Mesenchymal stem cells co-expressing CXCR4 and IL-10 and their use

Abstract

This invention provides mesenchymal stem cells (MSCs) with improved therapeutic efficacy by enhancing migration to inflammatory sites and increasing the release of immunosuppressive and anti-inflammatory cytokines. [Solution] The present invention provides MSCs characterized by being transduced into an integrated expression vector for stable co-expression of chemokine receptor type 4 (CXCR4) and interleukin IL-10. Furthermore, the present invention provides the use of the above MSCs as pharmaceuticals, particularly in the treatment of inflammatory and / or autoimmune diseases.

Description

[Technical Field]

[0001] The present invention relates to the medical field. In particular, the present invention relates to mesenchymal stem cells (MSCs) characterized by being transduced with an integrated expression vector for stable co-expression of chemokine receptor type 4 (CXCR4) and interleukin IL-10. Furthermore, the present invention relates to the use of the above MSCs as pharmaceuticals, particularly in the treatment of inflammatory diseases and / or autoimmune diseases. [Background technology]

[0002] MSCs are pluripotent adult stromal cells that have immunomodulatory effects on activated lymphoid cells, including T cells, B cells, natural killer cells, and dendritic cells. MSCs exhibit the ability to homing to inflammatory sites, where they can regulate inflammatory responses and contribute to the repair of damaged tissue.

[0003] In animal models, MSCs have demonstrated efficacy not only in regenerative medicine but also in inflammatory and autoimmune disease models. In Phase I / II clinical trials, MSCs have demonstrated a safety profile and provided preliminary evidence of clinical utility in various diseases, including, for example, steroid-resistant graft-versus-host disease (GVHD), severe systemic lupus erythematosus, complex perianal fistula, osteoarthritis of the knee, and chronic total paralysis. Despite the results obtained in animal models and early-stage clinical trials, only three Phase III clinical trials have shown statistically significant efficacy of MSCs compared to standard treatment. These include the treatment of complex perianal fistula (NCT00475410), steroid-refractory GVHD in children (NCT02336230), and chronic progressive ischemic heart failure (NCT01768702).

[0004] Among the parameters that can reduce the therapeutic efficacy of MSCs, it is worth mentioning that the ex vivo expansion of these cells has been shown to reduce the moderate expression of homing receptors observed in MSCs and further induce senescence in these cells.

[0005] Therefore, the present invention particularly focuses on improving the migration of MSCs to the site of inflammation, further improving the therapeutic efficacy of MSCs by secreting immunosuppressive cytokines and anti-inflammatory cytokines, and enhancing the therapeutic efficacy of standard non-modified MSCs.

Summary of the Invention

[0006] As described above, the present invention particularly focuses on improving the therapeutic efficacy of MSCs by enhancing the migration of MSCs to the site of inflammation and enhancing the release of immunosuppressive cytokines and anti-inflammatory cytokines as compared with standard non-modified MSCs.

[0007] To do so, the inventors of the present invention used MSCs transfected with an integrated expression vector co-expressing chemokine receptor type 4 (CXCR4) and interleukin IL-10.

[0008] Particularly, a lentiviral vector encoding for CXCR4 and IL-10 was constructed in the context of the present invention. This expression vector was used to transfect MSCs and co-express both CXCR4 and IL-10 in a stable manner.

[0009] Example 2.1 shows that MSCs transfected with CXCR4-IL10 mRNA have local inflammation These cells exhibit anti-inflammatory properties in a mouse model of the disease. Nevertheless, these cells do not show enhanced anti-graft-versus-host disease (GvHD) properties compared to WT MSCs (Example 2.2). Compared to MSCs transfected with CXCR4-IL10 mRNA, MSCs transfected with a lentiviral vector containing the CXCR4-IL10 sequence (Example 2.3) not only exhibited enhanced in vitro immunomodulatory properties (Examples 2.4 and 2.5) and local in vivo anti-inflammatory effects (Examples 2.3 to 2.6) compared to WT MSCs, but also, surprisingly, showed significant anti-GvHD activity, as shown in Example 2.7 of the present invention.

[0010] Indeed, in vitro experiments included in this invention demonstrate that stable co-expression of these molecules efficiently enhances the migration of MSCs to SDF-1 and improves the immunosuppressive properties of these cells. Furthermore, preferential homing of MSCs ectopically expressing CXCR4 and IL10 to inflammatory pads was demonstrated in a mouse model in which local pad inflammation was induced. In summary, these results demonstrate that stable co-expression of specific homing and anti-inflammatory molecules in human MSCs, such as CXCR4 and IL10, enhances the anti-inflammatory capacity of these cells compared to WT MSCs. The use of this new generation of MSCs transduced with an embedded expression vector co-expressing CXCR4 and IL10 will have a significant impact on clinical cell therapy for the treatment of inflammatory and / or autoimmune diseases.

[0011] In short, the use of MSCs transduced with an embedded expression vector co-expressing both CXCR4 and IL-10 as pharmaceuticals is proposed herein, particularly in the treatment of inflammatory and / or autoimmune diseases.

[0012] Therefore, the first embodiment of the present invention relates to an expression cassette (hereinafter referred to as the expression cassette of the present invention) comprising a DNA sequence that sequentially includes a) a promoter, b) a sequence encoding chemokine receptor type 4 (CXCR4), and c) a sequence encoding interleukin IL-10. In a preferred embodiment, the expression cassette further comprises a regulatory element for increasing transgene expression. In a preferred embodiment, the regulatory element is a woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof. In a preferred embodiment, the expression cassette further comprises a sequence encoding an autocatalytic peptide between the sequence encoding chemokine receptor type 4 (CXCR4) and the sequence encoding interleukin IL-10. In a preferred embodiment, the autocatalytic peptide is E2A. In a preferred embodiment, the promoter is a human phosphoglycerate kinase (PGK) promoter sequence or a functional homolog or variant thereof. In a preferred embodiment, the expression cassette comprises, in order from 5' to 3', a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homolog or variant thereof; b) a sequence encoding chemokine receptor type 4 (CXCR4); c) a sequence encoding autocatalytic peptide E2A; d) a sequence encoding interleukin IL-10; and d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0013] In a preferred embodiment, the expression cassette contains non-natural codon-optimized sequences of the human genes CXCR4 (SEQ ID NO: 1) and IL10 (SEQ ID NO: 3). In a preferred embodiment, the sequence encoding the autocatalytic peptide E2A is SEQ ID NO: 2, which is used to facilitate the co-expression of both molecules (CXCR4 and IL10).

[0014] A second embodiment of the present invention relates to a recombinant gene delivery vector (hereinafter referred to as the recombinant gene delivery vector of the present invention) comprising the expression cassette defined above. In a preferred embodiment, the recombinant gene delivery vector is a lentiviral vector. Preferred form In this context, the vector of the present invention is an integration vector that is permanently incorporated into the host chromosome.

[0015] A third embodiment of the present invention relates to cells comprising the expression cassette or recombinant gene delivery vector of the present invention (hereinafter referred to as "cells of the present invention"). In a preferred embodiment, the cells are MSCs derived from bone marrow, placenta, umbilical cord, amniotic membrane, menstrual blood, peripheral blood, salivary glands, skin and foreskin, synovial fluid, amniotic fluid, endometrium, adipose tissue, umbilical cord blood, and / or dental tissue.

[0016] A fourth embodiment of the present invention relates to a pharmaceutical composition comprising the recombinant gene delivery vector or cells of the present invention and optionally a pharmaceutically acceptable additive or carrier.

[0017] A fifth embodiment of the present invention relates to a gene delivery vector or cell of the present invention used as a pharmaceutical. In a preferred embodiment, the present invention relates to a gene delivery vector or cell of the present invention used in the treatment of inflammatory diseases and / or autoimmune diseases, such as graft-versus-host disease (GvHD), sepsis, or rheumatoid arthritis. Alternatively, this embodiment relates to a method for treating inflammatory diseases and / or autoimmune diseases, such as graft-versus-host disease (GvHD), sepsis, or rheumatoid arthritis, comprising administering to a patient a therapeutically effective dose or amount of the gene delivery vector or cell of the present invention, or a pharmaceutical composition containing the same.

[0018] For the purposes of this invention, the following terms are defined: "Comprising" includes everything that follows the word "comprising". This means that it is not limited to these. Therefore, the use of the term "includes" indicates that the enumerated elements are required or mandatory, while the other elements are optional and may or may not be present. "Consisting of" means "includes and is limited to" everything that follows it. Therefore, the phrase "consisting of" indicates that the listed elements are necessary or essential, and no other elements can be present. "Pharmacologically acceptable additives or carriers" refers to additives that may be optionally included in the pharmaceutical composition of the present invention and that do not cause significant adverse toxicological effects in patients. In this invention, “therapeutably effective dose or amount” refers to the situation in which cells or pharmaceutical compositions are administered as described above and produce a positive therapeutic response in subjects with inflammatory or autoimmune diseases. The exact required amount will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the mode of administration, etc. The appropriate “effective” dose in all individual cases can be determined by those skilled in the art using routine experiments based on the information provided herein. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows evidence of the in vivo efficacy of MSCs (Medical Stem Cells) transfected with bicistronic CXCR4-IL10 mRNA in a mouse model of local inflammation. Compared to wild-type (WT) MSCs, enhanced anti-inflammatory activity is observed in MSCs transfected with CXCR4-IL10 mRNA. [Figure 2] This figure shows the lack of in vivo efficacy of MSCs (Methodoxylated Stem Cells) transfected with bicistronic CXCR4-IL10 mRNA in a GVHD model mouse. A) Survival curve, B) Weight, and C) Clinical score. [Figure 3] (A) Design of a DNA bicistronic lentiviral vector used to co-express CXCR4 and IL-10. (B) CXCR4 levels, (C) IL-10 secretion, and (D) vector copy number per cell (VCN / cell) in CXCR4 / IL10-MSCs compared to WT-MSCs. ND: Undetectable. [Figure 4]This figure shows the in vitro characterization of MSCs transduced with the DNA PGK-CXCR4-IL10 lentiviral vector. A) Immunophenotype of CXCR4 / IL10-MSCs compared with WT-MSCs. B) Differentiation potential of CXCR4 / IL10-MSCs into bone tissue compared with WT-MSCs. C) Differentiation potential of CXCR4 / IL10-MSCs into adipose tissue compared with WT-MSCs. [Figure 5] This figure shows the enhanced migratory ability of MSCs transduced with the DNA PGK-CXCR4-IL10 lentiviral vector compared to WT-MSCs. A) Representative photographs of the migratory ability of WT-MSCs and CXCR4 / IL10-MSCs in response to SDF-1. B) Quantification of the migratory ability of WT-MSCs and CXCR4 / IL10-MSCs in response to SDF-1. [Figure 6] This figure shows the enhanced in vitro immunosuppressive activity of MSCs transduced with the DNA PG-CXCR4-IL10 lentiviral vector. A) Scheme of the experimental system used to evaluate the in vitro immunosuppressive activity of MSCs. B) CXCR4 / IL10-MSCs showed improved immunosuppressive activity compared to WT-MSCs. [Figure 7] This figure shows the enhanced in vivo efficacy of MSCs transduced with the DNA PGK-CXCR4-IL10 lentiviral vector in a mouse model of local inflammation. A) Scheme of the experimental system used to evaluate the in vivo anti-inflammatory effects of WT-MSCs and CXCR4 / IL10-MSCs. B) Enhancement of the anti-inflammatory effect of MSCs transduced with the PGK-CXCR4-IL10 lentiviral vector compared to WT-MSCs. [Figure 8] This figure shows the enhancement of anti-GvHD in MSCs transduced with DNA PGK-CXCR4-IL10 LV compared to WT-MSCs: an analysis of GvHD clinical signs. A) Scheme of the experimental system used to evaluate in vivo anti-GvHD in WT-MSCs and CXCR4 / IL10-MSCs. B) GvHD scores comparing different experimental groups. [Figure 9]This figure shows the enhancement of anti-GvHD in MSCs transduced with DNA PGK-CXCR4-IL10 LV compared to WT-MSCs. A) Flow cytometry analysis of human CD45+ cells in the peripheral blood of recipient mice showing reduced proliferation of xenodoner leukocytes in a GVHD humanized mouse model. B) Flow cytometry analysis of human CD45+ cells in the spleen of a GVHD humanized mouse model, confirming reduced infiltration of xenodoner leukocytes in this immune organ. [Figure 10] This figure shows the enhancement of anti-GvHD in MSCs transduced with DNA PGK-CXCR4-IL10 LV compared to WT-MSCs: analysis of infiltration of donor lymphocytes expressing IFN-g or IL10. A) Reduction in the content of INFg-secreting human T cells involved in GVHD disease in the spleen of NSG mice injected with CXCR4-IL10-MSC. B) Increase in the content of IL10-secreting human T cells in the spleen of NSG mice with GVHD treated with CXCR4-IL10-MSC. [Figure 11] Enhancement of anti-GvHD in MSCs transduced with DNA PGK-CXCR4-IL10 LV compared to WT-MSCs: This figure shows the quantification of human factors in recipient mice by qPCR. A) Analysis of pro-inflammatory factors (IFNg, IL-17, and IL-22) in the spleen of NSGs treated with WT-MSCs or CXCR4 / IL10-MSCs. B) Analysis of anti-inflammatory factors (IL-5 or FoxP3) in the spleen of NSGs treated with WT-MSCs or CXCR4 / IL10-MSCs. [Figure 12] This figure shows the changes in weight and GVHD clinical score in NSG mice transplanted with human mononuclear cells and injected with either WT or CXCR4 / IL10-MSC. (A) Changes in weight shown as a percentage over time, with day 0 weight assumed to be 100%. (B) Clinical disease scores determined over time in different transplantation groups. The overall GVHD score was evaluated in terms of weight loss, posture, activity, hair quality, skin integrity, and presence of diarrhea. *p<0.05, **p<0.01, ***p<0.001. [Figure 13]This figure shows flow cytometry analysis of circulating human cells in peripheral blood 3 weeks post-transplantation in NSG mice transplanted with human mononuclear cells and injected with either WT or CXCR4 / IL10-MSC. (A) Percentage of circulating human CD45+ cells. (B) Percentage of circulating human CD3+ T cells. (C) Characterization of CD3+ T cells as human CD4+, CD8+, or CD4+CD8+ T cells. Each bar represents mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. [Figure 14] This figure shows the phenotypic characteristics of circulating human CD4+ and CD8+ T cells (naive, effector, and memory T cells) in NSG mice transplanted with human mononuclear cells and injected with WT or CXCR4 / IL10-MSC. (A) Effector T / naive T cell ratio in CD4+ T cells. (B) Effector T / naive T cell ratio in CD8+ T cells. Each bar represents the mean ± SEM. *p<0.05, **p<0.01. [Figure 15] This figure shows flow cytometry analysis of activation markers in circulating human T cells in peripheral blood 3 weeks after transplantation of human mononuclear cells into NSG mice and injection of either WT or CXCR4 / IL10-MSCs. Activation profiles of T cells labeled as CD3+CD45+ are shown. Each bar represents the mean ± SEM of data from two different experiments using MNCs from two different donors (n=10-12 mice / group). *p<0.05. [Figure 16] This figure shows the analysis of exhaustion markers in circulating human CD3+ T cells in peripheral blood of NSG mice containing human mononuclear cells and injected with either wild-type (WT) or CXCR4 / IL10-MSCs, 3 weeks after administration. It also shows the inhibition profile of CD3+CD45+ human T cells. [Figure 17] This figure shows the levels of human cytokines and growth factors involved in GVHD in the serum of NSG-transplanted mice 3 weeks after transplantation of human mononuclear cells and infusion with either wild-type (WT) or CXCR4 / IL10-MSCs. Each bar represents the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. [Figure 18]This figure shows the flow cytometry analysis of human hematopoietic cells in the spleen of NSG mice carrying human mononuclear cells and injected with either wild-type (WT) or CXCR4 / IL10-MSCs, 3 weeks after transplantation. The percentages represent the distribution of circulating human CD45+ cells as CD3+, CD19+, CD56+, CD14+, and CD15+ cells. Each bar represents the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. [Figure 19] This figure shows the phenotypic characteristics of T cell subpopulations in the spleen. (A) Distribution of human CD4+ T cells, CD8+ T cells, or bipositive T cells within the CD3+CD45+ cell population. (B) Distribution of naive, effector, and memory subpopulations within the CD4+ T cell population. (C) Distribution of naive, effector, and memory subpopulations within the CD8+ T cell population. Each bar represents the mean ± SEM. [Figure 20] This figure shows flow cytometry analysis of activation profiles of human T cells in the spleen of NSG mice transplanted with human mononuclear cells and injected with either wild-type (WT) or CXCR4 / IL10-MSCs 3 weeks later. (A) Activation profile of CD3+CD45+ labeled T cells. (B) Activation profile of a subpopulation of CD4+ T cells. (C) Activation profile of a subpopulation of CD8+ T cells. Each bar represents mean ± SEM. *p<0.05, **p<0.01. [Figure 21] This figure shows flow cytometry analysis of exhaustion profiles of human T cells in the spleen of NSG mice transplanted with human mononuclear cells and injected with either WT or CXCR4 / IL10-MSCs 3 weeks prior. (A) Inhibition profile of CD3+CD45+ T cells. (B) Inhibition profile of a subpopulation of CD4+ T cells. (C) Activation profile of a subpopulation of CD8+ T cells. Each bar represents the mean ± SEM. *p<0.05. [Figure 22]This figure shows the phenotypic characteristics of a subpopulation of human CD19+ B cells in the spleen (naive B cells CD24-CD38-CD27-; transitional B cells CD24-low / +CD38+CD27-; memory B cells and plasma cells CD24-low / +CD38+CD27+). Each bar represents the mean ± SEM. *p<0.05. [Figure 23] Flow cytometry analysis of human B cell polarization relative to regulatory B cells in the spleen of NSG mice 3 weeks after transplantation of human mononuclear cells and injection of WT or CXCR4 / IL10-MSCs is shown. (A) Graph showing representative flow cytometry analysis and IL10+ transitional B cell percentage for each group. (B) Graph showing representative flow cytometry analysis and IL10+ memory B cell percentage for each group. Each bar represents the mean ± SEM. *p<0.05. [Figure 24] This figure shows the histopathological analysis of the lungs of NSG mice transplanted with human mononuclear cells and injected with either WT or CXCR4 / IL10-MSC. (A) Representative images of H / E staining (left), human anti-CD3 immunohistochemistry (center), and human anti-CD8 immunohistochemistry (right). (B) Quantification of infiltrating CD3+ T cells in the lung. (C) Quantification of infiltrating CD8+ T cells in the lung. Each bar represents the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 25] This figure shows the histopathological analysis of the livers of NSG mice transplanted with human mononuclear cells and injected with either WT or CXCR4 / IL10-MSC. (A) Representative images of H / E staining (left), human anti-CD3 immunohistochemistry (center), and human anti-CD8 immunohistochemistry (right). (B) Quantification of infiltrating CD3+ T cells in the liver. (C) Quantification of infiltrating CD8+ T cells in the liver. Each bar represents the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 26]This figure shows the experimental design for DSS-induced colitis. Different concentrations of dextran sulfate sodium (DSS) were administered as needed over 7 days in drinking water at concentrations ranging from 2.5% to 3%. On day 5, a single dose of either WT or CXCR4 / IL10-MSC (3 × 10⁶ cells / mouse) was administered intraperitoneally. For long-term evaluation, a re-induction study using a 7-day cycle of drinking water DSS was performed 12 weeks later. [Figure 27] This figure shows the state of DSS-induced colitis in mice after intraperitoneal administration of WT-MSC or CXCR4 / IL10-MSC. Disease activity index (DAI) (A), change in weight multiplier (B), and survival rate (C). Representative images of colon tissue 10 days after DSS treatment (magnification 4× and 10×) (D). Healthy, n=14; DSS, n=26; DSS+WT-MSC, n=21; and DSS+CXCR4 / IL10-MSC, n=26. Data are expressed as percentages over time, relative to day 0, using the mean and standard error of the disease activity index and change in weight multiplier. Survival rate is expressed as a percentage. Significance was analyzed by the Mann-Whitney U test and the Long Rank test, and expressed as *p<0.05 and ****p<0.0001 DSS vs. healthy; $p<0.05 DSS+WT-MSC vs. DSS;&p<0.05 and && p<0.01 DSS+CXCR4 / IL10-MSC vs. DSS and #p<0.05 DSS+CXCR4 / IL10-MSC vs. DSS+WT-MSC. The results correspond to five independent experiments. [Figure 28]This figure shows the status of DSS-induced colitis in mice 3 months after administration of WT-MSC or CXCR4 / IL10-MSC. The figures represent disease activity index (DAI) (A), weight multiplier change (B), and survival rate (C). Healthy, n=10; DSS, n=15; DSS+WT-MSC, n=15; and DSS+sCXCR4-IL10-MSC, n=15. Data are expressed as percentages over time, relative to day 0, using the mean and standard error of disease activity index and weight multiplier change. Survival rate is expressed as a percentage. Significance was analyzed by the Mann-Whitney U test and the Long Rank test, and expressed as follows: **p<0.01 and ****p<0.0001 DSS vs. healthy, & p<0.05 DSS+CXCR4 / IL10-MSC vs. DSS, and # p<0.05 and ## p<0.01 DSS+CXCR4 / IL10-MSC vs. DSS+WT-MSC. The results correspond to three independent experiments. [Modes for carrying out the invention]

[0020] The present invention is illustrated by the following examples without any intention to limit its scope of protection. [Examples]

[0021] Example 1. Materials and Method Example 1.1. Generation and proliferation of adipose tissue-derived MSCs (Ad-MSCs) Adipose tissue samples are obtained by surgical excision from a healthy donor after informed consent. The adipose tissue was deaggregated, and collagenase A (Serva, Germany) was used at a final concentration of 2 mg / ml. The cells were then digested at 37°C for 4 hours. The digested sample was filtered through a 100 μm nylon filter (BD Bioscience, USA) and centrifuged for 10 minutes. The cell pellet was then divided into 5% platelets. Dissolving solution (Cook Medical, USA), 1% penicillin / streptomycin (Gibco) and 1 Added human basic fibroblast growth factor (bFGF, Peprotech, USA) at ng / ml. The cells were resuspended in α-MEM (Gibco, USA). Cultured in a Corning flask (USA) at a concentration of 10,000 cells / cm³. 2 Cells were seeded at the specified concentration and cultured at 37°C. To promote Ad-MSC proliferation, the cell medium was changed every 2-4 days, and when the attached cells reached near confluence (70-90%), 0.25% trypsin / EDTA (Sigma-Aldrich, USA) was used. The cells were then continuously passaged. For in vitro and in vivo studies, Ad-MSCs were used after 4 to 8 passages.

[0022] Example 1.2. Characterization of WT-MSC and CXCR4 / IL10-MSC WT-MSCs and MSCs transduced with the CXCR4-IL10 lentiviral vector (CXCR4 / IL10-MSCs) were immunophenotypically characterized by flow cytometry (Fortessa, BD Bioscience, USA) as described in the Mesenchymal cell kit (Immunostep, Spain). These studies included... The noclonal anti-human antibodies were as follows: CD29, CD44, CD73, CD90, CD105, CD166, CD45, CD19, HLA-DR, CD14, and CD34. Data were analyzed using FlowJo version X (FlowJo LLC, USA).

[0023] The osteogenic and adipogenic differentiation capabilities of Ad-MSCs were evaluated using NH-OsteoDiff and NH-AdipoDiff Media (Miltenyi Biotec, Germany), respectively, by the manufacturer. The determination was made according to the protocol. Alkaline phosphatase deposition was observed after staining with Fast BCIP / NCP (Sigma-Aldrich), and lipid droplets were visualized using a light microscope (Nikon, Germany).

[0024] Example 1.3. Construction of a DNA CXCR4 / IL10 lentiviral vector A fragment (7362 bp) containing the lentiviral backbone and PGK promoter is introduced into a blank FANCA transgene with restriction sites at the 5' and 3' ends, respectively, for AgeI and SacII restriction enzymes (New England Biolabs, pCCL.PGK.FANCA.Wpre (USA) * The plasmid (9087 bp) was obtained by co-digestion. The digested lentiviral backbone, which did not contain the transgene, was purified from an agarose gel using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany). Fragments containing codon-optimized sequences of human CXCR4 and IL10 were obtained by polymerase chain reaction (PCR) using the pUC57 plasmid used for mRNA synthesis as a template. PCR of each plasmid was performed using two specific primers containing the AgeI and SacII restriction sites at the 5' end, as well as the first or last 20 bp of the CXCR4 or IL10 transgene. Amplification was performed according to the Herculase II Fusion Enzyme protocol (Agilent, USA), using dimethyl sulfoxide (DMS) depending on the target size. The procedure was performed without using O) and stabilized at 58°C, the annealing temperature. The PCR product was simultaneously digested with Age I and Sac II, and further purified by column chromatography using the NucleoSpin Gel and PCR Clean-up Kit.

[0025] The digested lentiviral backbone and target fragment were processed using T4 DNA Ligase (New England Biolabs) while maintaining a target:vector ratio of 5:1. Ligation was performed. The ligated product was introduced into Stable3 bacteria, and pCCL.PKG-CXCR4-IL10.Wpre * Plasmid obtained.

[0026] Example 1.4. Generation of a lentiviral vector All autoinactivated HIV-1 vectors used in this study were generated using a second-generation packaging system in HEK293T cells to obtain VSV-G pseudotyped viruses. The total volume was 12 × 10⁴ on a 150 mm diameter plate the day before. 6 Individual cells were plated. Gene transfer was performed on 70%-80% confluent cells in 150 mm diameter plates according to the CaCl2 DNA precipitation method described above. Briefly, one hour before gene transfer, the culture medium was changed to 10% HyClone (GE Healthcare, USA) and 1% penicillin / strep The cells were replaced with fresh DMEM-Glutamax containing tomycin. A fresh isomolecular mixture of three plasmids containing the transgene, viral genome, and packaging construct was prepared. HEK293T cells in each plate were transfused with 22.5 μg of the transgene plasmid, 12 μg of the pMD2.VSVg envelope plasmid (PlasmidFactory, Germany) containing a heterologous VSVg envelope, and 27.5 μg of the pCMVdR8.74 packaging plasmid (PlasmidFactory) containing the gag-pol-rev viral gene. These plasmid mixtures were prepared in 3.8 ml of ultrapure water (H2O) and carefully mixed with 450 μl of 2.5 M CaCl2. After incubation at room temperature for 5 minutes, 3.8 ml of 2 × Hanks equilibrium saline (HBS) buffer (100 mM HEPES (Gibco), 281 mM NaCl, 1.5 mM Na2HPO4, pH=7) was added. Add 13) drop by drop, Ca 2+ A precipitate was formed. This solution was added to HEK293T cells, and these precipitates were integrated. After 5 hours, the medium containing the precipitate was replaced with fresh medium. The supernatant was collected 48 hours after gene transfer. These were collected and filtered using a 0.22 μm pore size filter (Millipore, Merck KGaA, Germany), and concentrated by ultracentrifugation at 20,000 rpm at 4°C for 2 hours. The virus pellet was then suspended in DMEM at 4°C for at least 1 hour, centrifuged to discard cell debris, and stored in 100 μl aliquots at -80°C.

[0027] Example 1.5. Transduction of Ad-MSC Human Ad-MSCs were transduced using two different strategies: transduction of attached MSCs and transduction of MSCs in suspension. In this set of experiments, a transduction enhancer (TE) was added during the transduction process to increase the effectiveness of transduction.

[0028] Example 1.6. Co-expression of CXCR4 and IL-10 proteins The expression of CXCR4 on the cell surface of Ad-MSCs was determined by flow cytometry after labeling with a PE-conjugated anti-human CXCR4 antibody at 4°C for 30 minutes (Biolegend, USA). The IL10 levels secreted by Ad-MSCs were measured in the supernatant of cultured cells using the human IL10 Quantikine ELISA Kit (R&D System, USA).

[0029] Total protein extracts were separated from Ad-MSCs using RIPA buffer (ThermoFisher Scientific, USA) containing a protease inhibitor mixture (Merck Millipore, Germany). 20 micrograms of each cell lysate were cleaved in 4%–12% polyacrylamide gel (Bio-Rad, USA) and transferred to a PVDF membrane (Bio-Rad). The membrane was blocked with 5% (v / v) fat-free dried milk in 0.1% Tween-20 PBS. The cells were incubated with diluted rabbit monoclonal anti-human CXCR4 antibody (Abcam, UK) in the blocking solution. The samples were immunoblotted using the following method. Mouse anti-human vinculin (Abcam) was applied to the samples. It was used as a control. The blot was processed using ChemiDoc MP System and ImageLab software (Bio-Rad) for Clarity Western EC Visualization was performed using the L substrate (Bio-Rad).

[0030] Example 1.7. Cell migration assay The migration assay was performed in a Transwell equipped with an 8 μm pore polycarbonate membrane insert (Costar, Cambridge, MA). 5 × 10 3 One Ad-MSC was placed in the upper insert chamber of the Transwell assembly. Mouse or human SDF-1 (Peprotech, USA) at a final concentration of 100 ng / ml was contained in the lower chamber. 24 hours after vaping, the top of the membrane was gently scraped off with a cotton swab to remove non-migrating cells, and the membrane was washed with PBS. The membrane was fixed overnight at 4°C with 3.7%-4% formalin and stained with hematoxylin at room temperature for 4 hours. The number of migrating cells was measured under a Nikon Eclipse E400 microscope (10×) (Nikon, UK) using four random samples per well. Determined by area scoring, Leica DFC420 camera (Leica, UK) A photograph was obtained using this method.

[0031] Example 1.8. In vitro immunosuppressive assay Peripheral blood mononuclear cells (MNCs) were obtained from heparinized peripheral blood samples from healthy donors after informed consent, using a density gradient with Ficoll-Paque PLUS (GE Healthcare Bioscience, Sweden). Before co-culturing, MNCs were marked with the intracellular fluorescent dye CFSE (carboxyfluorescein diacetate succinimimidyl ester, Molecular Probe, USA) according to the protocol described above. WT-MSCs and CXCR4 / IL10-MSC, 5×10 4 The cells were plated in a 24-well plate at a concentration of 5 × 10⁶ cells / well. After 24 hours, 5 × 10⁶ cells were observed. 5 Each MNC was added to each well in the presence of 10 μg / mL of phytohemagglutinin (PHA) (Sigma-Aldrich) and T cells Proliferation was induced. After 3 days of incubation, cells collected from the culture wells were analyzed for cell proliferation by flow cytometry. The data were analyzed using ModFit LT (trademark) (Verity Software House, USA).

[0032] Example 1.9. Quantification of Secreted Cytokines and Factors WT-MSCs and CXCR4 / IL10-MSCs were seeded in 6-well plates at a concentration of 1×10 5 cells / well. After 4 hours of gene introduction, the supernatant was collected, and secreted PGE2 and TGFβ1 were quantified by ELISA (R&D System, USA). Secreted IL-6, IFNγ, and TNFα were quantified by flow cytometry using the LEGENDplex (trademark) Human Th Cytokine Panel (Biolegend, USA) according to the manufacturer's protocol. Therefore, it was quantified by flow cytometry.

[0033] Example 1.10. Gene Expression Analysis RNA from WT-MSCs and CXCR4 / IL10-MSCs was isolated using the RNAeasy (trademark) Plus Mini Kit and reverse transcribed using RETROscript (ThermoFisher Scientific, Waltham, USA). Quantitative real-time PCR (qPCR) was performed on the cDNA using the FastStart Uni versal SYBR Green Master Mix (Roche, Indianapolis, USA) and specific primers for human interleukin and different factors. qPCR was performed on a 7500 fast real-time PCR system (ThermoFisher Scientific). The results were normalized according to the 2 method with respect to human GAPDH expression -ΔΔCt and the expression of control samples. and normalized against the expression of control samples.

[0034] Example 1.11. LPS-Induced Inflammatory Pad Model FVB / NJ mice were housed at the animal facility (registration number ES280790000183) located in Madrid, Spain. The mice were regularly screened for pathogens according to FELASA procedures and were given free access to water and food. All experimental procedures were carried out in accordance with Spanish and European regulations (Spanish National RD53 / 2013 and Law 6 / 2013, European Directive 2010 / 63 / UE). The procedures were approved by the CIEMAT Animal Experiment Ethics Committee in accordance with approved biosafety and bioethics guidelines. Confirmed. FVB / NJ mice were sedated and given a single injection of 40 μg of E. coli LPS in 30 μl of PBS into the right pad. Similarly, as a control, 30 μL of PBS was injected into the left pad. Four hours after Ad-MSC gene transduction, 5 × 10⁶ mice were observed. 5 One WT-MSC or CXCR4 / IL10-MSC was intravenously administered via the tail vein. Pad inflammation was determined by measuring the thickness using a digital caliper at 24, 48, and 72 hours after LPS administration. At the end of the experiment, the mice were sacrificed by CO2 inhalation. Peripheral blood cells were collected and analyzed using a hematology analyzer Abacus (Diatron, USA) to analyze the mouse blood. We analyzed the fluidic parameters.

[0035] Example 1.12. Humanized mouse model of graft-versus-host disease (GvHD) in NSG mice To establish the model, NSG mice were irradiated with 2 Gy, and the following day, 5 × 10⁻¹⁶ mice were treated with irradiation. 6 Human MNCs were transplanted. Three days later, one million WT-MSCs or CXCR4 / IL10-MSCs were intravenously injected. The animals were weighed daily and monitored for symptoms of potential GVHD, such as weight loss, hunchback, disheveled fur, and diarrhea. The severity of GVHD was graded from 0 (no GVHD) to 8 (severe GVHD). The animals were humanely euthanized when they met the euthanasia criteria for GVHD (weight loss exceeding 20% ​​or a score of 6.5 or higher).

[0036] Example 1.13. Statistical Analysis Statistical analysis was performed using Graph Pad Prism 7.0 software (Graph Pad Software, USA). In vitro test data were expressed as mean ± standard deviation (SD), and in vivo test data were expressed as mean ± standard error of the mean (SEM). Normal distribution was analyzed using the Shapiro-Wilk test. To compare three or more groups, parametric tests (one-way ANOVA) were used for normal distributions, and nonparametric tests (Kruskal-Wallis test) were used for non-normal distributions. Appropriate post-hoc analyses were performed to compare means. A p-value < 0.05 was considered statistically significant.

[0037] Example 1.14. Histopathological analysis in a GVHD mouse model The lungs and liver were surgically removed and fixed overnight with formalin. After fixation, tissue samples were processed using standard methods and embedded in paraffin to form blocks. To evaluate the tissue morphology, 3 μm–5 μm sections of the paraffin blocks were prepared using a microtome and stained with hematoxylin-eosin using standard methods. The tissue was interpreted according to the previously established GVHD grading system.

[0038] Example 1.15. Immunohistochemical analysis in a GVHD mouse model Slides containing the samples were deparaffinized and rehydrated according to a standard protocol. Lung and liver samples were labeled with human CD3 and CD8. Antigen retrieval of CD3-labeled samples was performed using sodium citrate buffer (1.8 mM citrate monohydrate and 8.2 mM trisodium citrate dihydrate; pH 6) in a pressure cooker (Dako, Agilent Technologies). Tris-EDTA buffer (antigen retrieval buffer pH 9; Dako) and the same pressure cooker were used for retrieval of CD8-stained samples. Endogenous peroxidase was inhibited for 10 minutes with 0.2% hydrogen peroxide dissolved in methanol. Nonspecific epitopes were blocked with 10% horse serum dissolved in PBS at 37°C for 30 minutes. Primary antibodies were diluted in the inhibition solution and incubated overnight at 4°C. Biotin-conjugated secondary antibodies were diluted in the blocking solution and incubated at room temperature for 1 hour. To amplify the signal, a biotin-avidin-peroxidase system (VECTASTAIN elite ABC HRP kit, Vector Laboratories) is used. The mixture was incubated at room temperature for 30 minutes using (DAB Kit, Vector Laboratories). The signal was visualized using diaminobenzidine as the peroxidase substrate. The samples were counterstained with hematoxylin, dehydrated using standard procedures, and mounted using mounting adhesive (CV Mount, Leica Biosystems). Images were captured using an optical microscope (Olympus BX41) and a digital camera (Olympus DP21). The percentage of markings in each sample was analyzed using the ImageJ program.

[0039] Example 1.16. Induction and evaluation of dextran sulfate sodium (DSS)-induced colitis. Different concentrations of dextran sulfate sodium (DSS; 36,000 MW to 50,000 MW, MP Biomedicals, Irvine, CA, USA) were freely used in drinking water at concentrations ranging from 2.5% to 3% for 7 days. On day 5, natural or CXCR4 / IL-10 modified MSCs (3 × 10⁻¹⁰) were used. 6 A single dose of individual cells / mouse was administered by intraperitoneal injection.

[0040] For long-term evaluation, a re-induction test was performed using drinking water DSS in a 7-day cycle (Figure 26).

[0041] The Colitis Score or Disease Activity Index (DAI) was defined as follows: (1) Weight loss (0: no loss, 1: 1% to 5%, 2: 5% to 10%, 3: 10% to 20%, 4: weight loss greater than 20%, and 5: no survival), (2) Stool consistency (0: normal stool, 1: loose stool, 2: watery diarrhea, 3: watery diarrhea with blood, and 4: no survival), and (3) General physical activity (0: normal, 1-2: moderate activity, 3: no activity, and 4: no survival). The weight multiplier change was calculated as a percentage, based on the difference in weight at the defined time point, relative to the initial weight on day 0 immediately before the start of DSS treatment.

[0042] Colitis scores were also assessed by histological analysis of the colon. The colon was surgically removed and fixed with formalin overnight. After 48 hours, a 1 cm section of colonic tissue was excised, embedded in paraffin, and stained with hematoxylin / eosin. The section was examined under a light microscope for analysis of infiltrating mononuclear cells and the structure of the intestinal epithelium and submucosa.

[0043] Example 2. Results For clarification, please note that the results provided in Examples 2.1 and 2.2, which use MSCs transfected with bicistronic CXCR4-IL10 mRNA as a baseline, are included only as comparative examples to show how these results improved when MSCs were transfected with an integrated expression vector co-expressing CXCR4 and IL10 (Examples 2.3 to 2.7).

[0044] Example 2.1. MSCs transfected with bicistronic CXCR4-IL10 mRNA showed significant local anti-inflammatory activity. The in vivo efficacy of MSCs (Medical Stem Cells) transfected with bicistronic CXCR4-IL10 mRNA was investigated in a mouse model of local inflammation induced by LPS. Both WT-MSCs and CXCR4-IL10-RNA-MSCs showed significant anti-inflammatory effects, but MSCs transfected with bicistronic CXCR4-IL10 mRNA were significantly more effective than WT-MSCs. Figure 1 shows an analysis of the in vivo efficacy of MSCs transfected with bicistronic CXCR4-IL10 mRNA in a mouse model of local inflammation. Enhanced anti-inflammatory effects were observed in MSCs transfected with CXCR4-IL10 mRNA compared to WT-MSCs.

[0045] Example 2.2. Lack of in vivo efficacy of MSCs transfected with bicistronic CXCR4-IL10 mRNA in a graft-versus-host disease mouse model. Furthermore, in the graft-versus-host disease mouse model, bisistronic CXCR4-IL10 The in vivo efficacy of MSCs with mRNA-derived genes was verified. C57Bl / 6 A mouse model of haplotype-matched hematopoietic transplantation was derived by transplanting BM cells from donor mice into B6D2F1 recipient mice that had been pre-irradiated with a lethal dose of 11 Gy. All recipients received 10 × 10⁶ cells. 6 A total of 2 x 10⁶ BM donor cells were injected intravenously to induce graft-versus-host disease (GVHD) in the recipient. 8 One donor splenocyte was also administered. One day after GVHD induction, mice were given physiological saline (GVHD group), WT-MSCs or mRNA gene-transformed MSCs (1 x 10⁶). 6The drug was administered via the tail vein. The transplanted recipients were observed daily for symptoms of GVHD, such as weight loss, hunchback, unkempt fur, and diarrhea. The severity of GVHD was graded from 0 (no GVHD) to 8 (severe GVHD). Animals were humanely euthanized when they met the criteria for euthanasia for GVHD (weight loss exceeding 20% ​​or a score of 6.5 or higher). Figure 2 shows the in vivo results of MSCs transfected with bicistronic CXCR4-IL10 mRNA in a mouse model of GVHD. The analysis of vivo efficacy is shown. A) Survival curve, B) Weight, and C) Clinical score. As shown in Figure 2A, no difference in inhibiting GVHD was observed between WT-MSCs and CXCR4-IL10 mRNA MSCs.

[0046] Example 2.3. Generation of MSCs transduced with a bicistronic DNA CRCR4-IL10 lentiviral vector to improve the efficacy of WT MSCs in inhibiting graft-versus-host disease. In these studies, optimized sequences of the CXCR4 and IL10 genes were cloned into a bicistronic lentiviral vector under the human physiological promoter PGK, creating a lentiviral vector (Figure 3A).

[0047] After validating different methods of Ad-MSC transduction and different amounts of vector, a population of modified Ad-MSCs (CXCR4 / IL10-MSCs) was obtained. This population of CXCR4 / IL10-MSCs overexpressed CXCR4, and approximately 80% of the MSCs were positive for CXCR4. CXCR4 / IL10-MSCs secreted higher concentrations of IL10 compared to unmodified MSCs (WT-MSCs). Vector copy number in these CXCR4 / IL10-MSCs was analyzed by qPCR (Figure 3B).

[0048] Example 2.4. In vitro characterization of CXCR4 / IL10-MSC compared with WT-MSC MSCs modified using the bicistronic PGK-CXCR4-IL10 lentiviral vector were characterized according to the criteria established by the ISCT (International Society for Cell Therapy) for mesenchymal cells.

[0049] In vitro characterization showed that modification of MSCs using a bicistronic lentiviral vector did not affect their immunophenotype (Figure 4A) or their differentiation potential into bone (Figure 4B) and adipose tissue (Figure 4C) compared to unmodified mesenchymal cells (WT-MSCs).

[0050] Example 2.5. In vitro functionality of CXCR4 / IL10-MSC compared to WT-MSC To investigate the in vitro functionality of mesenchymal cells (CXCR4 / IL10-MSCs) modified using a bicistronic lentiviral vector, we first performed a Transwell migration assay in response to SDF-1, a ligand for CXCR4 (Figure 5A). The results of this assay showed enhanced migratory ability of CXCR4 / IL10-MSCs compared to WT-MSCs (Figure 5B).

[0051] The following in vitro functional characterization study consisted of an immunosuppressive assay, in which the ability of CXCR4 / IL10-MSCs to inhibit the proliferation of activated mononuclear cells (MNCs) was evaluated in comparison with that of WT-MSCs (Figure 6A).

[0052] As previously described, WT-MSCs showed a high ability to inhibit the proliferation of activated MNCs. However, this inhibition was significantly higher when MSCs were transduced with the PGK-CXCR4-IL10 lentiviral vector (Figure 6B). These studies demonstrate that transduction of MSCs with a bicistronic lentiviral vector significantly improves the immunomodulatory capacity of these cells compared to WT-MSCs.

[0053] Example 2.6. Enhancement of the in vivo efficacy of CXCR4 / IL10-MSC for inhibiting local inflammation compared to WT-MSC. To verify the in vivo efficacy of MSCs transduced with the PGK-CXCR4-IL10 lentiviral vector, the cells were examined in a mouse model of local inflammation induced by LPS.

[0054] LPS was injected into the right pad of each mouse. One day after LPS injection, different types of Ad-MSCs (WT-MSC and CXCR4 / IL10-MSC) were intravenously administered (n=7 to 14 mice / group). Inflammation was measured visually using a digital caliper in the left pad, which served as a control in each mouse (Figure 7A).

[0055] The results showed that 24 hours after Ad-MSC injection (48 hours after LPS injection), inflammation was controlled in all mice given Ad-MSC, while inflammation continued to grow in the group of mice given only LPS injection.

[0056] However, inflammation control was statistically superior in the mouse group given CXCR4 / IL10-MSC (Figure 7B).

[0057] Example 2.7. Improvement of the efficacy of MSCs transduced with a DNA bicistronic lentiviral vector in inhibiting graft-versus-host disease (GvHD) compared to WT MSCs. The therapeutic efficacy of MSCs transduced with bicistronic lentiviral vectors was investigated in a graft-versus-host disease (GvHD) mouse model based on the injection of peripheral blood human mononuclear cells (MNCs) into immunodeficient NSG mice (Figure 8A). To establish the model, mice were irradiated with 2 Gy and the following day injected with 5 × 10⁶ cells. 6 Human MNCs were transplanted. Three days later, one million WT-MSCs or CXCR4 / IL10-MSCs were intravenously infused. Animals were weighed daily and monitored for major signs of possible GVHD (Figure 8B).

[0058] As shown in Figure 8B, the GVHD score was significantly better in the NSG mouse group given CXCR4 / IL10-MSC, not only compared to the GVHD group but also to the WT-MSC group.

[0059] Two weeks after MNC injection, mice given only human MNC (GvHD group) began to show signs of disease (weight loss, hunchback). At this point, all three recipient mice were sacrificed, and human CD45 levels were measured in peripheral blood (PB) and spleen (SP). + The percentage of cells was analyzed. In mice given WT-MSCs, infiltrating human CD45 + A significant reduction in the percentage of cells was found. Nevertheless, the reduction observed in both the PB and spleen was significantly greater in mice injected with CXCR4 / IL10-MSC (Figures 9A and 9B).

[0060] Human CD45 is the cause of GVHD disease + CD3 + Cells were analyzed by flow cytometry in a humanized GVHD mouse model. Notably, NSG mice treated with CXCR4 / IL10-MSCs but not with WT-MSCs showed a higher proportion of pro-inflammatory T cells (CD3) compared to the GvHD control group. + IFNg + The percentage of ) A significant reduction was observed (Figure 10A). Furthermore, in the group that received CXCR4 / IL10-MSCs but not WT-MSCs, anti-inflammatory T cells (CD3) were reduced compared to the GvHD control group. + IL10 + A statistically significant increase was observed in the percentage of (Figure 10B).

[0061] These data observed by flow cytometry were confirmed by qPCR. Pro-inflammatory factors such as IFNg, IL-17, and IL-22 were significantly reduced in mice that received CXCR4 / IL10-MSC but not WT-MSC compared to the GvHD control group. Quantification of the expression levels of anti-inflammatory factors such as IL-5 or FoxP3 showed that these factors were statistically increased compared to the GvHD control group in mice that received CXCR4 / IL10-MSC but not WT-MSC (Figure 11).

[0062] Example 2.8. In vivo efficacy of CXCR4 / IL10-MSCs verified in a humanized model of graft-versus-host disease (GvHD). To validate the in vivo efficacy of CXCR4 / IL10-MSCs against WT-MSCs, a humanized model of graft-versus-host disease (GvHD) was developed. The greatest weight loss was observed in the GvHD group that did not receive any type of Ad-MSC. Furthermore, no weight loss was observed in the CXCR4 / IL10-MSC group compared to the significant weight loss observed in the GvHD group and the WT-MSC group (Figure 12A). In addition, the WT-MSC group exhibited lower GvHD scores (more moderate clinical signs) compared to non-MSC treated mice, while GvHD scores were significantly lower in mice treated with CXCR4 / IL10-MSC (Figure 12B).

[0063] Analysis of human leukocytes in the peripheral blood of transplanted mice showed a significant reduction in mice given Ad-MSCs (%hCD45 cells; Figure 13A). Nevertheless, mice treated with CXCR4 / IL10-MSCs showed the lowest percentage of human leukocytes, most of which were human CD3 in all cases. + It is a T cell (Figure 13B), CD4 + T cells, CD8 + There was no difference between T cells or bipositive T cells (Figure 13C).

[0064] Human CD4 in naive, effector, and memory T cells + T cells or CD8 + Analysis of T cell distribution revealed CD4 cells exhibiting an effector phenotype in mice given CXCR4 / IL10-MSC. + T cells and CD8 + This showed a significant reduction in the percentage of T cells (Figures 14C and 14F).

[0065] The activation profile of circulating human T cells in the peripheral blood of mice was investigated. All groups that received any type of Ad-MSC showed CD25 + This showed an increase in the percentage of T cells, which was statistically higher in mice treated with CXCR4 / IL10-MSCs. Furthermore, these cells were CD25 + CD4 + These were lymphocytes, suggesting the presence of circulating regulatory T cells in this group (Figure 15).

[0066] Also, circulating human CD3 in peripheral blood + The T cell exhaustion profile was analyzed using CTLA4, PD1, TIGIT, and TIM3 markers. Three weeks after MNC transplantation, circulating CD3 positive for CTLA4 was observed. + Cellular enlargement was observed in both groups given Ad-MSCs, and this was significantly greater in mice given CXCR4 / IL10-MSCs (Figure 16).

[0067] Circulating human cytokines and factors involved in the development of GvHD were analyzed in the serum of these mice. As shown in Figure 17, all groups treated with any type of Ad-MSC showed signs of GvHD. Compared to the control group (D), the levels of circulating pro-inflammatory human cytokines such as IFNγ, IL17A, IL1α, IL8, IL12, or TNFα were statistically significantly reduced. Furthermore, these two groups given Ad-MSCs showed increases in circulating human anti-inflammatory factors such as IL10, TGFβ, or IL6. Notably, the shift in cytokine secretion from a pro-inflammatory to a more anti-inflammatory profile was statistically more pronounced in mice given CXCR4 / IL10-MSCs compared to mice given WT-MSCs (Figure 17).

[0068] These results demonstrated that infusion of CXCR4 / IL10-MSCs resulted in a significant reduction in the percentage of circulating human T cells in peripheral blood, relative to values ​​corresponding to mice treated with WT-MSCs. Furthermore, peripheral blood T cells exhibited a more immunosuppressive profile after infusion of CXCR4 / IL10-MSCs compared to WT-MSCs.

[0069] In summary, this data indicates that CXCR4 / IL10-MSCs induce a significant reduction in the inflammatory environment and an enhancement of the immunomodulatory environment at a systemic level in NSG immunodeficient mice transplanted with human leukocytes.

[0070] We investigated the distribution of different human hematopoietic lineages in the spleen: CD3 + T cells, CD19 + B cells, CD56 + NK cells, CD14 + Monocytes and CD15 + Granulocytes. Human CD45 observed in the spleen 3 weeks post-transplant in the GvHD group. + Approximately 70% of the cells are human CD3 + While T cells were present (64.98±4.14%), this percentage decreased in the group given WT-MSCs (59.22±4.56%) and more significantly in the group given CXCR4 / IL10-MSCs (48.67±3.58%). Furthermore, human CD19 +When the percentage of B cells was analyzed in the spleen, a significant increase in this population was detected in the group treated with CXCR4 / IL10-MSCs (14.62±1.52%) compared to either the GvHD group (6.73±1.03%) or the group given WT-MSCs (8.99±1.53%). Finally, CD56 in the spleen of transplanted mice. + NK cells, CD14 + Monocytes and CD15 + The percentage of granulocytes was very low, and there was no difference between the different study groups.

[0071] CD4 in the spleen + cells, CD8 + No significant differences were found between the study groups in the distribution of T cells among naive, effector, and memory T cells (Figure 19A). The most distinctive subpopulation was human CD4 among naive, effector, and memory T cells. + T cells or CD8 + No differences were observed in the distribution of T cells between the different study groups (Figures 19B and 19C).

[0072] The activation patterns observed in the spleen were very similar to those observed in peripheral blood. Differences between groups were found only in CD25 expression in the spleen (Figure 20A). All groups administered Ad-MSCs compared CD25 expression to the GvHD group. + A significant increase in the percentage of T cells was observed, and this was even higher in mice treated with CXCR4 / IL10-MSCs compared to the group given WT-MSCs. These cells were particularly CD25 + CD4 + These are T cells (Figure 20B), and these CD4 cells in the spleen + This demonstrated the immunomodulatory phenotype of T cells.

[0073] Analysis of inhibitory receptors in the spleen of NSG-transplanted mice 3 weeks after MNC transplantation was performed using the GvHD control group of mice not treated with AdMSC (Figure 21) as a baseline, and comparing the results for mice treated with CXCR4 / IL10-MSC to human CD3 +In T cells (Figure 23A), and further in CD4 + Cells or CD8 + In cells (Figures 21B and 21C, respectively), TIM3 + A significant increase in T cells was observed. No differences were observed between the groups compared to other fatigue markers analyzed.

[0074] Flow cytometry analysis of B cells in the spleen showed no change in the naive B cell subpopulation between the non-MSC treated group and the group given any type of AdMSC. However, the percentage of transitional B cells that had not yet differentiated into antibody-producing B cells was higher in mice given CXCR4 / IL10-MSCs (34.78±7.09%) compared to the group given WT-MSCs (24.3±5.18%) and the GvHD control group that did not receive any MSCs (17.47±2.21%). Finally, a slight decrease in the percentage of fully differentiated B cells was observed only in the group given CXCR4 / IL10-MSCs (Figure 22).

[0075] These results suggest that WT-MSCs, and more prominently, CXCR4 / IL10-MSCs, maintained the B cell population in a transitional state without completing their differentiation into memory B cells or plasma cells.

[0076] The percentage of Breg cells in the transitional B cell population was higher in mice given WT-MSCs (Figure 23A). Furthermore, this percentage was statistically higher in mice injected with CXCR4 / IL10-MSCs. The same pattern was observed among the IL10-secreting memory B cell populations (Figure 23B).

[0077] In summary, these results suggest that infusion of CXCR4 / IL10-MSCs not only significantly promotes the development of T cells with immunomodulatory phenotypes relative to WT-MSCs, but also improves the development of B cells that have a beneficial effect against the progression of GvHD.

[0078] During the final stages of acute human GvHD, donor effector T cells mediate tissue damage in different organs through direct cytotoxic activity or inflammatory cytokine production. Histopathological signs of GvHD were analyzed in target organs of this disease, such as the lungs or liver. Histological analysis of the lungs showed that mice given CXCR4 / IL10-MSCs exhibited a significant reduction in human T cell infiltration in the parenchyma compared to two other groups that showed similar structures to disease-free control groups (Figure 24A). Human CD3 + T cells and CD8 + By quantifying the presence of T cells, it was found that infusion of any type of Ad-MSC reduced the percentage of both cell types in the lungs. Notably, in mice treated with CXCR4 / IL10-MSCs, human CD3 + Cells (Figure 24B) and CD8 + The reduction was far more significant for both types of cells (Figure 24C).

[0079] Histopathological analysis of the livers of transplanted mice revealed levels of human T cell infiltration in the parenchyma, as well as perivascular inflammation, which were significantly reduced after injection of any type of Ad-MSC. Furthermore, this inflammation was virtually absent in mice treated with CXCR4 / IL10-MSC (Figure 25A). On the other hand, Ad-MSC administration reduced human CD3 in the liver. + Cells and CD8 + The presence of cells was significantly reduced, and this reduction was far more significant when mice were given CXCR4 / IL10-MSCs (Figures 25B and 25C).

[0080] Example 2.9. Enhancement of the efficacy of CXCR4 / IL10-MSCs that stably express CXCR4 and IL10 in an experimental model of dextran sulfate (DSS)-induced inflammatory bowel disease (IBD). Furthermore, we investigated the in vivo efficacy of genetically modified MSCs expressing CXCR4 and IL10 in a novel experimental model of inflammation: DSS-induced inflammatory bowel disease (IBD).

[0081] According to the experimental design (materials and methods) shown in Figure 26, the disease activity index (DAI) in colitic mice treated with a single dose of CXCR4 / IL10-MSC was measured. ) is compared to either mice not treated with MSCs or mice treated with WT-MSCs. In comparison, the figures were significantly lower (Figure 27A). Furthermore, significant differences were observed in weight loss (Figure 27B) and survival rate (Figure 2FC) of CXCR4 / IL10-MSC-treated mice during the first 7-day DSS cycle compared to the WT-MSC-treated group and the non-MSC-treated group. Compared to non-MSC-infused colitis mice, histologically better preserved colonic morphology and reduced leukocyte infiltration were observed in CXCR4 / IL10-MSC-treated colitis mice (Figure 27D).

[0082] To investigate the long-term effects induced by CXCR4 / IL10-MSC in mice with colitis, experiments were conducted according to the experimental design shown in Figure 26. After a 3-month incubation period following the first DSS treatment, a second induction test was performed using a 7-day DSS cycle. Injected CXCR4 / IL10-MSC induced a significant reduction in DAI (Figure 28A) and a less significant decrease in body weight (Figure 28B) and survival rate (Figure 28C) compared to non-MSC-injected mice with colitis.

[0083] These data demonstrate that CXCR4 / IL10-MSCs exhibit enhanced immunomodulatory properties compared to WT-MSCs in a DSS-induced model of colitis, suggesting that these genetically modified MSCs may be more potent MSC-based cell therapy products for the treatment of inflammatory bowel disease compared to WT-MSCs.

Claims

1. a. Promoter and, b. Sequences encoding chemokine receptor type 4 (CXCR4), c. The sequence encoding interleukin IL-10, A DNA expression cassette, including...

2. The expression cassette according to claim 1, characterized in that the sequence encoding the chemokine receptor type 4 (CXCR4) is sequence number 1, and the sequence encoding the interleukin IL-10 is sequence number 3.

3. The expression cassette according to claim 1 or 2, further comprising a regulatory element for increasing the expression of the introduced gene.

4. The expression cassette according to any one of claims 1 to 3, wherein the regulatory element is a woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof.

5. The expression cassette according to any one of claims 1 to 4, further comprising a sequence encoding an autocatalytic peptide between the sequence encoding the chemokine receptor type 4 (CXCR4) and the sequence encoding the interleukin IL-10.

6. The expression cassette according to any one of claims 1 to 5, wherein the autocatalytic peptide is E2A, preferably SEQ ID NO:

2.

7. The expression cassette according to any one of claims 1 to 6, wherein the promoter is a human phosphoglycerate kinase (PGK) promoter sequence or a functional homolog or variant thereof.

8. In order from 5' to 3', a. Human phosphoglycerate kinase (PGK) promoter sequence or its functional homolog or variant, b. The sequence encoding the chemokine receptor type 4 (CXCR4), c. The sequence encoding the autocatalytic peptide E2A, d. The sequence encoding interleukin IL-10, e. Woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs), An expression cassette according to any one of claims 1 to 7, including the following:

9. In order from 5' to 3', a. Human phosphoglycerate kinase (PGK) promoter sequence or its functional homolog or variant, b. Sequence ID No. 1, which encodes the chemokine receptor type 4 (CXCR4), c. Sequence ID No. 2, which encodes the self-catalyzed peptide E2A, d. Sequence ID 3, which codes for interleukin IL-10, e. Woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs), An expression cassette according to any one of claims 1 to 8, comprising:

10. A recombinant gene delivery vector comprising an expression cassette according to any one of claims 1 to 9.

11. The recombinant gene delivery integration vector according to claim 10, characterized in that it is a lentiviral vector.

12. A cell comprising an expression cassette according to any one of claims 1 to 9, or a recombinant gene delivery vector according to claim 10 or 11.

13. The cells according to claim 12, characterized in that they are mesenchymal stem cells derived from bone marrow, placenta, umbilical cord, amniotic membrane, menstrual blood, peripheral blood, salivary glands, skin and foreskin, synovial fluid, amniotic fluid, endometrium, adipose tissue, umbilical cord blood, and / or dental tissue.

14. A pharmaceutical composition comprising a recombinant gene delivery embedding vector according to claim 10 or 11, or cells according to claim 12 or 13, and optionally a pharmaceutically acceptable additive or carrier.

15. A gene delivery integration vector according to claim 10 or 11, or a cell according to claim 12 or 13, which is for pharmaceutical use.

16. A gene delivery vector according to claim 15, 10, or 11, or a cell according to claim 12 or 13, for the treatment of an inflammatory disease.

17. A gene delivery integration vector according to claim 15, 16, 10, or 11, or a cell according to claim 12 or 13, for the treatment of graft-versus-host disease (GvHD), sepsis, rheumatoid arthritis, or inflammatory bowel disease.

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