueous DSPE-PEG solution (10 mg/ml) (final concentration 1.26 mg PEG per ml exosome). The sample was then incubated at 37°C for 1 h and stirred on a vibrating stirrer every 10 min to prevent aggregation of PEG molecules or exosomes. After incubation, the sample was suspended in more PBS and ultimately subjected to ultracentrifugation at 120,000×g for 75 min to wash out unbound PEG molecules. The exosome pellet was then resuspended in 50 μl PBS and stored at -80°C.

Nanoparticle Trajectory Analysis (NTA)

Samples of both PEGylated and non-PEGylated exosomes were analyzed by nanoparticle trajectory analysis (NTA) (Malven UK) using the NTA NanoSight NS 500, version 3.2 launcher using optimized and validated protocols (Maguire et al., 2017, Hole et al., 2013, Gerlach et al., 2017). All samples were analyzed using a NanoSight NS 500 equipped with a 405 nm laser and a 430 nm long wavelength filter. All samples were stored on dry ice prior to analysis. Dilutions for NTA were made in DPBS buffer (Gibco) which was certified particle-free by NTA immediately prior to measurement. The samples were mixed on a vibratory mixer for a short time before loading to ensure sufficient mixing and destruction of loosely bound exosome clusters. Each sample was manually diluted in PBS to obtain the optimal particle concentration suitable for NTA (20 to 70 exosomes per field of view), with each dilution factor recorded in automatically generated reports. A total of six x 60 second videos were recorded for each exosome sample and the detection threshold during analysis was chosen to ensure that only clearly visible nanoobjects were analyzed to ensure that artifacts were removed.

Zeta Potential Measurement

Exosome zeta potential was measured using Litesizer 500, a light scattering particle analyzer (Litesizer™ 500, Anton Paar Ltd, UK). The zeta potential was measured in the glide plane (15°) of the vesicle-exosomes, and it was ensured that the concentration of particles in each dilution was high enough to provide reliable measurements by generating an average detectable light intensity above 20 kim pulses/s.

Exosome sample analysis based on flow cytometry (with bead binding)

For analysis based on flow cytometry, exosomes (total 2×10 ⁹ particles) isolated from MSCs labeled with far red dye were incubated with 10 μl of aldehyde latex beads (0 = 4 μm) in PBS for 15 min at room temperature without rotation. Then the sample was mixed with 950 μl of PBS and incubated overnight at 4°C without rotation. The remaining exosome-bead binding sites were then blocked by adding 1 M glycine in PBS for 30 min at room temperature. The exosome-coated bead samples were then centrifuged at 1700 RCF (relative centrifugal force) for 5 min, and the pellet was resuspended in 0.5% BSA (bovine serum albumin) in PBS solution. Samples were washed 3 times in the same manner and resuspended in a final volume of 1 ml of 0.5% BSA in PBS. The exosome-coated bead samples were then either conjugated with PE-conjugated mouse anti-human antibody to detect CD9, CD63, and CD81, or left untreated as controls. Isotype controls were also made by incubating exosome-coated beads with PE-conjugated anti-mouse IgG1 isotype 1 or 2. All samples were then analyzed using BD FACS CANTO and data analyzed using FlowJo software.

Transmission electron microscopy (TEM)

Morphological study of both PEGylated and non-PEGylated exosomes was carried out using an electron microscope (EM). Exosomes were fixed with 2% paraformaldehyde (PFA), washed three times with PBS before being adsorbed onto 200 mesh gold EM grids coated with formvar film/carbon. EM meshes were then blocked with PBS/50 mM glycine and PBS/5% BSA prior to treatment with primary mouse anti-human antibodies to either CD63 (cat. no. sc5275, Santa Cruz) or TSG-101 (cat. no. sc7964, Santa Cruz). ) (PBS/0.1% BSA secondary control only) overnight and followed by 30 minutes of secondary goat anti-mouse IgG gold-conjugated antibody (Cat. No. G7652, Sigma). The meshes were then fixed with 1% glutaraldehyde and incubated in 2% phosphotungstic acid in PBS to add contrast to the exosome membrane. The exosome grids were then washed in PBS and allowed to air dry before being analyzed with a TEM (transmission electron microscopy) microscope.

Mucus penetration and cellular internalization experiments with cystic fibrosis epithelial cells cultured at an air-liquid interface

Cystic fibrosis (CF) is a genetic disorder in which compromised host innate defenses lead to recurrent severe respiratory tract infections. Airway epithelial cell culture (AEC) at the air-liquid interface differentiates into cells commonly found in bronchial epithelium (ciliated epithelial cells and goblet cells) and produces thick, viscous dehydrated mucus on the apical surface, which is in contact with air. The air-liquid interface model is thus an exact recapitulation of the conditions that aerosol-delivery-based gene therapy would encounter in an actual CF-affected lung. Surprisingly, the inventors found that mucus penetration and cellular internalization was achieved by applying high concentrations of untreated exosomes to the mucus surface of ALI (air-liquid interface) cultures. 25 μl of treated exosomes and untreated exosomes were then applied to the apical surface of the mucus of the ALI cultures, with the dose of treated exosomes approximately half the dose of untreated exosomes. (2.65E plus 0.7 exosomes per microliter of untreated control and 1.22E plus 07 exosomes per microliter of treated test composition). After 8 hours of incubation, samples were washed 3 times with PBS, fixed in 4% PFA, and stained with phalloidin (tight contacts) and Hoechst stain (nuclei).

Analysis based on confocal fluorescence microscopy with a Zeiss LSM 710 confocal microscope. Scan to analyze xyz images of epithelial cells in treated and untreated samples.

results

Exosomes isolated from human bone marrow MSCs were characterized using various methods to quantify their size, to measure their surface charge, and to identify them in the presence of certain surface markers. Both PEGylated and non-PEGylated exosomes were used for characterization (with the exception of flow cytometry). The inventors isolated exosomes using differential ultracentrifugation, since this method remains the most widely used standard method for isolating exosomes from various biological fluids or cell culture supernatants (Chia et al., 2017, Chia et al., 2016, Thery et al., 2006) . Analysis of the trajectories of nanoparticles (NTA) and non-PEGylated and PEGylated exosomes showed that their average size was 143.2 plus/minus 3.5 nm and 165.6 plus/minus 6.0 nm, respectively (Fig. 1A and B). On the other hand, when PEG molecules separately suspended in PBS were analyzed by NTA, they failed to meet the quality control criteria. This indicates that the number of particles shown in the PEGylated exosome sample was not an artifact caused by PEG molecules. Since NTA measures the hydrodynamic particle diameter, the measured diameter of both exosome samples obtained by transmission electron microscopy (TEM) was smaller than that measured by NTA analysis.

As shown by NTA analysis, the average size of PEGylated exosomes was slightly higher than that of non-PEGylated exosomes. This difference in size is due to the incorporation of PEG molecules into the surface of exosomes. In addition, the zeta potential distribution was measured using the Litesizer 500. The surface charge was measured multiple times and the zeta potential of non-PEGylated exosomes was about -16.5 mV and that of PEGylated exosomes was about -2.6 mV (Fig. 2A and B ). The y-axis shows the relative frequency, which gives the probability of a particular charge being recorded over a particular time period - in this case, the exosome charge was recorded as 1000 frames/minute. This clearly indicates that the exosomes were effectively PEGylated, and the presence of PEG molecules on the exosome membrane almost neutralized the surface charge. In addition, since exosome samples required an additional ultracentrifugation step to remove excess PEG molecules, the actual number of particles after exosome isolation may be higher than the number obtained after the PEGylation procedure. TEM analysis of immunogold labeled exosomes confirmed the presence of CD63 and TSG-101 markers (FIG. 3), and most of the exosomes were less than 100 nm in size. On FIG. 3A-3F shows transmission electron microscopy (TEM) images of immunogold-labeled (CD63 and TSG 101) exosomes (PEGylated and non-PEGylated) (black line, 100 nm). As in FIG. FOR, and in Fig. Only the secondary exosomes of the control group, non-PEGylated and PEGylated, respectively, are represented in the pollutants; On FIG. GS are PEGylated CD63 exosomes; in FIG. 3D shows non-PEGylated CD63 exosomes; in FIG. 3E shows PEGylated TSG 101 exosomes; and in FIG. 3F shows non-PEGylated TSG101 exosomes. However, the number of CD63 and TSG-101-labeled PEGylated exosomes was significantly less than that of non-PEGylated exosomes, but they did not show any observable change in morphology. This may indicate effective PEGylation of the exosome surface, which prevented antibodies from binding to their respective antigen. In addition, the labeling status of both MSCs and exosomes with far red dye was confirmed by flow cytometry analysis (FIG. 4). On FIG. 4A-E show detection of Far-Red+ (far red dye) MSCs and bead-conjugated exosomes by flow cytometry. On FIG. 4A-4C show images of beads coated with Far-Red+exosomes, and FIG. 4D and 4E show Far-Red+positive MSCs (population signaling above threshold). On FIG. 4A shows the bead population (population signaling above the threshold), and FIG. 4B and 4C show the above threshold signaling population for Far-Red+exosome coated beads.

Thus, the inventors effectively modified the surface of exosomes derived from human bone marrow by a simple incubation method with low molecular weight DSPE-PEG. The inventors used DSPE-PEG (namely, lipid cross-linked PEG) for this experiment to incorporate a lipid moiety into the exosome bilayer with polymer anchoring.

In this example, the inventors used "post-inclusion" methods used for PEGylation of preformed liposomes (Nag et al., 2013) to anchor the polymer to the surface of exosomes after exosome isolation by ultracentrifugation. In this method, the inventors mixed a suspension of fluorescently labeled exosomes with a certain concentration of DSPE-PEG to ensure the incorporation of PEG into the exosome membrane. In liposome preparations, samples are typically incubated at a higher temperature to achieve faster and maximum PEG incorporation. However, higher temperature conditions are not compatible with the production of exosomes, so the inventors carried out PEGylation procedures at 37°C. PEGylated and non-PEGylated exosomes were characterized in different ways, and the inventors' results indicate that the surface of the exosomes is PEG-modified in the PEGylated samples, as indicated by an increase in mean hydrodynamic diameter, an almost neutralized surface charge, and a reduced level of TSG 101 and CD 63-positive exosomes. compared to non-PEGylated exosomes.

Analysis of samples by confocal fluorescence microscopy clearly shows that PEGylated exosomes stained with fluorescent far red dye could be taken up by epithelial cells underlying the mucus layer at a dose less than half that of 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 for exosome-based therapies. The localization of exosomes in areas surrounding the cell nuclei is also obvious, namely, where the cell's protein translation apparatus is located, that is, on the ribosome covering the endoplasmic reticulum. The degree of cellular uptake clearly provides for the possibility that smaller viral vectors may be completely retained in these mucus layers. Therefore, the invention contemplates that the modified exosomes will be able to modulate gene expression, for example, the introduction of only about 100 microRNA molecules is required to provide detectable changes in the cell. Similarly, the mRNA transcripts that form the protein in the cell only average about 40 transcripts in the case of the CFTR protein, which is also readily achievable with the internalization of exosomes by target cells demonstrated by the present invention.

With reference to FIG. 5, mucus penetration and cellular internalization experiments demonstrate the ability of extracellular surface modulated vesicles, in particular exosomes, to pass through mucus and enter apical cells. The cell nucleus is stained purple and cell membranes can be identified by tight junctions stained green. It is clear that the red spots, namely the exosomes, have entered the cells and clustered around the nuclei. Thus, this ability of PEGylated exosomes has been demonstrated.

Fig. 5A-5C are confocal microscopy images of cultures of highly differentiated bronchial epithelial cells affected by cystic fibrosis. 25 µl of exosomes were applied to the surface of the mucus layers lying on the surfaces of the apical cells before washing, fixing and staining the samples. For Fig. 5A 3.05×10 ⁸ exosomes in 25 μl PBS (exosomes treated to be thickly coated with DSPE-PEG ₂₀₀₀ and with a surface charge of -2.5 mV) were applied to the surface of the mucus layer. For Fig. 5 In 6.63×10 ⁸ exosomes in 25 μl PBS (exosomes are untreated exosomes, no surface modification and a surface charge of -19.5 mV) were applied to the surface of the mucus layer. As can be seen, comparable mucus penetration and cellular internalization of treated exosomes was achieved with less than half of the applied exosomes. Fig. 5C is a labeled enlargement showing exosomes in the apical compartments of epithelial cells and localized near nuclei (endoplasmic reticulum and ribosomal protein translation apparatus).

Surface engineered exosomes may have a beneficial therapeutic application when delivered without any genetic modification of their contents, or genetic modification of parental cells may optimize the contents of exosomes for delivery to target cells and treatment of specific diseases. More studies are planned on the kinetics of diffusion through mucus and cellular internalization, as the rate of delivery is an important factor that will influence the degree of intracellular delivery in vivo. Surface-modified exosomes exhibit accelerated uptake that is of significant benefit in avoiding mucociliary clearance mechanisms, and this can be demonstrated by comparing the penetration kinetics of equal "doses" of surface-modified and unmodified exosomes applied to the mucus surface at multiple concentrations via confocal-based video analysis. microscopy in real time.

The required doses of exosomes needed for aerosol delivery can also be approximated based on in vitro mucus penetration experiments. A known number of exosomes were applied to a known surface area of mucus-coated epithelial cells (60 mm ² ) and very significant uptake by the epithelial cells was demonstrated. Some key assumptions:

1. Viscosity of a given concentration of the exosome composition and achievable aerosol yield at a given viscosity:

2. These surface engineered exosomes will permeate relatively quickly through the mucus layer, which would reasonably suggest that concentration would significantly affect this through concentration driven diffusion, and relatively highly diluted exosome formulations were used in pilot studies (0.00063928 % (v/v) surface-modified exosomes).

The following table illustrates the exosome loading capacity of an aerosol drop of different exosome sizes, @ 1% exosome concentration (w/v):

For example, in the case of cystic fibrosis, the target area of the lung is the total airway surface area from the trachea to the bronchioles, which is approximately 2.471 plus/minus 320 cm ² in an adult lung. This surface area could be treated using a deposited aerosol volume of approximately 70 microliters of a 1% (w/v) exosome composition.

The surface area of the distal lung is much larger with about 18 times more alveolar cells than bronchial epithelial cells, and targeting this area is desirable, for example, for the treatment of emphysema and idiopathic pulmonary fibrosis. It is expected that the level of absorption of exosomes by cells is much higher in these areas of the lung due to the absence of a mucosal barrier.

In conclusion, the inventors showed that exosomes were efficiently PEGylated using lipid-conjugated PEG by a simple incubation method, and the resulting exosomes penetrated the viscous CF mucus and were internalized by epithelial cells, demonstrating a pulmonary exosome-based gene therapy platform, or simply as a therapeutic useful exosomes for the treatment of inflammatory diseases of the lung.

PEGylation also results in a sterically stabilized exosome composition that has a significantly reduced viscosity compared to non-PEGylated compositions. Formation of an aerosol containing such exosomes having a thick hydrophilic "crown" to protect against agglomeration caused by hydrophobic interaction is therefore facilitated, which means that aerosols to deliver exosomes at an acceptable and useful concentration are now facilitated and available at higher concentrations. PEGylated exosomes show improved mucus penetration, which property then facilitates their passage through lung mucus into lung tissue, further enhancing the effectiveness of PEGylated exosomes in therapeutic applications against lung disease. Finally, PEGylated monoclonal antibodies and nanoparticles may also exhibit enhanced "protective" properties that help them avoid uptake and clearance by phagocytes, such as alveolar macrophages, which are found in increased numbers in inflammatory lung diseases.

Thus, the invention provides coated vesicles, such as exosomes, which can be used in gene therapy for CF, COPD, adenocarcinoma, and other pathological conditions of the lung.

Example 2

The objectives of this example were to show that modification of the surface of exosomes can be efficiently achieved and that populations of genetically modified mesenchymal stem cells obtained by bioengineering techniques overexpressing the CFTr gene can be successfully generated.

Methods for cultivating BT-20 and hMCK cell lines

BT-20 cells obtained as BT-20 human epithelial cells from ATCC® NTV-19™ were cultured in DMEM (Dulbecco modified Eagle's medium) supplemented with 10% FBS. Fetal Bovine Serum) and 100 IU/ml penicillin/streptomycin as complete conditioned medium (CCM). Human mesenchymal stem cells, donor #096, were used and cultured in Alfa Gibco MEM medium with 10% FBS and 100 IU/ml penicillin/streptomycin and 1 ng/ml basic human fibroblast growth factor (bFGF).

Cells were fed 3 days a week using an aseptic technique that consisted of removing spent media from the culture flask by pipetting and adding to the waste vessel, followed by replenishing the culture flask(s) with an appropriate volume of fresh medium (including additives) to the top of the culture flask. culture in such a way that the cell culture (monolayer) is not disturbed by the flow. The flask was then removed from the fume hood, tilting the flask so that its lid was facing inward, and placed in an incubator at 37° C. with 5% CO ₂ . Cells were routinely examined under a light microscope for signs of contamination such as turbidity, cells in suspension, discoloration of the medium, large clumps or particles, etc.

To separate the cells every 7-10 days and before the cells reached 80-90% confluence, the cells were washed with 3-5 ml of PBS, so that it was delivered to the opposite wall of the flask in a way that did not damage the cells, followed by the addition of 5 ml warmed T/E (corresponding concentration, 37°C). The flask was then gently rocked on its axis for 30 seconds, making sure that all cells were covered, and the excess T/E was poured into the waste vessel with a pipette (leaving approximately 1 ml in the flask). Cells were incubated with the remaining small amount of T/E at 37°C for 3-5 min, with the separation of cells from the bottom of the flask. The separated cells were resuspended in 4-6 ml of complete medium (volume depends on the expected number of cells). The cell suspension is then transferred up and down with a 10 ml pipette to ensure homogeneity and removed into a 15 ml falcon. The flasks were incubated at 37°C.

Obtaining EV-Free Environments

EV-free media is a prerequisite for EV isolation protocols for purified EV release from cells in culture. We are talking about the exclusion of contamination of EV isolates of cultured cells with microvesicles outside the experimental setup.

Received 130-140 ml of 20% FBS plus basic medium (1:5) in autoclaved sterile glass bottle, and the corresponding amount of the above was subjected to ultracentrifugation at 110,000 × g for 18 hours, at 4°C. After ultracentrifugation, the supernatant was carefully aspirated into a new 50 ml Falcon tube, making sure that the pellet was not disturbed. The 50 ml falcon supernatant was filtered into a freshly autoclaved glass container. To this was added an equal portion of the basic medium to bring to a ratio of 1:1, bringing the total concentration of FBS in the above solution to 10%. This resulted in a 10% EV-free medium to be used for cell culture to isolate EV. Conditioned media were stored at 4° C. for up to 4 weeks with the addition of 100 IU/ml P/S (or other supplements) prior to use for the EV isolation protocol.

Cell Culture for EV Isolation and Obtaining Conditioned EV Enriched Media

EVs were isolated according to MISEV2018 guidelines and additional improved protocols (Thery et al., 2006). BT-20 cells were made up to a sufficient number of 3-4 T-175 cm ² flasks to seed 12 x T-175 cm ² T-flasks in EV-free media (2 x 10 ⁶ cells/flask). Cells were trypsinized and resuspended in EV depleted media and seeded in 12×T-175 cm ² T-flasks in 10 ml EV depleted media for 24 hours to ensure that the cells adhered to the bottom of the flask. The media were then replaced with an equal amount of fresh EV-free media (conditioned media). The flasks were left to incubate for 48 hours.

After 48 hours, the conditioned media were then collected from each flask and pooled in 50 ml falcons, and the cells in these flasks were trypsinized and the counted number of cells removed from the total cell lysate (the corresponding number of cells were then passaged/frozen/discarded). The culture supernatant of EV-free media was poured from T-flasks into 50 ml falcons (4 flasks per 50 ml falcons). Then, 50 ml filcones containing the supernatant were subjected to centrifugation to remove cell debris at 300 x g for 10 min, temperature -21°C. The supernatant was then aspirated using a 25 ml pipette, leaving an undisturbed residue of 2-3 ml at the bottom, into new 50 ml falcons, respectively. The supernatant was then centrifuged at 2000×g for 10 min at -21°C. After centrifugation, the supernatant was collected, leaving a sediment of non-living cells in the balance of 2-3 ml at the bottom. Then, using a sterile 0.2 μm filter and syringe, the top 25 ml of the supernatant was aspirated and collected in freshly autoclaved ultracentrifuge tubes. The filtered conditioned media were then ultracentrifuged at 110,000×g for 70 min at 4°C on a Hitachi Micro ultracentrifuge. After ultracentrifugation, 1 ml of the 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 the overflow container without scraping off the precipitate. The pellet containing EV was resuspended with 1 ml of PBS in each UCT (Ultracentrifuge Tube) and pooled together into one UCT. She was subjected to ultracentrifugation at 110,000 × g for 70 min at 4°C. After ultracentrifugation, 1 ml of the supernatant was withdrawn into a 1.5 ml eppendorf tube from the UCT and stored at -80° C. (supernatant analysis 2). The rest of the supernatant was discarded into the overflow container without scraping off the precipitate. 85 µl of sterile PBS was pipetted into the UCT containing EV and the marked side was scraped off for about 5 minutes with a 100 µl pipette tip. After 5-8 min scraping, all resuspended PBS was collected. All residue, including bubbles, was collected in a conical tube for future PEGylation. It was subjected to pulse centrifugation for 10 seconds in order to get rid of the bubbles, and the supernatant was mixed in LFH with a pipette. 15-20 µl was pipetted (NTA/Zeta) and 10 µl into a tube for protein analysis. All isolated EV samples were stored at -80°C. All steps were performed using aseptic techniques and all centrifugation steps were performed at -4°C.

Modification of EV after isolation with DSPE-PEG 2000 (AMIN) Amine DSPE-PEG (2000) (Avanti Polar Lipids, Inc) was purchased from Sigma Aldrich (Wicklow, Ireland). PEGylated exosomes were obtained based on previously published data on the production of PEGylated liposomes (Kooijmans et al., 2016). A final 1:4 volume ratio of isolated EV suspension (50 μl) was obtained by mixing the concentrated EV suspension with PBS in a plastic or glass vial for samples 1, 2, and 6 and samples 4, 5, and 7, respectively. To do this, an equal ratio (or desired percentage) of 1:1 (µg protein:µg protein) of DSPE-PEG 2000 amine (10 mg/ml) was added, which was kept for incubation at 37°C for 1 h, by placing the EV samples in temperature-controlled tube rack on top of the shaker at low speeds to prevent aggregation of PEG molecules or exosomes. After incubation, the EV-PEG suspension was injected into 16-20 ml PBS in an ultracentrifuge tube. Ultracentrifugation was performed at 110,000×g for 70 min to wash out unbound PEG molecules. The EV pellet was then resuspended in 75 µl PBS and stored in appropriately sized glass containers with lids, and from this 20 µl aliquot for zeta potential analysis in separate containers. They were stored at -80°C or -20°C for samples 1, 2, 6 and samples 4, 5, 7, respectively. Nanoparticle Trajectory Analysis (NTA)

Samples of both PEGylated and non-PEGylated exosomes were analyzed by nanoparticle trajectory analysis (NTA) (Malvern UK) using the NTA NanoSight NS 500, version 3.2 launcher using optimized and validated protocols (Maguire et al., 2017, Hole et al., 2013, Gerlach et al., 2017). All these samples were analyzed using a NanoSight NS 500 equipped with a 405 nm laser and a 430 nm long wavelength transmission filter. All samples were stored on water ice prior to analysis. Dilutions for NTA were performed in D-PBS [-MgCl ₂ ,-CaCl ₂ ] (Gibco) which was certified particle-free by NTA immediately prior to measurement. Samples were pipetted prior to loading to ensure sufficient mixing and disruption of loosely bound EV clusters. Each sample was manually diluted in D-PBS to obtain the optimal particle concentration suitable for NTA (10 to 100 particles per field of view), with each dilution factor recorded in automatically generated reports. A total of six × 60 second videos were recorded for each EV sample and the detection threshold was selected by the operator during analysis to ensure that only clearly distinguishable nano-objects were analyzed and to ensure that artifacts were removed. Since NTA measures the hydrodynamic particle diameter, namely the size of the solvated particle, which is estimated to be approximately spherical, the measured diameters may be larger than those obtained by electron microscopy (EM) based techniques. In general, exosomes have diameters in the size range of 30-150 nm. All samples were stored on dry ice prior to analysis. Dilutions for NTA were made in DPBS buffer (Gibco) which was confirmed to be particle-free by NTA just prior to measurement. The samples were mixed on a vibratory mixer for a short time before loading to ensure sufficient mixing and destruction of loosely bound exosome clusters. Each sample was manually diluted in PBS to obtain the optimal particle concentration suitable for NTA (20 to 70 exosomes per field of view), with each dilution factor recorded in automatically generated reports. A total of six x 60 second videos were recorded for each exosome sample, and the detection threshold during analysis was chosen to ensure that only clearly distinguishable nanoobjects were analyzed to ensure that artifacts were removed.

Zeta Potential Measurement

Zeta potential EV was measured using Litesizer 500, a light scattering instrument for particle analysis (Litesizer™ 500, Anton Paar Ltd, UK). Zeta potential was measured by diluting EV, PEGylated/non-PEGylated, PBS samples 1:100 or 1:16 (Sample EV-PBS) in an Anton Paar Calliope Core Omega cuvette using a Smoluchowski fit with a Debye factor of 1.5 with time to equilibrate 1 minute and a target temperature of 25°C with water as the dispersion medium selected in the software, since there was no option to select PBS as the dispersion medium. Samples intended for zeta potential analysis were thawed in a refrigerator/container and transferred to a laboratory for zeta potential analysis, where samples were stored at 4°C and taken out when used for analysis, the dilution was carried out 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, the first time with Dl-water (from Deionized Water) and then three times with sterile PBS, and then the sample was added using a sterile syringe. The maximum and minimum loading capacity for each cuvette was 350 µl and 300 µl, respectively. The particle concentration at each dilution was high enough to provide reliable measurements by generating the maximum intensity for the largest part of the particle zeta potential distribution (outliers included in the results).

Lentiviral transduction of hMCKs with EGFP gene constructs Lentiviral transductions were performed on donor hMCK #096. The introduction of EGFP-CFTR and EGFP-NEG controls was performed to express the amplified CFTR protein in EGFP-CFTR-transduced cells for the production of the CFTR gene and encapsulated RNA mRNA, which should be further characterized once a viable population has been reached. Transductions were completed using the rotation protocol.

hMCK were separated in the usual way and resuspended in 5 ml of complete media. The resuspended MSCs were pelleted by centrifugation at 1000 x g for 4 min, after which the complete media supernatant was discarded. Cells were carefully resuspended in 5 ml of basic medium (no FBS, no antibiotic) and cell counts were performed.

1×10 ⁶ (or approximately) cells were additionally diluted in approximately 5 ml of basic medium in separate 50 ml falcon tubes for each EGFP lentivirus, positive control and negative control (does not receive lentivirus). To each tube, experimental and positive control, was added MOI (from the English. multiplicity of infection - multiplicity of infection) 3-4 (approximately 18 μl) lentivirus. The negative control received no lentivirus and all 50 ml falcon tubes were centrifuged for 90 min at 2000×g at 37° C. after centrifugation, the media were discarded and the cell pellet resuspended in complete media. The cells were then seeded in T-flasks of the appropriate size (T175 or T-75) depending on the number of cells transduced and incubated in complete media supplemented with bFGF (1 ng/ml). After 48 hours of incubation, the media were changed to bFGF complete media and supplemented with 4 μg/ml puromycin to select transduced cells for 7-14 days, after which the cells were switched to normal complete media without puromycin. Cells were dissociated every 7-10 days after transduction.

results

For the initial experiments, the human breast cancer cell line BT-20 was used for its high proliferative capacity and ease of cultivation. Cells were transferred into 12×T-175 cm ² T-flasks. EV was isolated from the above using differential centrifugation followed by two ultracentrifugation steps according to MISEV guidelines according to the most widely used and validated methods, and then different methods were performed to characterize EV including size distribution by nanoparticle trajectory analysis and surface charge analysis. using zeta potential studies using the Litesizer™ 500 instrument. Both PEGylated and non-PEGylated EVs were characterized using established protocols and best practice methods.

EVs isolated from BT-20 cells were characterized using various methods to quantify their size and to measure their surface charge. The inventors isolated exosomes using differential ultracentrifugation, since this method remains the most widely used standard method for isolating exosomes from various biological fluids or cell culture supernatants (Chia et al., 2017, Thery et al., 2006). In nanoparticle trajectory analysis (NTA) of both PEGylated and non-PEGylated exosomes, it was found that NTA analysis is not a viable choice for diluted fractions of PEGylated EVs. PEGylated EVs could undergo surface modification through interaction with plastic surfaces in ultracentrifuge tubes or through degradation in plastic containers, for this the inventors used glass containers to store the PEGylated EV fractions in the case of sample 4, 5 and 7 and performed a zeta potential analysis for characteristics of these samples instead of NTA analysis.

Overview of cell culture BT-20

All parameters related to cell culture are summarized below in Tables 1a and 1b, which show cell count values, protein concentration of EV isolates, percentage of DSPE-PEG:Protein in the case of PEG treatment, readings of zeta potential and NTA measurements for fractions of isolated EVs. In the case of NTA analysis readings, the values for Sample 1 are not reliable due to the low number of particles per frame (namely, less than 3 particles per frame). This also applies to the post-PEG NTA regimen for samples 2 and 6. Less than 10 particles per frame were also observed with the pre-PEG NTA regimen for samples 3, 4 and 7. However, these results meet the quality criteria. All data are presented as number of cells/ml for cell count, µl for volume, mV for zeta potential, and nM for size distribution type for NTA assay results.

NTA analysis:

Tables 2a and 2b below show the measured values for rereading the NTA assay as follows: particle size distribution type (nm), total particle concentration, and particle concentration between 30-150 nm in NP/mL for EV- fractions isolated from BT-20 cells cultured in 12XT-175 cm T-flasks according to the EV isolation (MISEV) protocol. Data are presented as mean and SEM (Standard error mean). Sample 1 and all PEGylated samples provided unreliable data as less than 3 particles per frame were detected, which did not meet the NTA quality assessment criteria. Samples 2 and 3 provided less than 10 particles per frame, although their measurement met the quality assessment criteria. Sample 7 also has less than 10 particles per frame in SEM, but again meets the quality criteria.

1. The calibration of the system before each day of analysis was checked using the size standard based on polystyrene nanospheres NIST (from the English National Institute of Standards and Technology - National Institute of Standards and Technology), 100 nm, (100 nm NIST) and gold citrate nanoparticle, 30 nm, (30 nm AuNP).

2. All samples in the table below met the quality assessment criteria as discussed.

3. Samples #1, PEGylated Sample #1, PEGylated Sample #2, and PEGylated Sample #6 provided unreliable results due to the low number of particles per frame (namely, less than 3 particles per frame, which is classified as "particle-free"). For this reason, they did not meet the quality assessment criteria.

4. Samples #3 and #4 provided a low number of particles per frame (namely, less than 10). However, their measurements met the quality assessment criteria.

5. Samples #7 provided the SEM with their respective number of particles per frame reaching less than 10. However, their measurements met the quality assessment criteria, this may be the reason for the minor consideration of measuring their concentrations and should be reflected. Controls:

On FIG. 6a and 6b show nanoparticle trajectory analysis (NTA) readings for dPBS and 30 nm gold nanoparticles (Au) as controls for particle size (nm) on the x-axis and particle concentration distribution (particles/mL) on the y-axis. dPBS shows less than 1 particle per frame, i.e. a solution containing no particles as expected, and AuNPs show a definite peak with an average size of 34 plus/minus 0.5 nm and meet the quality control criteria. Table 3 below suggests the corresponding values in terms of mean and SEM.

sample 1

On FIG. 7a and 7b show the distribution according to the nanoparticle trajectory analysis for sample-1. EV samples for particle size (nm) on the x-axis and particle concentration distributions (particles/mL) on the y-axis. Both non-PEGylated and PEGylated samples had less than 3 particles per frame and did not meet the quality control criteria for the NTA assay, i.e. particle-free solution. The average particle size distribution and particle concentration are set forth in Table 4a below, and the total cell number and particle distribution of the original cell lysate are set forth in Table 4b. Data are presented as mean, SEM, particles/cell and particles/given volume, respectively.

Sample 2

On FIG. 8a and 8b show the nanoparticle trajectory analysis (NTA) distribution for Example 2. EV samples for particle size (nm) on the x-axis and particle concentration distributions (particles/mL) on the y-axis. The non-PEGylated sample showed a distinct peak at 110.5 plus/minus 5 nm and met the NTA assay quality criteria, although the PEGylated samples had less than 3 particles per frame and did not meet the NTA assay quality control criteria, i.e. a particle-free solution . The average size distribution and particle concentration are shown in Table 5a, and the total cell number and particle distribution of the original cell lysate are shown in Table 5b. Data are presented as mean, SEM, particle/cell and particle/sample volume, respectively.

Sample 3

On FIG. 9 shows the distribution according to nanoparticle trajectory analysis (NTA) for Example 3. EV samples for particle size (nm) on the x-axis and particle concentration distributions (particles/mL) on the y-axis. The non-PEGylated sample showed an average size distribution of 1.56 plus/minus 43.5 nm and still met the NTA assay quality criteria but had less than 10 particles per frame for the NTA assay. The average particle size distribution and concentration are shown in Table 6a below, and the total cell number and particle distribution of the original cell lysate are shown in Table 6b. Data are presented as mean, SEM, particle/cell, and particle/sample volume, respectively.

Sample 4

On FIG. 10 shows the distribution according to nanoparticle trajectory analysis (NTA) for Example 4. EV samples for particle size (nm) on the x-axis and particle concentration distributions (particles/mL) on the y-axis. The non-PEGylated sample exhibited an average size distribution of 120 plus/minus 26 nm and, although it met the NTA assay performance criteria, had less than 10 particles per frame for the NTA assay. The average particle size distribution and particle concentration are shown in Table 7a below, and the total cell number and particle distribution of the original cell lysate are shown in Table 7b. Data are presented as mean, SEM, particle/cell, and particle/sample volume, respectively.

Sample 5

On FIG. 11 shows the distribution according to nanoparticle trajectory analysis (NTA) for Example 5. EV samples for particle size (nm) on the x-axis and particle concentration distributions (particles/mL) on the y-axis. The non-PEGylated sample exhibited an average size distribution of 96 plus/minus 3.5 nm and, although it met the NTA assay performance criteria, had less than 10 particles per frame for the NTA assay. The average size distribution and particle concentration are shown in Table 8a below, and the total cell number and particle distribution of the original cell lysate are shown in Table 8b. Data are presented as mean, SEM, particle/cell, and particle/sample volume, respectively.

Sample 6

On FIG. 12a and 12b show the nanoparticle trajectory analysis (NTA) distribution for sample 5. EV samples for particle size (nm) on the x-axis and particle concentration distributions (particles/mL) on the y-axis. The non-PEGylated sample showed a distinct peak at 112 plus/minus 5 nm and met the NTA assay quality criteria, although the PEGylated sample had less than 3 particles per frame and did not meet the quality control criteria for the NTA assay, i.e. a particle free solution. The average particle size distribution and particle concentration are shown in Table 9a below, and the total cell number and particle distribution of the original cell lysate are shown in Table 9b. Data are presented as mean, SEM, particle/cell, and particle/sample volume, respectively.

Sample 7

On FIG. 13 shows the distribution according to nanoparticle trajectory analysis (NTA) for Example 7. EV samples for particle size (nm) on the x-axis and particle concentration distributions (particles/mL) on the y-axis. The non-PEGylated sample exhibited an average size distribution of 93.5 plus/minus 6 nm and met the performance criteria for the NTA assay, meeting the criteria for the purified EV sample, but had less than 10 particles per frame for SEM. The average particle size distribution and particle concentration are shown in Table 10a below, and the total cell number and particle distribution of the original cell lysate are shown in Table 10b. Data are presented as mean, SEM, particle/cell, and particle/sample volume, respectively.

Zeta Potential Analysis Sample 1 (PEG 100%)

On FIG. 14a and 14b show the mean zeta potential values for sample 1 of isolated PEGylated and non-PEGylated EVs (PEG 100%). Fig. 14a is a graph showing a series of measurement readings for a sample of isolated PEGylated EVs with mean zeta potential and distribution peak (mode) value for each run (particle concentration; 1:100 in PBS). On FIG. 14b shows zeta potential intensity distributions for three separate measurement readings for a sample of isolated PEGylated EV 1 (particle concentration: 1:100 in PBS) with relative frequency (%) on the y-axis and zeta potential (mV) on the x-axis. Different curves represent three successive measurements of individual dilutions of samples from the same E\/ isolate. Zeta potentials were determined using the Smoluchowski approximation. Data are presented as Mean and SD (standard deviation). All data are expressed in mV.

Sample 2 (PEG 50%)

Fig. 15a and 15b are the equivalents of FIG. 14a and 14b for Sample 2. Sample 4 (PEG 50%)

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

Sample 5 (PEG100%)

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

Sample 6 (PEG 75%)

On FIG. 18a and 18b show the mean zeta potential values for a sample of PEGylated and non-PEGylated EV 6 isolate (PEG 100%). Fig. 18a is a graph showing a series of measurement readings for a PEGylated EV isolate sample with average zeta potential and peak (mode) distribution for each run (particle concentration; 1:100 in PBS). On FIG. 18b shows zeta potential intensity distributions for three separate measurement readings for a PEGylated EV6 isolate sample (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 successive measurements of separate dilutions of samples from the same E\/ isolate. Zeta potentials were determined using the Smoluchowski approximation. Data are presented as Mean and SD. All data are expressed in mV.

Sample 7 (PEG 150%)

On FIG. 19 shows mean zeta potential values for a sample of PEGylated and non-PEGylated EV 7 isolate (PEG 100%). Zeta potential intensity distributions for three separate measurement readings for a sample of isolated PEGylated EV 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 show single run measurement readings for a sample of non-PEGylated EV isolate with an average zeta potential, followed by specific values of the same sample after PEGylation. SD and distribution peak (mode) values for each run (particle concentration 1:100 in PBS). Zeta potentials were determined using the Smoluchowski approximation. Data are presented as mean and SD. All data are expressed in mV.

MSC transduction

Transduction of hMCK, donor #096, was performed using established protocols. Lentiviral particles were purchased from Genecopoeia Ltd.

The inventors currently have 4 batches of transduced hMCK cells listed below, some of the initial attempts to transduce hMCK with MO11-2 were unsuccessful and are not included.

Lentivirus titer volumes

Transduction volumes for hMCK transduction

Fluorescence images are shown in Fig. 20 and 21.

Discussion

In this study, the modification of EV fractions produced by cells was investigated. As shown by NTA analysis in a previous study, the average size of PEGylated exosomes was slightly larger than that of PEGylated exosomes, although these experiments could not establish any particular relationship between the difference in hydrodynamic size of PEGylated EVs versus non-PEGylated EVs. This may be due to 1) the inclusion of PEG molecules on the EV surface, which changes their behavior in suspension; 2) washout of PEGylated EVs from plastic storage/incubation tubes; 3) washout of EV from ultracentrifuge tubes during the washing step of ultracentrifugation after PEGylation; 4) precipitation of PEGylated EVs using plastic/silicone pipettes. In addition, the zeta potential measured using the Litesizer showed that the surface charge of the non-PEGylated EVs ranged from -6.65 to -18.6 mV for a series of readings where a maximum of 1000 runs were done with the Anton Paar software until until the algorithm for performing operations on sets for phase quality control was reached, or the average of 1000 runs was taken into account automatically by the instrument, while in the case of PEGylated EVs in a number of runs, similar to the above results for samples 1, 2 and 6, the maximum average was observed zeta potential -0.40 mV to a minimum of -23.6 mV, although subsequent readings of the zeta potential measurement were observed to have a marked trend of significantly lower values than the initial readings, suggesting a modification in the surface characteristics of PEGylated EVs by applied voltage and EV PEGylation effectively neutralizing the EV surface charge. This phenomenon may indeed help explain the fact that the surface modification of exosomes is reversible, which is a possible explanation for the observation that the surface modification with low molecular weight PEG did not interfere with cellular transmembrane uptake of exosomes, as the inventors expected.

Another zeta potential analysis via repeated measurements of the same sample was performed in single run assays, although results were inconclusive, possibly due to storage capacity or PEG-plastic interactions. To further reduce the interaction of plastic tubes with PEG particles during PEGylation, the incubation step was carried out by using glass vials, although the ultracentrifugation step using plastic ultracentrifuge tubes could not be bypassed, the use of plastic/silicone pipettes during DSPE transfer could not be bypassed either. -PEG or PEGylated EV. In the future, the inventors will modify their experimental procedures accordingly, by using glass vessels and instruments such as pipettes, and modifying the purification procedure for EV after PEGylation by switching to SEC chromatography (from the English. Size-exclusion chromatography - Exclusion chromatography). It was also observed that DSPE-PEG was possibly degraded when some of these experiments were performed, although the inventors observed positive values for modes in some of the samples, indicating that efficient PEGylation was possible using more PEG. This is a clear indication that some of the EVs were PEGylated, as PBS alone would not cause a positive peak shift in the distribution, and that the presence of PEG molecules on the exosome membrane nearly neutralized the surface charge. In addition, since exosome samples required an additional ultracentrifugation step to remove excess PEG molecules, the actual number of particles after exosome isolation may be higher than the number obtained after the PEGylation procedure.

hMCK was also transduced, although with initial delay, stable transduction was achievable at about MOI of 4 or greater using a rotation protocol. Fluorescence images from transduced cells show GFP fluorescence in all transduced cells, which is the result of a CFTR-EGFP and control lentivirus EGFP fusion protein. This is shown in FIG. 22-25 show GFP-positive MSCs photographed under a fluorescence microscope after seeding the cells on glass slides, fixing and staining nuclei with DAPI (blue). This confirms the presence of cells, not just autofluorescence. Two magnifications are provided showing GFP expression in all cells.

In conclusion, it has been shown that EVs can be PEGylated to confer mucus permeability without interfering with intracellular EV trafficking - as has been suggested, further PEGylation testing using improved and specific tools is possible - and EVs can be characterized - again this can be more research should be done for large-scale production. Characterization of EVs and proteins from CFTR-overexpressing cells for CFTR protein overexpression using RNA, gene and protein analysis will further show efficient transduction and proof of concept. The functional transfer of CFTR expression mediated by these exosomes (versus negative controls) will be evaluated in air-liquid interface models of CF epithelial cells, since this model reproduces the conditions, including the mucosal barrier, that the inhaled aerosol will encounter once the aerosol droplets have settled. in a lung affected by CF. These experiments are expected to demonstrate significant improvements over the state of the art viral vector and nanoparticle mediated gene transfer approaches, namely higher transduction efficiency, functional transfer of CFTR protein and mRNA to virtually all cells in culture, and demonstration of functionality. active agents administered to CF epithelial cells by evaluating chloride ion transport in transduced cell culture experiments using a Ussing chamber.

In addition, increasing EV recovery using high cell density bioreactor-based technologies and using more efficient and commercially scalable EV recovery routes such as tangential flow filtration and size exclusion chromatography can increase EV yield. The use of immortalized stem cells that are transduced with lentivirus overexpressing CFTR to create a master cell bank that is capable of reliably producing overexpressed recombinant nucleic acids over an increased number of passages is also extremely useful for generating EV on an industrial scale. Further optimization of the PEGylation protocol can neutralize the charge of EVs isolated from CFTR-transduced mesenchymal stem cells without much loss, especially storage and transfer of DSPE-PEG and PEGylated samples using an organic solution storage device.

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Claims (18)

1. Aqueous composition for delivering cargo to lung cells, capable of aerosolization, containing exosomes from mesenchymal stem cells (MSCs) having a surface coating of a hydrophilic polymer of polyethylene glycol (PEG), moreover, the exosomes carry the specified cargo containing microRNA (miR), anti- miR, mRNA, long non-coding RNA, cRNA, small interfering RNA, short hairpin RNA, piwi-interacting RNA, regular-spaced clustered short palindromic repeat (CRISPR) RNA sequence, modifications of the above or engineered nucleic acid sequences, protein, cytokine or a lipid, where the PEG has a molecular weight of less than 5 kDa, the surface coating covers at least 65% of the surface of the exosomes. 2. The composition according to claim 1, in which the hydrophilic polymer contains distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). 3. A composition according to claim 1 or 2, wherein the surface coating polymer has a molecular weight of less than 3 kDa. 4. The composition according to any one of paragraphs. 1-3, in which exosomes have a cargo containing microRNA (miR). 5. Composition according to claim 4, wherein miR is selected from miR-125b-5p, miR-125b-1-3p, miR-513a-5p and miR-17. 6. Composition according to any one of paragraphs. 1-3, in which exosomes have a cargo containing mRNA or its translated protein. 7. The composition of claim 6 wherein the mRNA is a modified cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. 8. The composition according to any one of paragraphs. 1-7 for use in therapy of lung disease. 9. Composition according to claim 8 for use in cancer therapy. 10. The composition of claim. 8, where the therapy is for the treatment of cystic fibrosis, chronic obstructive pulmonary disease (COPD), lung cancer or other pathological condition of the lung. 11. The composition of claim 10, wherein the exosomes contain a therapeutic cargo that contains at least one exogenous mRNA, protein, miR, or anti-miR. 12. The composition according to any one of paragraphs. 10, 11, where exosomes originate from mesenchymal stem cells genetically modified to overexpress specific miR, anti-miR, mRNA or other nucleic acid and mature protein as therapeutic cargo. 13. A method of treating a patient with a lung disease, including: providing a composition according to any one of paragraphs. 1-7; the formation of an aerosol of exosomes of this composition; And administration of this aerosol to the patient. 14. The method of claim. 13, including the formation of an aerosol using a nebulizer. 15. The method of claim. 14, including the formation of an aerosol using a vibrating mesh nebulizer.

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