Antifibrotic cells, pharmaceutical preparations containing said cells and methods for obtaining said cells

Abstract

The method for producing modified mesenchymal cells, particularly for the treatment of renal fibrosis, comprises modifying cells with a viral vector so that the modified cells encode a protein that is decorin. The mesenchymal cells obtained in this manner are modified with a modifying agent consisting of a viral vector associated with the mesenchymal cells, such that the modified mesenchymal cells express decorin. The medicament for the treatment of renal fibrosis and pulmonary fibrosis, in particular, comprises mesenchymal cells modified with a modifying agent and expressing decorin.

Description

[Technical field]

[0001] The present invention relates generally to modified cells that can be used to prevent or treat the formation of fibrosis in the lungs and kidneys, to pharmaceutical products comprising the modified cells, and to methods for obtaining the modified cells. [Background technology]

[0002] Fibrosis and dysfunction in various tissues, such as the lungs and kidneys, are known to have many causes, but what they have in common is that repeated consistent lesions limit the organ's ability to regenerate itself.

[0003] These lesions trigger the activation and migration of fibroblasts, leading to the deposition of extracellular matrix and proteins, primarily collagen, and the secretion of cytokines that mediate with cells of the immune system in a process known as fibrosis (Mullins, L. et al; “Disease Models and mechanisms”; 2016.9.1419-1433).

[0004] TGF-β, also called TGF-Beta, is one of the most important cytokines involved in the fibrotic process.

[0005] This cytokine induces the conversion of epithelial cells to fibroblasts through a process known as "epithelial-mesenchymal transition", which results in an increase in fibroblast numbers and significant collagen deposition (Kalluri et al.; J Clin Inv. 2003).

[0006] Based on this state of the art, the use of decoy receptors that can specifically recognize and interact with TGF-β and block its binding to target epithelial cells could be a potential strategy to contrast fibrotic processes (Lan HY; International Journal of Biological Sciences 2011), especially in kidney and lung tissues (Border WA et al. Nature. 1992; https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3812949 / Yue X et al. Curr Enzym Inhib. 2010).

[0007] Mesenchymal cells (MCs) are ideal cellular vehicles to deliver and release TGF-β decoy receptors in fibrotic kidneys and lungs, as both tissues have been reported to be associated with several pathophysiological events (https: / / journals.physiology.org / doi / full / 10.1152 / ajplung.00152.2021; Bollenbecker S et al. American Journal of Physiology. Lung Cellular and Molecular Physiology 2022; https: / / link.springer.com / article / 10.1007 / s40620-018-00563-1; Sorino C et al. Journal of Nephrology 2018).

[0008] MCs have the ability to migrate and colonize sites of injury, having been recruited from soluble molecules released into the bloodstream by injured leukocytes and cells (Di Marino AM. Front Immunol. 2013).

[0009] They are present in many tissues (including adipose tissue, bone marrow, and umbilical cord), are easy to isolate, can be genetically manipulated, and do not induce immunogenic responses, making them ideal vectors for introducing therapeutic compounds, such as TGF-β decoy receptors, into cell-based therapeutic approaches.

[0010] This current situation has several drawbacks.

[0011] One drawback is that renal fibrosis is treated with invasive and damaging therapies such as dialysis.

[0012] Dialysis is known to help the kidneys filter the blood from waste products, but it does not cure people who undergo dialysis.

[0013] In fact, dialysis is a non-curative treatment that must be performed weekly or daily, depending on the severity of the patient's condition.

[0014] Another drawback is that, in addition to being very painful for the patient, dialysis is also psychologically disruptive because it is unpleasant for the patient and causes physical discomfort that can lead to low blood pressure, itching, insomnia, pain and sexual disorders.

[0015] Moreover, dialysis is a hospital-only treatment, which means patients must travel from their homes for regular treatment, a further disadvantage for those with walking difficulties.

[0016] Indeed, dialysis is a complex treatment that requires specialized machinery and the assistance of medical personnel skilled in its use.

[0017] Another drawback is that dialysis, in addition to being the only treatment for kidney fibrosis, is a very expensive treatment, costing the European Community health system 14 to 15 billion euros for machines, reagents, labor and hospital beds.

[0018] Furthermore, patients with severe kidney disease are forced to undergo transplants, which are not always possible and involve side effects and high social and medical costs.

[0019] Finally, another drawback must be highlighted: when the state of renal fibrosis is very advanced, dialysis cannot be used, since it brings no benefit to the function of the kidney.

[0020] Similarly, pulmonary fibrosis has a dramatic but incurable impact on cardiopulmonary medicine and has extremely important health and social implications (https: / / openres.ersjournals.com / content / 4 / 2 / 00045-2017: Hilberg O et al. ERJ Open Research 2018).

[0021] One of the crucial drivers of fibrosis is TGF-β, which plays a role in acute and chronic kidney and lung diseases, as well as in cancer (https: / / www.sciencedirect.com / science / article / pii / S1359610105001103: Bierie B & Moses HL. Cytokine & Growth Factor Reviews 2006). For this reason, attempts to counteract the action of TGF-β have been previously described.

[0022] US-A1-2019 / 125804 discloses a method for treating cancer in a subject by administering human umbilical cord perivascular cells (HUCPVC) genetically modified to increase the expression of oligonucleotides, antibodies or polypeptides, in particular decorin, as a TGF-β decoy binder (https: / / www.sciencedirect.com / science / article / pii / S0021925820621263: Ferdous Z et al. JBC 2007). The drawback of this solution is related to the modification of HUCPVC with recombinant adenovirus, which induces decorin production in HUCPVC, since this modification is not stable. Moreover, from the point of view of immunogenicity, undesirable side effects may occur due to the immunogenicity of adenovirus (https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2020.00909 / full: Coughlan L. Frontiers in Immunology 2020).

[0023] Furthermore, US-A1-2019 / 117701 discloses mesenchymal stem cells that may have therapeutic effects on various conditions, including fibrosis. In particular, those mesenchymal stem cells express at least one cell surface marker selected from the group consisting of CD201, CD46, CD56, CD147 and CD165. Mesenchymal stem cells expressing such markers are also positive for CD29, CD73, CD90, CD105 and CD166 and maintain an undifferentiated state. US-A1-2019 / 117701 asserts that, among a wide variety of additional molecules, mesenchymal stem cells spontaneously secrete decorin. The document does not suggest that mesenchymal stem cells be forcibly modified to produce (or enhance production of) decorin. Furthermore, the main characteristic of mesenchymal stem cells is that they express at least one cell surface marker selected from the group consisting of CD201, CD46, CD56, CD147 and CD165, but not decorin. Moreover, with regard to US-A1-2019 / 125804, the claimed cells are derived from umbilical cord, fat or bone marrow, and there is minimal suggestion of the use of MCs from ET, which is particularly different since, as can be seen in Figure 2 of the present application, cells derived from ET do not spontaneously express decorin. Summary of the Invention [Problem to be solved by the invention]

[0024] The aim of the present invention is to overcome the above-mentioned drawbacks and to provide modified MCs for the treatment of renal and pulmonary fibrosis, as well as a method for obtaining modified cells that make it possible to overcome the above-mentioned drawbacks, in particular to definitively treat fibrosis, leading to the elimination of the pathology.

[0025] Another technical object of the present invention is to complete a new treatment for renal fibrosis, the treatment of which is not accompanied by pain or psychological disturbance for patients.

[0026] A further technical object of the present invention is to arrive at a new treatment for pulmonary fibrosis, which is associated with significant levels of morbidity and mortality.

[0027] Yet another technical object of the present invention is that it is easily accessible to people with mobility problems and high oxygen requirements, so that it can be administered as a medicine without the need for the patient to go to a hospital.

[0028] Another technical objective of the invention is to significantly reduce costs to the health care system by eliminating the need for specialized and expensive machines to perform dialysis, 24-hour hospital specialists, specialized reagents, and, as yet unsolved, expensive anti-fibrotic drugs.

[0029] Another technical object of the present invention is to enable the treatment of patients, particularly those with advanced renal fibrosis, who are no longer treatable by dialysis. [Means for solving the problem]

[0030] According to one aspect of the present invention there is provided mesenchymal cells modified with a modifying agent according to claim 1 for use in the treatment of renal or pulmonary fibrosis.

[0031] According to an embodiment, the mesenchymal cells are derived from endometrial tissue.

[0032] This differs from prior art solutions, e.g. known from US-A1-2019 / 125804, in which the cells are derived from umbilical cord perivascular tissue.

[0033] According to an embodiment, the mesenchymal cells are viable cells harvested from endometrial slough necrotic material.

[0034] According to another aspect of the invention, there is provided a method for producing a MC modified with a modifying agent according to the features of claim 12.

[0035] According to another aspect of the invention, in accordance with claim 14, the modifying agent is selected from decorin isoform A, decorin isoform B, decorin isoform C, decorin isoform D, decorin isoform E.

[0036] According to another aspect of the present invention, there is provided a method for producing MCs modified with a modifying agent and expressing a molecule called HLA-G, so as to have antifibrotic and immunosuppressive effects, according to the features of claim 17.

[0037] According to another aspect of the present invention, there is provided a medicament, in particular for preventing or slowing renal fibrosis, according to the features of claim 10.

[0038] According to another aspect of the invention there is provided a medicament for use in preventing or delaying renal fibrosis.

[0039] Also disclosed is a mesenchymal cell modified with a stable modifying agent, where the modifying agent is a viral vector selected from a lentivirus and a retrovirus.

[0040] The present invention provides the following advantages: To treat patients with renal fibrosis and achieve complete recovery even in advanced stages of the disease. It enables patients to be treated without the need for specific machines, specialists or reagent materials, significantly reducing treatment costs. Patients no longer need to travel back and forth between home and the hospital. Reduce kidney transplant surgery. Eliminate discomfort and pain for the patient being treated.

[0041] According to another aspect of the invention there is provided a medicinal product, in particular for preventing or slowing pulmonary fibrosis, according to the features of claim 11.

[0042] According to another aspect of the invention there is provided a medicament for use in preventing or delaying pulmonary fibrosis.

[0043] The present invention further provides the following advantages: To treat patients with pulmonary fibrosis and achieve complete recovery even in advanced disease states. It allows for the treatment of patients without the need for specific machines, specific anti-fibrotic drugs, specialists / caregivers, or reagent materials, significantly reducing the cost of treatment. Patients no longer need to travel back and forth between home and the hospital. Eliminate discomfort and oxygen dependency for treated patients.

[0044] Other features and advantages of the present invention will become apparent from the detailed description of some preferred, but not exclusive, embodiments of the modifying agent modified MC, the method for producing the modifying agent modified MC, and the medicament, in particular the medicament for preventing renal and pulmonary fibrosis, all of which are illustrated by way of non-limiting examples in the accompanying drawings. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 is a diagram of the viral vector used to modify MCs with a gene encoding decorin. [Diagram 2] FIG. 2 is a graph showing the amount of decorin secreted into the culture medium by three different sources of MCs (two individuals for each source) at three different times. [Figure 3A] FIG. 3A is a representative cytofluorometric analysis of a sample of control endometrial mesenchymal cells to assess positivity for the decorin signal sequence. [Figure 3B] Figure 3B is a representative cytofluorometric analysis of infected ET-MC samples to assess positivity for the decorin signal sequence. Transgenic samples were positive in 89.5% of cases. [Figure 4] FIG. 4 is a graph of decorin m-RNA levels normalized to the basal expression level of ET-MC not overexpressing decorin. [Diagram 5] FIG. 5 is a graphic representation of the amount of decorin secreted into the culture medium of control cells versus cells engineered to overexpress decorin, detected at three different times. [Figure 6A]Figure 6A is a representative cytofluorometer analysis of a sample of MCs isolated from adipose tissue (AT-MCs), which was used as a control to assess positivity for the decorin signal sequence. [Figure 6B] Figure 6B shows a representative cytofluorometric analysis of a control AT-MCs sample to assess positivity for the decorin signal sequence. The transgenic sample is positive in 54.5% of the cultured cells. [Figure 7] FIG. 7 is a graph showing the amount of decorin mRNA normalized based on the basal expression level in MCs not overexpressing the target protein. [Figure 8] FIG. 8 is a graph showing the amount of decorin secreted into the culture medium at three different time points in control AT-MC cells compared to AT-MC cells engineered to overexpress decorin. [Figure 9] FIG. 9 is a graphic representation of the number of idiopathic pulmonary fibrosis (IPF) CC-7231 derived fibroblasts that migrated in the presence / absence of empty vector ET-MC and ET-MC expressing decorin after 48 hours of culture, based on assays performed on cells isolated from three donors. [Figure 10] FIG. 10 shows a graph of Ki-67 gene expression as a proliferation marker in fibroblasts co-cultured with empty vector ET-MC and ET-MC expressing decorin for 48 hours, based on assays performed in two donors. [Figure 11] FIG. 11 is a graphic representation showing expression of the aSMA gene, a marker of profibrotic activity of fibroblasts, in 48 hour co-cultures with empty vector ET-MC and ET-MC expressing decorin. [Figure 12] Figure 12 is a graphical representation summarizing the scanning electron microscopy (SEM) analysis of biomimetic 3D models of dermal and kidney fibrosis obtained by bioprinting technology. [Figure 13]FIG. 13: Evaluation of fibronectin protein expression in a biomimetic 3D model of fibrosis obtained by bioprinting technology with dermis-derived fibroblasts. [Figure 14] FIG. 14: Evaluation of fibronectin protein expression in a biomimetic 3D model of fibrosis obtained by bioprinting technology using kidney fibroblasts. [Figure 15] FIG. 15 shows HLA-G expression in EDT-MCs with or without decorin introduction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] In accordance with the present invention, a method for producing modified mesenchymal cells (MCs) provides for modifying MCs with a modifying agent, in particular a modifying agent consisting of a viral vector encoding a protein, said protein being decorin, which modified MCs express.

[0047] Advantageously, the step of modifying the MC consists of infecting the MC with a viral vector.

[0048] The decorin is selected from decorin isoform A, or isoform B, or isoform C, or isoform D, or isoform E.

[0049] The viral vector is preferably selected from a lentivirus or a retrovirus, as these are associated with stable decorin production.

[0050] The present invention also provides MCs modified with a modifying agent obtained by the above method, in particular, the modifying agent comprises a retroviral vector that infects MCs, and the MCs infected with the retroviral vector are infected MCs that express decorin.

[0051] The decorin is selected from decorin isoform A, or isoform B, or isoform C, or isoform D, or isoform E, more preferably decorin isoform A.

[0052] MC can be selected from MC of human origin or animal origin.MC can also be selected from autologous or allogeneic MC.It can also be provided that MC is selected from MC of adipose tissue, or MC of bone marrow, MC of endometrial tissue, MC of placental tissue, MC of peripheral blood, MC of umbilical cord blood, MC of amniotic fluid and / or MC of derivative.In a preferred embodiment, MC is derived from endometrial tissue.

[0053] Advantageously, the decorin expressed by the modified mesenchymal cells is recurrently expressed decorin.

[0054] The modified MC as described above can be provided for use in treating pulmonary and renal fibrotic processes.In particular, the present invention provides the MC modified with a viral vector selected from lentivirus or retrovirus and expressing decorin as described above for use as a medicament for treating fibrosis.

[0055] In particular, the present invention also provides a pharmaceutical comprising a MC modified with a modifying agent as described above, wherein the modifying agent comprises a viral vector selected from a lentivirus or a retrovirus that infects the modified MC comprising a modified cell expressing decorin.

[0056] The isoform of decorin previously called decorin isoform A was selected and will be referred to by this name below for ease of explanation, but it is also acronymized as DCN A. DCN A is the most well-characterized of the decoy receptor isoforms of TGF-β (Zhang L et al, Aging, 2021).

[0057] In the present invention, we have referred to DCNA which is best characterized from a biological point of view.

[0058] 1 - Materials and Methods 1.1 - Isolation and culture of MCs 1.1.1-MCs from endometrial tissue (ET): Blood from endometrial tissue (referred to as menstrual blood) was collected from healthy volunteer donors (n=2, designated ET-MCs donor #1 and ET-MCs donor #2) during the first few days of the menstrual cycle.

[0059] Each donor was provided with a menstrual cup (DivaCup, Diva International, San Francisco, CA, USA) to collect blood, which was then transferred to saline (PBS, PAA Laboratories, Pasching, Austria) containing 1% penicillin / streptomycin (10,000 U / mL Penicillin, 10 mg / mL Streptomycin 0.9% NaCl solution, PAALaborators), 35 mg / mL fluconazole (Diflucan, Pfizer, New York, NY, USA), and heparin (500 U / mL, Sigma, St. Louis, MO, USA).

[0060] Samples were stored at 4°C for 24–48 h after collection until delivery to the specialized laboratory. Endometrial tissue, if present, was discarded and the remaining blood was passed 20 times through a 19G needle and homogenized using a 10 mL syringe.

[0061] This cell suspension was cultured as described below.

[0062] 1.1.2 - Adipose tissue (AT) derived MCs: AT-MCs were obtained from lipoaspirate samples (n=2, AT-MC donor #1 and AT-MC donor #2) harvested by liposuction from the subcutaneous tissue of healthy donors.

[0063] The samples were used within the framework of the project entitled "Development of novel anti-tumor therapies based on the use of mesenchymal / stromal progenitors isolated from adipose tissue" (Protocol No. 0004827 / 20) with the approval of the local ethical committee (Azienda Universitario-Ospedaliera, Policlinico di Modena) and with the patient's informed consent.

[0064] Lipoaspirates (1 cc) were washed extensively with an equal volume of saline (PBS Code: 14190-094, GIBCO, Thermo Scientific, Waltham, MA, USA) and then digested with 0.075% collagenase at 37°C for 30 min to obtain AT-MCs precursors.

[0065] Then, the enzyme activity was neutralized with DMEM (Code: 41966-029. GIBCO, Thermo Scientific, Waltham, MA, USA), 10% heat-inactivated FBS (Code: SH30070.03. HyClone Laboratories, Inc, Logan, Utah, USA) was added, and the mixture was centrifuged at 1200 g for 10 min to obtain a cell pellet.

[0066] The pellet was suspended in 160 mM NH4Cl (Code: A9434, Sigma Aldrich Inc, USA) and incubated at ambient temperature for 10 min to lyse contaminating red blood cells.

[0067] The resulting cells were then harvested by centrifugation, filtered through a 100 μm filter to remove cell debris, and cultured overnight at 37°C / 5% CO2 in control medium (DMEM, 10% FBS).

[0068] After this incubation, the plates were washed several times with PBS (Code: 14190-094, GIBCO, Thermo Scientific, Waltham, MA, USA) to remove any remaining non-adherent red blood cells.

[0069] 1.1.3 - MCs from bone marrow (BM): BM samples were taken from the posterior iliac crest.

[0070] Samples (designated BM-MCs donor #1 and BM-MCs donor #2) were aspirated into a 10 mL Luer-lock syringe containing 0.5–1 mL of Na-citrate (38 mg / mL) (code: PHR1416, Sigma Aldrich Inc, USA) and processed as follows: BM was diluted 1:1 (v:v) with sterile PBS without Ca2 / Mg2 (PBS code: 14190-094, GIBCO, Thermo Scientific, Waltham, MA, USA) and passed 20 times through a 10 mL sterile syringe with a 19 G needle (Becton Dickinson Plastipak, Drogheda, Ireland). This cell suspension was cultured as described below. Samples were used with the informed consent of the patients and with the approval of the local ethical committee (Azienda Universitario-Ospedaliera, Policlinico di Modena, “Cellular therapies for cancer”, protocol code nr. 335 / CE).

[0071] <1.2 - Cell culture method> AT-MCs and ET-MCs were cultured in αMEM (Code: 22561-021. GIBCO, Thermo Scientific, Waltham, MA, USA) supplemented with 2.5% PLP (Human Platelet Lysate, Code: BC0190030 Macopharma Italy SRL, Milan, Italy), 2 mM L-glutamine (Code: ECB3000D. Euroclone SpA, Italy), 1 international unit (IU) / mL heparin (Code: H3149. Sigma Aldrich Inc, USA), and 10 μg / mL ciprofloxacin (Code: A15571 / AIT. Fresenius Kabi Italia Srl, Verona, Italy).

[0072] BM-MCs were cultured in αMEM (Code: 22561-021, GIBCO, Thermo Scientific, Waltham, MA, USA) supplemented with 8% PLP (Human Platelet Lysate, Code: BC0190030 Macopharma Italy SRL, Milan, Italy), 2 mM L-glutamine (Code: ECB3000D, Euroclone SpA, Italy), 1 IU / mL heparin (Code: H3149. Sigma Aldrich Inc, USA), and 10 μg / mL ciprofloxacin (Code: A15571 / AIT. Fresenius Kabi Italia Srl, Verona, Italy).

[0073] Human embryonic kidney cells 293T were cultured in DMEM (Code: 41966-029. GIBCO, Thermo Scientific, Waltham, MA, USA) supplemented with 10% FBS heat-inactivated defined serum (Code: SH30070.03. HyClone Laboratoires, Inc, Logan, Utah, USA), 2 mM L-glutamine (Code: ECB3000D. Euroclone SpA, Italy), and 1% penicillin / streptomycin (pen / strep, Code: MS00581009. Carlo Erba Reagents Srl, Cornaredo, Italy).

[0074] Human fibroblasts isolated from non-pathological kidneys were obtained as cryopreserved cells (Code: H6016, Cell Biologics Inc., USA). After thawing, they were cultured in complete fibroblast medium (M2267 - Kit, Cell Biologics Inc., USA) consisting of base medium plus the following supplements: FGF 0.5 mL, hydrocortisone 0.5 mL, L-glutamine 5 mL, antimycotic antibiotic solution 5 mL, and FBS 50 mL. The volume correlates to 500 mL of culture medium. The cells were cultured at 37°C for 20-30 min in flasks pretreated with gelatin-based conditioning solution (Code: 6950, Cell Biologics Inc, USA), which can enhance the adhesion ability of cells.

[0075] Human fibroblasts isolated from the dermis were obtained as cryopreserved cells from ATCC. After thawing, the cells were cultured in DMEM (Code: 41966-029. GIBCO, Thermo Scientific, Waltham, MA, USA) supplemented with 10% FBS (Code: SH30070.03. HyClone Laboratoires, Inc, Logan, Utah, USA), 2 mM L-glutamine (Code: ECB3000D. Euroclone SpA, Italy), and 1% penicillin / streptomycin (Code: MS00581009. Carlo Erba Reagents Srl, Cornaredo, Italy).

[0076] Idiopathic pulmonary fibrosis-derived human lung fibroblasts were obtained in frozen vials (Cat: CC-7231; Lonza Group Ltd.) at passage 2 (hereafter referred to as CC7231).

[0077] Idiopathic pulmonary fibrosis-derived hypertrophic fibroblasts were chosen as the target cells for the study because, unlike renal hypertrophic fibroblasts, they are commercially available and because they share with the latter the same biological mechanisms underlying the fibrotic process and the target of decorin (TGFβ; Ong CH et al., European Journal of Pharmacology, 2021).

[0078] Upon receipt, cells were thawed in DMEM (Code: 41966-029. GIBCO, Thermo Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated FBS (Code: SH30070.03. HyClone Laboratoires, Inc, Logan, Utah, USA), 2 mM L-glutamine (Code: ECB3000D. Euroclone SpA, Italy), 1% penicillin / streptomycin (pen / strep, Code: MS00581009. Carlo Erba Reagents Srl, Cornaredo, Italy), seeded at a density of 2500 / cm2, and cultured in the following medium: Clonetics™ FGM™-2 BulletKit™ (Cat: CC-3132; Lonza Group Ltd.).

[0079] 1.3 - Virus production and infection of MCs 293T cells were transfected with a combination of expression vector and helper plasmid: a viral vector (manufactured by OriGene Technologies Inc.) using the transfection reagent JetPEI DNA (Code 101-40N. Polyplus transfection, Illkirch, France).

[0080] 1 mL of the retroviral supernatant produced from the transfected 293T was supplemented with 6 μg / mL polybrene (Code TR1003. Sigma Aldrich Inc. USA) and incubated at 45,000 / cm 2 ET-MC and AT-MC were transduced at early passage (p2 / p4). Infection was repeated twice, followed by cell growth and selection in 2 μg / mL puromycin. Viral supernatants were used fresh or frozen at -80°C.

[0081] 1.4 - Cytofluorometer Analysis Intracellular staining of MYK in wild-type and transformed ET-MCs and AT-MCs was performed using the Becton Dickinson Cytofix / Cytoperm Kit (code: 554714, BD, Franklin Lakes, NJ, USA).

[0082] To assess MYK expression, MCs were labeled with the primary antibody mouse anti-human MYK (Code: TA150121, OriGene Technologies Inc.) and the secondary antibody APC Goat AntiMouse Ig (Code: 550826. BD, Franklin Lakes, NJ, USA).

[0083] To assess HLA-G expression, ET-MCs were labeled with a primary anti-HLA-G FITC-conjugated monoclonal antibody (MoAb) (87G, Exbio, Praha, Czech Republic) APC Goat AntiMouse Ig polyclonal multiple adsorption (Cod:550826. BD, Franklin Lakes, NJ, USA).

[0084] Data were collected using a FACS Aria III (BD) flow cytometer and analyzed using FACS Diva software (BD).

[0085] [2 - Molecular Biology] Total RNA was isolated using TRIzol™ (Code: 15596026. Invitrogen, Carlsbad, MN, USA) according to the manufacturer's instructions.

[0086] Then, cDNA was synthesized from 2 μg of total RNA using a kit called RevertAid H minus first-strand cDNA synthesis (Code: K1622. Fermentas ThermoFisher, Waltham, MA, USA).

[0087] The cDNA was quantified using a spectrophotometer (Beckman Coulter DU™ 730, Pasadena, Calif., USA).

[0088] Quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems StepOne™ Real-Time PCR System and the reagent Fast SYBR™ Green Master Mix.

[0089] The qRT-PCR reaction (10 μl) consisted of 50 ng cDNA, Fast SYBR Green Master Mix (Code: 4385612, Applied Biosystems, Foster City, CA, USA), and 300 nM of forward and reverse primers.

[0090] The sequences of each primer are listed in Table 1. The relative expression level of the target gene was calculated by the 2-ΔΔCt method using human β-actin and GAPDH genes as control genes.

[0091] [Table 1]

[0092] 2.1 - ELISA Assay DCNA levels in ET-MCs and AT-MCs samples were measured using a Duo Set Elisa kit (DY143. R&D Systems, 614 McKinley Place NE, Minneapolis, MN, USA) according to the manufacturer's instructions. This assay is based on an enzyme-linked immunosorbent assay (ELISA).

[0093] 2.2 - Migration Assay On day 1, ET-MCs were plated in 24-well plates at 10,000 / cm 2 The cells were seeded in culture medium at a concentration of 100 μg / ml (3 wells for each condition).

[0094] CC-7231 is 10000 / cm 2 (3 wells per condition) on Transwell Permeable Supports (Code: 3422, Costar Corning Incorporated, USA) in DMEM (Code: 41966-029. GIBCO, Thermo Scientific, Waltham, MA, USA) containing 2.5% heat-inactivated NuSerum™ IV culture supplement (Code: 355104, BD Biosciences), 2 mM L-glutamine (Code: ECB3000D. Euroclone SpA, Italy) and 1% penicillin / streptomycin (pen / strep, Code: MS00581009. Carlo Erba Reagents Srl, Cornaredo, Italy).

[0095] On day 4, the supports were removed and washed with DPBS 1X (Code: 14190-094, Gibco) before being fixed in cold methanol. After fixation, they were further washed with distilled water, the layer of non-migrating cells was removed with buffer, and the remaining ones were stained with crystal violet 0.4% (Code: C0075 Sigma Aldrich). After further washing with distilled water, they were allowed to dry before being observed under a microscope and counted manually.

[0096] 2.3 - Proliferation Assay Density 10000 / cm 2 CC-7231 (6 wells per condition) were seeded in 24-well plates in DMEM (code: 41966-029, GIBCO, Thermo Scientific, Waltham, MA, USA) containing heat-inactivated 2.5% NuSerum™ IV culture supplement (code: 355104, BD Biosciences), 2 mM L-glutamine (code: ECB3000D, Euroclone SpA, Italy), and 1% penicillin / streptomycin (pen / strep, code: MS00581009, Carlo Erba Reagents Srl, Cornaredo, Italy).

[0097] ET-MCs and idiopathic fibrosis fibroblasts were co-cultured in a 1:1 ratio. In detail, ET-MCs were seeded at the bottom of MW wells and fibroblasts were seeded in transwells placed above the ET-MC cultures. The transwells have a porosity of 3um, allowing the exchange of medium and the solutes contained therein. The co-culture thus obtained was continued for 48 hours, after which the cells were lysed and RNA was extracted with Trizol (code: 15596026, Invitrogen, Carlsbad, MN, USA) according to the manufacturer's instructions.

[0098] Then, cDNA was synthesized from 2 μg of total RNA using a kit called RevertAid H minus first-strand cDNA synthesis (Code: K1622. Fermentas ThermoFisher, Waltham, MA, USA).

[0099] The cDNA was quantified using a spectrophotometer (Beckman Coulter DU™ 730, Pasadena, Calif., USA).

[0100] Quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems StepOne™ Real-Time PCR System and the reagent Fast SYBR™ Green Master Mix.

[0101] The qRT-PCR reaction (10 μl) consisted of 50 ng of cDNA, Fast SYBR Green Master Mix (code: 4385612, Applied Biosystems, Foster City, CA, USA), and 300 nM of forward and reverse primers for the Ki-67 gene.

[0102] [3 - Assay to assess aSMA expression] CC-7231 at 10000 / cm 2 (6 wells for each condition) in 24-well plates in DMEM (Code: 41966-029. GIBCO, Thermo Scientific, Waltham, MA, USA) containing 2.5% heat-inactivated NuSerum™ IV culture supplement (Code: 355104, BD Biosciences), 2 mM L-glutamine (Code: ECB3000D, Euroclone SpA, Italy), and 1% penicillin / streptomycin (pen / strep, Code: MS00581009, Carlo Erba Reagents Srl, Cornaredo, Italy).

[0103] ET-MCs were co-cultured with idiopathic fibrosis-derived fibroblasts in a 1:1 ratio. Specifically, ET-MCs were seeded in the bottom of MW wells, and fibroblasts were seeded in transwells placed above the ET-MC cultures. The transwells have a porosity of 3 microns that allows the exchange of medium and the solutes contained therein. After 24 and 48 hours of culture, cells were lysed and RNA was extracted with TRIZOL.

[0104] Subsequently, cDNA was synthesized from 2 μg of total RNA using a kit called RevertAid H minus first-strand cDNA synthesis (Code: K1622. Fermentas ThermoFisher, Waltham, MA, USA).

[0105] The cDNA was quantified using a spectrophotometer (Beckman Coulter DU™ 730, Pasadena, Calif., USA).

[0106] Quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems StepOne™ Real-Time PCR system and the reagent Fast SYBR™ Green Master Mix.

[0107] The qRT-PCR reaction (10 μl) consisted of 50 ng of cDNA, Fast SYBR Green Master Mix (Code: 4385612. Applied Biosystems, Foster City, CA, USA), and 300 nM of forward and reverse primers for the aSMA gene.

[0108] [4 - SEM microscopy analysis of biomimetic 3D models of fibrosis produced by bioprinting technology] The biomimetic models obtained by bioprinting consisted of fibroblasts isolated from the dermis or kidney grown in a three-dimensional matrix of natural origin. The analysis was performed using a SEM (TM4000 Plus II, tabletop microscope, Hitachi Technologies Co., Ltd., Japan). The interaction of the electron beam with the atoms of the examined sample allows the generation of very high magnification images and the analysis of changes in the tissue microstructure. The micrographs were obtained at 1000x magnification with a secondary electron detector, under medium vacuum conditions, with an accelerating voltage of 10 kV for the electron beam.

[0109] [5 - Fibronectin Immunofluorescence (IF) Reaction] Paraffin-embedded biomimetic models were microtomed and cut into 4-micron-thick sections. After drying at 37°C, sections were deparaffinized and rehydrated through a graded series of increasing alcohol concentrations. After unmasking with citric acid (Code: 403727, Carlo Erba Reagents spa, Arese, Milan), sections were incubated with anti-fibronectin primary antibody (Code: ab2413, Abcam) at a concentration of 1:100 for 1 h at room temperature, then with donkey anti-rabbit secondary antibody IgG-h+I Dylight 594 conjugated anti-rabbit (Code: A120-108D4, Bethyl) at a concentration of 1:700 for 1 h at room temperature. Nuclei were stained with the dye DAPI (Code: 10236276001, Roche) at a concentration of 1:200 for 5 min at ambient temperature. Sections were then mounted in buffered glycerin. Analyses were performed using an AxioZoom V16 microscope (Zeiss) with a 1x objective lens (Plan NeoFluar Z 1x / 0.25 FWD 53.1mm, Zeiss) and 180x digital magnification.

[0110] Signal quantification was performed with the ImageAnalysis plug-in of ZEN Pro Zeiss software.

[0111] The analysis results were calculated by assuming the negative control to be 100% expression and μm 2 and reported as a percentage.

[0112] [6 - Results] The gene for DCN A was cloned into a bicistronic viral vector into which the gene for puromycin was also inserted (OriGene Technologies Inc. NM_001920).

[0113] The viral vector carrying the gene (preferably, but not limited to, a lentivirus type in this case) shown in Figure 1 was amplified in bacteria capable of producing large amounts of the target DNA, and an empty vector was used as a negative control.

[0114] The bacterial products were purified and then sequenced to characterize the genetic sequence inserted into the lentiviral vector, and sequence analysis confirmed that no mutations had occurred during amplification.

[0115] To identify the correct source of MCs to carry the therapeutic target, we isolated MCs from adipose tissue (AT), bone marrow (BM), and endometrial tissue (ET), with two donors from each source.

[0116] After isolation, the amount of decorin isoform A physiologically produced by AT-MC, BM-MC, and ET-MC wild-type cells was analyzed at different times (24 h, 48 h, and 72 h) of early passage (P2) in supernatants taken from two donors for each cell type, as shown in Figure 2 .

[0117] AT-MC was shown to secrete the greatest amount of DCN A, ranging from 22085 to 99774 pg / mL.

[0118] In contrast, ET-MC secreted low amounts of DCN A, ranging from 206 to 688 pg / mL, more than 30-fold less than AT-MC.

[0119] BM-MCs have been shown to secrete amounts intermediate to the previously mentioned sources of DCN A, with values ​​varying between 19,425 and 24,863 pg / mL.

[0120] These data proved unexpected for those skilled in the art, since until the present invention, DCNA secretion in MCs had never been clearly stimulated, nor had it been suggested to those skilled in the art, directly or indirectly, by the literature or previous publications.

[0121] [7 - Endometrial mesenchymal cells (ET-MC)] Starting with MCs and known basal secretion of DCN A by a sequenced bicistronic vector (the lowest of any source analyzed so far), three ET-MC donors were infected.

[0122] To purify ET-MCs after infection and obtain a completely pure population for DCNA, cells were selected with puromycin for 96 h.

[0123] The dose for selecting the cells was optimized ranging from 0.5 μg / mL to 5 μg / mL of antibiotic.

[0124] To verify and quantify the efficiency of infection with the DCN A vector, we analyzed ET-MC for positivity for MYK, a signal sequence inserted into the plasmid and correlated with expression of the DCN A gene.

[0125] This signal sequence allows the amount of induced protein in engineered cells to be assessed and distinguished from the endogenous version.

[0126] ET-MC donors induced to express DCN A showed a positive rate of 89.5% compared to the empty vector-expressing control, thus indicating good infection efficiency (Figures 3A, 3B).

[0127] To quantify the expression of DCN A genes in infected ET-MCs, their m-RNA was collected, retranscribed into cDNA, and analyzed by RT-PCR.

[0128] Figure 4 shows how DCN A genes were overexpressed in post-infection samples, with the corresponding expression levels (RQ.ET-MCs) being 32.38 ± 10.45-fold higher compared to the empty vector control.

[0129] An ELISA assay was performed to compare the amount of protein released into the medium by empty vector ET-MCs or ET-MCs infected with DCNA.

[0130] As shown in Fig. 5 , the amount of isoform A released into the culture medium increased to a 230-fold difference between infected ET-MCs and empty vector.

[0131] Furthermore, the simultaneous production of HLA-G by ET-MC cells was evaluated by FACS analysis. From the analysis performed, it was inferred that ET-MC cells modified with decorin expressed a greater level of HLA-G compared to ET-MC empty vector.

[0132] [8 - Adipose tissue-derived mesenchymal cells (AT-MC)] Starting from a known basal secretion rate (highest among previously analyzed sources) of MC and DCN A with a sequenced bicistronic vector, two donors of AT-MC were infected.

[0133] To purify AT-MCs after infection and obtain a completely pure population for DCNA, cells were selected with puromycin for 96 h.

[0134] The dose for selecting cells was optimized in the range of 0.5 μg / mL to 5 μg / mL of antibiotic.

[0135] To verify and quantify the efficiency of infection with the DCN A vector, AT-MC was analyzed for positivity for MYK, a signal sequence inserted into the plasmid, and correlated with expression of the DCN A gene.

[0136] This signal sequence allows the amount of induced protein in engineered cells to be assessed and distinguished from the endogenous version.

[0137] AT-MC donors induced to express DCN A showed a 54.5% positive rate compared to the empty vector-expressing control, thus indicating good infection efficiency (Figures 6A-6B).

[0138] To quantify the expression of decorin isoform A genes in transduced AT-MCs, their m-RNA was collected, retranscribed into cDNA, and analyzed by RT-PCR.

[0139] Figure 7 shows how the DCN A gene is overexpressed in post-infection samples, with the corresponding expression (RQ.AT-MCs) being 3.05 ± 1.14-fold higher compared to the empty vector control.

[0140] ELISA assays were performed to compare the amounts of protein released into the medium by empty vector AT-MCs or DCN A-infected AT-MCs.

[0141] As shown in Figure 8, the amount of decorin isoform A released into the culture medium is increased in a statistically significant manner, even though it does not reach the absolute amount reached after the above-mentioned modification of ET-MC. This phenomenon may be caused by the already high basal secretion of AT-MC (compared to the basal secretion of MC isolated from other sources), which interferes with the production and / or stability of proteins induced by the vector of interest.

[0142] After verifying the difference in the absolute amount of secreted protein between ET-MC and the aforementioned DCN A modified AT-MC and confirming that ET-MC can produce more DCN A (value expressed in pg / mL), we evaluated the effectiveness of DCN A produced by ET-MC against target cells, i.e., hypertrophic fibroblasts.

[0143] [9 - Functional Studies] Idiopathic pulmonary fibrosis-derived hypertrophic fibroblasts were chosen as the target cells for the study because, unlike renal hypertrophic fibroblasts, they are commercially available and share the same biological mechanisms underlying the fibrotic process and the same target of DCN A, TGFβ (Ong CH et al., European Journal of Pharmacology, 2021).

[0144] After proliferation, the enlarged fibroblasts were analyzed for their ability to migrate and proliferate in the presence of ET-MC engineered to express DCNA or empty vector.

[0145] Migration tests performed 48 hours after seeding ET-MC on a plate and seeding fibroblasts on top of it on a grid developed for the above study showed that the migration ability of CC-7231 cells cultured without ET-MC was almost zero. When cultured on empty vector ET-MC, the migration ability was increased due to factors released in the medium, but when cultured on ET-MC expressing DCN A, the migration ability was halved in all donors examined. One hypothesis is that fibroblasts are attracted to ET-MC thanks to the release of chemoattractant factors from ET-MC, including activation by TGFβ, one of the main activators of fibroblast migration in the fibrotic process (Frangogiannis NG et al. J Exp Med. 2020). However, this phenomenon is slowed down in the presence of ET-MC releasing DCN A. This is because DCN A binds to TGFβ, preventing it from binding to its receptor and reducing the number of migrating fibroblasts, as shown in Figure 9.

[0146] The study of proliferation of hypertrophic fibroblasts (CC-7231) was performed by co-culturing empty vector ET-MC and ET-MC expressing decorin at a 1:1 ratio.

[0147] Figure 10 shows the expression levels of the Ki-67 gene normalized with the culture of fibroblasts shown as a control. From this data, a statistically significant increase of the KI67 gene was observed in co-culture with the empty vector ET-MC, compared to the control represented by the culture medium alone, indicating a proliferation stimulation by factors released from the MC. In co-culture with ET-MC modified with decorin, this proliferation stimulation was considerably reduced. This biological response could be attributed to the reduction of TGFβ available in the specific culture medium used to maintain the cell culture. This is due to the lack of binding to the receptor due to previous binding with decorin secreted by the modified ET-MC. The obtained results suggest a role for decorin in reducing proliferation and migration.

[0148] Finally, aSMA gene expression was evaluated, which is a good marker of myofibroblast activation by fibroblasts, a typical phenotype of fibrosis in which cells with contractile capacity secrete extracellular matrix, exacerbating the pathology (Kuhn and McDonald, Am J Pathol 1991; Flaherty et al. J. Respir. Crit. Care Med. 2003; White et al. J. Pathol. 2003; Hinz, Proc. Am. Thorac. Soc 2012).

[0149] The results on aSMA gene expression shown in Figure 11 after co-culture of idiopathic fibroblasts CC-7231 with empty vector ET-MC and decorin-expressing ET-MC in a 1:1 ratio highlight the role of modified MCs. At both 24 and 48 hours of co-culture, a statistically significant decrease in the expression of aSMA was observed in fibroblasts co-cultured with decorin-expressing ET-MC.

[0150] These results indicate a role for decorin in reducing the activation state of hypertrophic fibroblasts.

[0151] Having gathered evidence regarding the role of decorin in commercially available fibroblasts isolated from idiopathic pulmonary fibrosis, we wished to evaluate its effects on biomimetic matrices of healthy and fibrotic dermis, and healthy and fibrotic renal interstitial tissue.

[0152] With the printing technique, fibroblasts (isolated from both the dermis and the kidney) were immersed in a three-dimensional matrix capable of mimicking fibrotic pathologies (Figure 12). The untreated model (CNTRL) shows a dense and compact surface with collagen fibrils encapsulated by abundant extracellular matrix (left column). The supernatant obtained from ET-MC containing the empty vector gives the model a solid surface characterized by partial remodeling of collagen fibrils and poor deposition of extracellular matrix. On the other hand, the supernatant obtained from ET-MC expressing decorin significantly reduces the density of the printed tissue without extracellular matrix, and the fibrils appear thinner and more fragmented.

[0153] Figure 13 shows immunofluorescence detection of fibronectin protein in a three-dimensional model obtained using dermis-derived fibroblasts, as a further indicator of fibrosis (O'Connell et al Fibronectin: Current Concepts in Structure, Function and Pathology 2012).

[0154] The intensity and localization of the signal associated with the presence of fibronectin is particularly strong in the CNTRL cases, whereas this protein is almost absent in the cases stimulated with MC supernatants.

[0155] In particular, the lowest expression level was observed when the model was stimulated with the supernatant of ET-MCs overexpressing decorin.

[0156] Quantification of staining confirmed that cases treated with ET-MCs and supernatants from ET-MCs expressing decorin had statistically significant reduced fibronectin expression levels compared to control cases (p-value < 0.05). Biomimetic models treated with supernatants from ET-MCs expressing decorin showed significantly lower expression levels not only compared to control cases but also in relation to ET-MCs.

[0157] Similar analyses were performed on three-dimensional models obtained with renal fibroblasts.

[0158] In FIG. 14, the fluorescence (red) indicates the presence of fibronectin protein in the model.

[0159] The intensity and localization of the signal associated with the presence of fibronectin are particularly strong in the CNTRL cases. In contrast, the protein is almost absent in the supernatants of ET-MC with empty vector and in cases treated with ET-MC overexpressing decorin. Quantification of the staining using an image analysis plugin (ZEN PRO, Zeiss) confirmed that the expression levels of fibronectin were statistically significantly decreased in cases treated with the supernatants of ET-MC with empty vector and in cases treated with ET-MC overexpressing decorin (p value < 0.05). In detail, the expression levels are significantly decreased in cases treated with the supernatants of ET-MC overexpressing decorin, not only compared to the control cases, but also compared to cases treated with the supernatants of ET-MC with empty vector.

[0160] In Figure 15, HLA-G was tested in both EDT-MC empty vector and EDT-MC modified to express decorin. Testing was performed by FACS using FITC or APC-conjugated anti-HLA-G monoclonal antibodies. Two different MC donors were considered. As can be seen, all samples expressed HLA-G without differences (p>0.05) related to the modification of the decorin gene.

[0161] [10 - Conclusion] According to the present invention, the endogenous secretion of DCNA by MCs from different sources was investigated. Each source was shown to secrete different levels of protein, and of these, ET-MC were selected as weakly secreting cells and AT-MC as strongly secreting cells of the identified decoy receptor to better understand the effect of the genetic modifications on the cells themselves. The infection method using a viral vector containing DCN A and puromycin genes was optimized to achieve efficient infection.

[0162] Following infection, MCs did not show signs of suffering, changes in morphology, and / or altered expression of typical markers of MCs.

[0163] Protocols for gene quantification by Real Time-PCR and signal sequence quantification by cytofluorometric analysis were optimized, and an increase in the DCN A gene was demonstrated.

[0164] Protein secretion was demonstrated by ELISA assay, showing increased release in MCs induced to produce DCN A, confirming the data obtained by molecular analysis showing greater expression of the DCN A gene, and by cytofluorometric analysis showing increased positivity in MCs induced to express DCN A compared to MCs carrying the empty vector.

[0165] Furthermore, a comparison was made between different sources of MC, and ET-MC was selected as the best source, as it showed higher positivity for the MYC signal sequence by cytofluorometric analysis, leading to higher mRNA expression and, consequently, higher amounts of secreted protein. For these reasons, ET-MC is the optimal carrier for the purposes of the present invention, expressed in the cell therapy and gene therapy approaches possible in the present invention.

[0166] To verify the functionality of engineered ET-MCs in a fibrotic environment, we designed a co-culture model and evaluated their proliferation, migration ability, and metabolic activity.

[0167] Commercially available hypertrophic fibroblasts derived from idiopathic pulmonary fibrosis were cultured with engineered ET-MCs (expressing either empty viral vector or DCN A). This co-culture was performed to observe the effect on the proliferative and profibrotic phenotype associated with ET-MCs and to evaluate the functionality of the released TGFβ decoy on cell behaviors such as cell proliferation, migration and metabolic activity.

[0168] The DCN A protein produced by the modified ET-MC not only interferes with fibrotic fibroblast proliferation, fibroblast migration, and fundamental events in the fibrotic process in vivo, but also interferes with fibroblast metabolic activity.

[0169] Moreover, surprisingly, blockade of TGF-β does not cause damage at the level of MCs, which are known to use TGF-β as a growth factor, and does not impair the performance of MCs in vitro.

[0170] Those skilled in the art will appreciate that the methods of producing MCs modified with modifying agents as described herein and the modified MCs obtained thereby can also be used to treat, by way of non-limiting examples, renal fibrosis, cardiac fibrosis, liver fibrosis, pulmonary fibrosis, or fibrosis occurring in other tissues and organs, such as the joints, bone marrow, brain, eye, intestine, peritoneum and retroperitoneum, pancreas, and skin.

[0171] In practice, it has been found that the invention achieves the intended purpose. The invention as conceived is susceptible to modifications and variations, all of which are within the scope of the inventive concept. Moreover, all details can be replaced with other technically equivalent elements. In practical implementation, the materials used, as well as their shapes and sizes, can be of any type according to requirements, without thereby departing from the scope of protection of the following claims.

Claims

1. A mesenchymal cell modified with a modifying agent for use in treating lung or kidney fibrosis, wherein the modifying agent comprises a viral vector encoding decorin that infects the mesenchymal cell, and the infected mesenchymal cell expresses decorin.

2. The cell for use according to claim 1, wherein the decorin is selected from decorin isoform A, or isoform B, or isoform C, or isoform D, or isoform E.

3. The cell for use according to claim 2, wherein the decorin is decorin isoform A.

4. The cells for use according to claim 1 , wherein the mesenchymal cells are selected from mesenchymal cells of human or animal origin.

5. The cells for use according to claim 1 , wherein the mesenchymal cells are selected from autologous or allogeneic mesenchymal cells.

6. 2. The cells for use according to claim 1, wherein the mesenchymal cells are selected from mesenchymal cells derived from adipose tissue, bone marrow, endometrial tissue, placental tissue, peripheral blood, umbilical cord blood, amniotic fluid, and / or derivatives.

7. The cells for use according to claim 1 , wherein the mesenchymal cells are derived from endometrial tissue.

8. The cell for use according to claim 1 , wherein the decorin expressed by the modified mesenchymal cell is recurrently expressed decorin.

9. The cell for use according to claim 1 , wherein the viral vector is selected from a lentivirus or a retrovirus.

10. A pharmaceutical for preventing or delaying the process of kidney fibrosis, comprising mesenchymal cells modified with the modifying agent of claim 1, wherein the modifying agent comprises a viral vector selected from a lentivirus or a retrovirus that infects the modified mesenchymal cells, the modified cells being cells that express decorin.

11. A pharmaceutical for preventing or delaying the process of pulmonary fibrosis, comprising mesenchymal cells modified with the modifying agent of claim 1, wherein the modifying agent comprises a viral vector selected from a lentivirus or a retrovirus that infects the modified mesenchymal cells, the modified cells being cells that express decorin.

12. A method for producing mesenchymal cells modified with a modifying agent for use as described in claim 1 and obtaining modified mesenchymal cells, characterized in that the modifying agent comprises a viral vector stably encoding a protein, the protein being decorin, and the modified mesenchymal cells express the decorin.

13. 13. The method of claim 12, wherein the modification comprises infecting the mesenchymal cells with the viral vector.

14. The decorin is decorin isoform A, or isoform B, or isoform C.

13. The method of claim 12, wherein the isoform is selected from form C, or isoform D, or isoform E.

15. The method of claim 14, wherein the decorin is decorin isoform A.

16. 13. The method of claim 12, wherein the viral vector is selected from a lentivirus or a retrovirus.

17. The method of claim 12, wherein the mesenchymal cells express HLA-G molecules.

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