Genetically modified mesenchymal stem cells expressing alpha-1 antitrypsin (aat)

Abstract

The present invention relates to genetically modified mesenchymal stem cells for use as a medicament in the treatment of a medical condition associated with inflammation and / or an unwanted immune response in a subject who does not have alpha-1 antitrypsin (AAT) deficiency, wherein the stem cells comprise an exogenous nucleic acid comprising (i) an alpha-1 antitrypsin (AAT) coding region operably linked to (ii) a promoter or a promoter / enhancer combination.

Description

Technical Field

[0001] This invention relates to genetically modified mesenchymal stem cells used as a medicament for treating medical conditions associated with inflammation and / or unwanted immune responses in subjects without α-1 antitrypsin (AAT) deficiency, wherein the stem cells comprise exogenous nucleic acids comprising (i) an α-1 antitrypsin (AAT) coding region operatively linked to (ii) a promoter or promoter / enhancer combination. Background Technology

[0002] Mesenchymal stem cells (MSCs) are non-hematopoietic cells found in bone marrow and other tissues. MSCs are generally considered pluripotent adult progenitor cells with the ability to differentiate into a limited number of cell lineages such as osteoblasts, chondrocytes, and adipocytes. Based on this ability to directly differentiate into these terminal phenotypes, the use of MSCs as therapeutic entities has been investigated, including their potential to promote or enhance bone repair and for repairing cartilage defects (Vilquin and Rosset, Regenerative Medicine 2006:1,4, p 589 and Veronesi et al., Stem Cells and Development 2013;22, p 181). The isolation and culture of MSCs for numerous therapeutic indications have been described, and this represents a promising approach for treating inflammation-related disorders (e.g., WO 2010 / 119039).

[0003] MSCs are known to exhibit immune escape properties after administration to patients. They have been shown to demonstrate favorable immunomodulatory effects in the case of transplantation of allogeneic donor material (Le Blanc et al., Lancet 2004:363, p 1439), thereby reducing potentially pathogenic allogeneic reactivity and rejection. Furthermore, MSCs are known to have antitumor effects, such as against Kaposi's sarcoma (Khakoo et al., J Exp Med 2006:203, p 1235). MSC therapy can also play a therapeutic role in wound healing. MSCs can be delivered therapeutically via systemic injection, followed by homing and migration to the site of injury (Kidd et al., Stem Cells 2009:27, p 2614). While the regenerative effects of MSCs on injured tissue are evident, their use as delivery vectors for therapeutic transgenic proteins of interest has not been fully explored.

[0004] Inflammatory diseases and diseases associated with unwanted immune responses represent a significant cause of health problems and mortality worldwide. For example, inflammatory diseases (such as lung diseases or autoimmune diseases) represent medical conditions where improved inflammation regulation is needed to provide effective treatment.

[0005] Lung diseases (such as respiratory diseases) can severely impact a patient's health and encompass a wide range of pathological conditions affecting the organs and tissues of the lungs. As an example of lung diseases, respiratory diseases include conditions of the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, pleura and pleural cavity, as well as the nerves and muscles involved in breathing. Respiratory diseases are among the leading causes of death worldwide. Lung infections (such as pneumonia and tuberculosis), lung cancer, and chronic obstructive pulmonary disease (COPD) caused 9.5 million deaths worldwide in 2008, accounting for one-sixth of all deaths globally. According to the World Health Organization, four respiratory disease categories appear among the top ten causes of death globally, accounting for one-sixth of all deaths and one-tenth of lost disability-adjusted life-years. In the 28 member states of the European Union, these diseases account for one-eighth of all deaths. To reduce inflammation, treatment often involves the use of anti-inflammatory drugs (such as corticosteroids). Given the prevalence of lung diseases, new treatment strategies are needed.

[0006] Autoimmune diseases are medical conditions associated with an individual's immune response against the cells, substances, or tissues of their body. A small percentage of the general population suffers from autoimmune diseases, which can be acute or chronic and often lead to prolonged deterioration. In both autoimmune and inflammatory diseases, such as autoimmune diseases targeting the body's own tissues or proteins, the medical condition typically arises from a response of the human adaptive and / or innate immune systems.

[0007] A significant example of an autoimmune disease is type 1 diabetes. Type 1 diabetes accounts for 5% to 10% of all diabetes cases. Globally, approximately 80,000 young adults are diagnosed with type 1 diabetes each year, and an estimated 3 million people in the United States have it (Chiang et al., Diabetes Care 2014:37, pp 2034-55). In patients with type 1 diabetes, pancreatic beta cells are destroyed by an autoimmune response involving beta cell autoantigens, macrophages, dendritic cells, B lymphocytes, and T lymphocytes. Because these patients lack pancreatic beta cells, insulin production is reduced, leading to elevated glucose levels in the blood and urine. Current treatments typically rely on regulating insulin levels rather than directly addressing the underlying immune system pathology.

[0008] An important example of an inflammatory disease is arthritis, such as gout (gouty arthritis). Gout is a medical condition characterized by recurrent, acute inflammatory arthritis, typically in a specific location, causing swelling and joint pain. The metatarsophalangeal joint at the base of the big toe is a commonly affected area. Gout is generally recognized as being caused by elevated levels of uric acid in the blood, leading to uric acid crystals that deposit in the joints, tendons, and surrounding tissues of the patient's body. Treatment typically involves the use of anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs) or corticosteroids, or medications that inhibit uric acid production.

[0009] Many anti-inflammatory agents are known, such as steroids and nonsteroidal agents, for treating conditions like arthritis. However, many of these medications frequently cause unwanted side effects, especially with prolonged use. The need for further anti-inflammatory agents is evident, particularly in cases of inflammatory or autoimmune diseases that respond poorly to mild or short-term treatment and / or cause unwanted side effects in patients requiring long-term treatment.

[0010] α-1 antitrypsin (also known as A1AT, AAT, PI, SERPINA1) is a glycoprotein of approximately 52 kDa and is one of the most abundant endogenous serine protease inhibitors (SERPIN superfamily). AAT is considered an acute-phase protein, and its concentration can increase many times over during acute inflammation.

[0011] Alpha-1 antitrypsin deficiency (A1AD) is a medical disorder that causes a defect in the production of alpha-1-antitrypsin (A1AT), resulting in reduced A1AT activity in the blood and lungs and excessive deposition of abnormal A1AT protein in hepatocytes. More than 75 different known mutations exist in SERPINA1, and 90% of A1AD cases are due to the PI*Z missense mutation: Glu342Lys. AAT deficiency leads to chronic, uninhibited tissue breakdown, causing neutrophil elastase to freely break down alveolar interstitial elastin, resulting in respiratory complications such as airway inflammation and emphysema.

[0012] Despite the development of gene therapy methods for recombinant functional AAT, treating AAT deficiency remains a challenge. For example, US2014 / 0142161A describes a gene therapy method for treating AAT deficiency by using a recombinant adeno-associated virus (rAAV)-based vector to enhance AAT expression.

[0013] Li et al. (J Hepatol 2011:54, pp 930-8), Ghaedi et al. (Tissue and Cell 2010:42, pp 181-9), and Li et al. (Mol Ther 2010:18, pp 1553-8) described genetically modified mesenchymal stem cells expressing AAT and discussed their potential use in treating AAT deficiency, particularly in treating liver diseases. Given the severity of diseases caused by A1AD, novel strategies are needed to enhance AAT production in affected subjects.

[0014] Despite recent preliminary progress in treating AAT deficiency, the therapeutic potential of AAT in cell therapy remains largely unexplored in subjects without AAT deficiency. Ghaedi et al. (J Gene Med 2011:13, pp 171-80) demonstrated the cytotoxic effects of AAT-expressing mesenchymal stem cells on human umbilical vein endothelial cells (HUVECs) and discussed the potential use of said mesenchymal stem cells as an inhibitor of angiogenesis. Based on the inventors' understanding, the application of transgenic AAT expression via MSCs in cell therapy for inflammatory or autoimmune diseases has not been previously proposed. Summary of the Invention

[0015] In view of the prior art, the technical problem based on the present invention is to provide alternative and / or improved means of treating inflammatory diseases and diseases associated with unwanted immune responses.

[0016] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0017] Therefore, the present invention relates to genetically modified mesenchymal stem cells used as a medicament for treating medical conditions associated with inflammation and / or unwanted immune responses in subjects without α-1 antitrypsin (AAT) deficiency, wherein the stem cells comprise exogenous nucleic acids containing (i) an α-1 antitrypsin (AAT) coding region operatively linked to (ii) a promoter or a promoter / enhancer combination. Such MSCs may be referred to as “AAT-modified MSCs”.

[0018] In a preferred embodiment, the present invention relates to genetically modified mesenchymal stem cells (AAT-modified MSCs) as described herein, said genetically modified mesenchymal stem cells being used as a drug to treat medical conditions associated with inflammation and / or unwanted immune responses in subjects without α-1 antitrypsin (AAT) deficiency, wherein the medical conditions are selected from the group consisting of inflammatory lung diseases, gout, chronic fibrosis, and autoimmune diseases (especially type 1 diabetes).

[0019] By unifying the various medical indications for treatment with AAT-modified MSCs according to the invention (especially inflammatory diseases of the lungs, gout, chronic fibrosis, and autoimmune diseases (especially type 1 diabetes)) through the common characteristics of inflammation and / or unwanted immune responses in subjects without α-1 antitrypsin (AAT) deficiency. Considering that the use of AAT-modified MSCs has not previously been proposed for the treatment of medical diseases unrelated to α-1 antitrypsin (AAT) deficiency, the various uses of the invention represent a unified group of medical indications.

[0020] What is particularly surprising is that AAT-modified MSCs were able to treat medical conditions associated with unwanted inflammation and / or immune responses in subjects who did not show AAT deficiency.

[0021] AAT supplementation for subjects with AAT deficiency has been proposed, and such supplementation appears to represent a useful approach to treating many pathological conditions caused by reduced functional AAT levels. However, it has not previously been proposed that increased local delivery of AAT to sites of inflammation via MSC-mediated delivery could provide a therapeutic option for unwanted inflammatory or immune responses in patients with functional AAT. Instead, those skilled in the art might assume that administering MSC-AAT to subjects without AAT deficiency could lead to potential side effects due to AAT overdose. Furthermore, the beneficial effects achieved by the present invention, based on AAT replacement therapy for subjects with AAT deficiency, would not be considered obvious, as the provision of excessive AAT would not be presumed to provide a therapeutic benefit in patients with functional AAT.

[0022] According to the present invention, the term α1-antitrypsin deficiency (A1AD) refers to any condition that results in a deficiency of α1-antitrypsin (A1AT), such as a genetic disorder that leads to decreased A1AT activity in the blood and lungs, and the deposition of multiple abnormal A1AT proteins in hepatocytes. For example, AAT deficiency can be characterized by one or more of 75 known different mutations in SERPINA1, thereby 90% of A1AD cases are due to the PI*Z missense mutation: Glu342Lys.

[0023] Therefore, subjects without such AAT deficiency are the preferred prospective subjects for the medical treatments described in this article.

[0024] Due to their ability to migrate to inflamed areas, MSCs represent a suitable tool for delivering therapeutic agents. Without being bound by theory, the MSCs of this invention represent drug delivery carriers for the efficient delivery of therapeutic agents (i.e., AAT protein) expressed in said MSCs by exogenous nucleic acids. Furthermore, the combination of MSCs and AAT provides an unexpected synergistic effect. MSCs themselves exhibit beneficial anti-inflammatory properties regarding inflamed areas after homing and / or transplantation into inflamed tissues, and when MSCs are conjugated with AAT as a therapeutic transgene, MSCs provide enhanced anti-inflammatory function.

[0025] Surprisingly, systemic administration, preferably intravenous administration, of the MSCs described herein leads to MSC migration to sites of inflammation. In addition to the enhanced local anti-inflammatory effect of AAT from the expression of transgenes present in AAT-modified MSCs, MSCs are able to migrate and / or transplant into areas of inflamed tissue, thereby providing anti-inflammatory signals from within the MSCs themselves. The MSCs used in this invention can also circulate in the bloodstream after systemic or intravenous administration and can transplant or bind to inflamed areas in the body, thereby functioning locally. The combination of MSCs showing increased AAT expression (due to AAT transgenes) in the treatment of medical conditions defined by unwanted inflammation and / or unwanted immune responses provides an unexpected synergistic effect, whereby MSCs homing to areas of elevated inflammation provide a greater synergistic therapeutic effect than the sum of each independent effect when considered individually, in addition to the anti-inflammatory properties of both the MSCs themselves and the AAT transgenes.

[0026] For example, when AAT is administered systemically as a therapeutic protein or as a viral vector in gene therapy, only limited benefits are obtained due to the lack of targeting of inflamed areas in vivo and the potential for unwanted off-target effects. When MSCs are administered systemically, some positive effects can be obtained from the MSCs themselves because they are localized to inflamed tissues, although the intensity of the anti-inflammatory response induced by unmodified MSCs may not be sufficient to demonstrate a significant therapeutic effect. Combinations of MSCs expressing AAT as a transgene under constitutive or conditional promoters can synergistically combine the inherent properties of AAT and MSCs in a targeted and effective manner. For example, the local effects of AAT are beneficial for treating various inflammatory diseases, such as those characterized by unwanted immune responses. AAT can protect tissues from enzymes of inflammatory cells and can reduce the production of inflammatory cytokines.

[0027] Local delivery of excess AAT via MSCs provides the desired therapeutic benefit in patients without AAT deficiency, while minimizing potential side effects from excessive AAT due to off-target effects. In this process, the immunomodulatory properties of MSCs synergize with the anti-inflammatory effects of AAT to produce a therapeutically effective dose of AAT. Thus, AAT-modified MSCs avoid and / or minimize potential side effects resulting from systemic administration of AAT proteins or nucleic acid carriers encoding AAT. Due to the homing ability of MSCs to inflamed tissues, using MSCs as a carrier for AAT administration provides local production of AAT in the diseased area of ​​the body.

[0028] The AAT coding region is preferably any nucleic acid encoding a naturally occurring or synthetic AAT protein sequence that exhibits AAT function, having reduced, identical, similar, or increased activity compared to human AAT, or is functionally similar to AAT. The amino acid sequence of AAT is available from NCBI database accession number 1313184B. Those skilled in the art of molecular biology or genetics can provide the corresponding nucleic acid sequence encoding AAT. This invention covers the use of sequence variants of AAT that exhibit functional similarity to the unmodified human form of AAT.

[0029] The SERPINA1 coding sequence (CDS) published at http: / / www.ncbi.nlm.nih.gov / nuccore / NM_000295.4 is a preferred embodiment, and it includes bases 262 to 1518 of the full sequence (SEQ ID NO1):

[0030]

[0031] In some embodiments of the present invention, the CDS is codon-optimized to improve protein production. The codon-optimized coding sequence is preferably read as follows (SEQ ID NO 2):

[0032]

[0033] GGCATCACCAAGGGTTCAGCAACGGCGCCGATCTGAGCGGCGTGACAGAAGAGGCCCCTCTGAAGCTGTCCAAGGCCGTGCACAAAGCCGTGCTGACCATCGACGAGAAGGGCACCGAAGCCGCTGGCGCC ATGTTTCTGGAAGCCATCCCCATGAGCATCCCCCCTGAAGTGAAGTTCAACAAGCCCTTCGTGTTCCTGATGATCGAGCAGAACACCAAGAGCCCCCTGTTCATGGGCAAGGTCGTGAACCCCACCCAGAAA

[0034] The nucleotide sequence of SEQ ID NO 1 and / or 2 encodes the human AAT protein according to the amino acid sequence of SEQ ID NO 3, which is preferred in this invention:

[0035] MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGL NFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALV NYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLP KLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

[0036] Therefore, this invention covers genetically modified MSCs as described herein, said genetically modified MSCs comprising nucleic acid molecules selected from the group consisting of:

[0037] a) A nucleic acid molecule comprising a nucleotide sequence encoding an AAT protein, such as the AAT protein according to SEQ ID NO 3, preferably encoded by a nucleotide sequence according to SEQ ID NO 1 or 2;

[0038] b) Nucleic acid molecules complementary to the nucleotide sequence according to a);

[0039] c) A nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity, wherein the nucleotide sequence having sufficient sequence identity is functionally similar to / equivalent to the nucleotide sequence according to a) or b), preferably having at least 70%, 80%, preferably 90%, more preferably 95% sequence identity with the nucleotide sequence according to a) or b).

[0040] d) As a result of the genetic code, it is degenerated into nucleic acid molecules based on the nucleotide sequences of a) to c); and

[0041] e) Nucleotide sequences modified according to a) through d) by deletion, addition, substitution, translocation, inversion and / or insertion, and functionally similar / equivalent to nucleotide sequences according to a) through d).

[0042] Therefore, this invention covers the genetically modified MSCs described herein, said genetically modified MSCs comprising a nucleotide sequence encoding the amino acid sequence according to SEQ ID NO 3. This invention further covers sequence variants of SEQ ID NO 3, particularly sequence variants having at least 70% sequence identity with SEQ ID NO 3, preferably sequence variants having at least 80%, 85%, 90%, or at least 95% sequence identity with SEQ ID NO 3. Such sequence variants are preferably functionally similar to or equivalent to the human AAT disclosed herein. This invention also covers variations in protein length while maintaining functional equivalence to the human AAT of SEQ ID NO 3. For example, truncation or elongation of protein lengths up to 50 amino acids, 40, 30, 20, or 10 amino acids can maintain AAT activity and are therefore covered in this invention.

[0043] Functionally similar sequences are those encoding functional AAT gene products and capable of achieving the same or similar functional effects as human AAT. AAT function can be determined by its ability to inhibit various proteases (such as trypsin) in vitro or by its ability to inhibit neutrophil elastase (described below). Appropriate assays for determining protease activity or for determining neutrophil elastase activity are known to those skilled in the art.

[0044] Protein modifications of AAT proteins, which can occur through substitutions in the amino acid sequence, and the nucleic acid sequences encoding these molecules are also included within the scope of this invention. A substitution, as defined herein, is a modification of the amino acid sequence of a protein, whereby one or more amino acids are replaced by the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from the original protein. In some embodiments, this modification does not significantly alter the function of the protein. As with additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be performed without significantly altering the function of a protein. This is particularly true when the modification involves “conserved” amino acid substitutions, which are substitutions of one amino acid for another amino acid with similar properties. Such “conserved” amino acids can be natural or synthetic amino acids, and because of their size, charge, polarity, and conformation, they can be substituted without significantly affecting the structure and function of the protein. Typically, many amino acids can be substituted with conserved amino acids without adversely affecting the function of the protein. Generally, the groups of conserved amino acids are: nonpolar amino acids Gly, Ala, Val, Ile, and Leu; nonpolar aromatic amino acids Phe, Trp, and Tyr; neutral polar amino acids Ser, Thr, Cys, Gln, Asn, and Met; positively charged amino acids Lys, Arg, and His; and negatively charged amino acids Asp and Glu. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys can be interchanged, even if they belong to different groups.

[0045] Methods for genetically modified MSCs are known to those skilled in the art. Examples of suitable methods for genetically modified MSCs are disclosed in WO 2010 / 119039 and WO2008 / 150368.

[0046] In one embodiment, the genetically modified cells as described herein are characterized in that the cells are obtained from bone marrow, umbilical cord, adipose tissue, or amniotic fluid.

[0047] In one embodiment, the genetically modified cells as described herein are characterized in that the cells are CD34 negative.

[0048] In one embodiment, the genetically modified cell as described herein is characterized in that the cell is a human cell.

[0049] In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized in that the exogenous nucleic acid comprises a viral vector, such as a viral vector in the form of a viral expression construct, more preferably a retroviral vector, particularly a gamma retroviral vector.

[0050] In one embodiment, genetically modified mesenchymal stem cells as described herein are characterized in that the exogenous nucleic acid is or includes a non-viral expression construct.

[0051] In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized in that the cells further include (iii) a selective marker gene operatively linked to a constitutive promoter or a promoter / enhancer combination.

[0052] In one embodiment, genetically modified mesenchymal stem cells as described herein are characterized in that the promoter or promoter / enhancer combination is a constitutive promoter.

[0053] In another embodiment, genetically modified mesenchymal stem cells as described herein are characterized in that the promoter or promoter / enhancer combination is a CMV or EF2 promoter.

[0054] In a preferred embodiment, the genetically modified mesenchymal stem cells as described herein are characterized by having an EFS promoter as their constitutive promoter.

[0055] In a preferred embodiment, the genetically modified mesenchymal stem cells as described herein are characterized by having a constitutive promoter that is the PGK promoter.

[0056] In a preferred embodiment, the genetically modified mesenchymal stem cells as described herein are characterized by having an EF1α promoter as their constitutive promoter.

[0057] In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized in that the promoter or promoter / enhancer combination is an inducible or conditional promoter.

[0058] In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized in that the promoter is inducible upon differentiation of the cells after administration. In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized in that the promoter is the Tie2 promoter.

[0059] In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized by an inflammation-specific promoter, preferably wherein the promoter is induced by inflammatory mediators or inflammatory cytokines and / or when the genetically modified mesenchymal stem cells are near inflamed tissue.

[0060] The inducible form of the promoter was designed to exhibit inflammation-specific and / or localized expression of the AAT protein. This, combined with the homing and / or migratory properties of MSCs, resulted in a synergistic effect that allowed very small amounts of AAT protein to be expressed or produced in regions of the subject distinct from diseased tissues or organs.

[0061] In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized by having a RANTES promoter.

[0062] In one embodiment, the genetically modified mesenchymal stem cells as described herein are characterized by having an HSP70 promoter.

[0063] Surprisingly, given the existing technology, expression of the inducible promoter described herein leads to adequate expression of the therapeutic protein AAT upon appropriate stimulation of the inflammatory site. The promoter provided herein exhibits suitable inducible properties to rapidly and strongly express AAT upon proximity to inflamed tissue. Furthermore, the anti-inflammatory and immunomodulatory effects of AAT on tissue are particularly pronounced when AAT expression is controlled by the said inducible promoter. This results in a synergistic effect, thereby making the treatment of patients with medical conditions associated with unwanted inflammatory and / or immune responses particularly effective when administered genetically modified MSCs expressing AAT under Tie2 and / or RANTES control.

[0064] In a further aspect, the present invention relates to AAT-modified cells themselves, and not to specific medical applications. In this regard, the various structural features of AAT-modified cells described herein, such as the transgenic sequences, promoters, additional vector components, and combinations thereof, as described below and in the examples, represent a contribution to the prior art not previously described.

[0065] In a further aspect, the present invention relates to genetically modified mesenchymal stem cells used as medicines as described herein.

[0066] In one embodiment, genetically modified mesenchymal stem cells used as a drug as described herein are characterized by administering the cells by introducing a therapeutically effective number of cells into the patient's bloodstream.

[0067] In a further aspect, the present invention relates to genetically modified mesenchymal stem cells used as medicaments, as described herein, for the treatment of lung diseases.

[0068] In one embodiment, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the lung disease is an inflammatory disease of the lung.

[0069] In one embodiment, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the lung disease is a respiratory disease.

[0070] In a further embodiment, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the lung disease is acute lung injury, chronic obstructive pulmonary disease (including chronic bronchitis, emphysema, bronchiectasis, and bronchiolitis), acute respiratory distress syndrome, asthma, sarcoidosis, allergic pneumonia, and / or pulmonary fibrosis.

[0071] In one embodiment, genetically modified mesenchymal stem cells used as a drug as described herein are characterized by administering the cells by introducing a therapeutically effective number of cells into the patient's lungs via inhalation, optionally combined with introducing the cells into the patient's bloodstream.

[0072] The lung diseases described in this invention may include, but are not limited to, one or more of the following: acute bronchitis, acute respiratory distress syndrome (ARDS), asbestosis, asthma, bronchiectasis, bronchiolitis obliterans with organizing pneumonia (BOOP), bronchopulmonary dysplasia, cottonseed deposition, chronic bronchitis, coccidiasis (Cocci), COPD, cryptogenic organizing pneumonia (COP), cystic fibrosis, emphysema, Hantavirus pulmonary syndrome, histoplasmosis, human metapneumovirus, allergic pneumonia, influenza, lymphangioma, mesothelioma, Middle East respiratory syndrome, nontuberculous mycobacterial infection, pertussis, pneumoconiosis (black lung disease), pneumonia, primary ciliary dyskinesia, primary pulmonary hypertension, pulmonary hypertension, pulmonary fibrosis, pulmonary vascular disease, respiratory syncytial virus, sarcoidosis, severe acute respiratory syndrome, silicosis, sleep apnea, sudden infant death syndrome, or tuberculosis.

[0073] In a preferred embodiment, the lung disease is selected from inflammatory lung diseases or restrictive lung diseases, respiratory infections and / or pulmonary vascular diseases or conditions.

[0074] Inflammatory lung diseases are typically characterized by high neutrophil counts, such as asthma, cystic fibrosis, emphysema, chronic obstructive pulmonary disease, or acute respiratory distress syndrome. Restrictive lung diseases are a group of respiratory disorders characterized by loss of lung compliance, leading to incomplete lung expansion and increased lung stiffness, such as in infants with respiratory distress syndrome.

[0075] Respiratory infections can affect any part of the respiratory system. They are conventionally categorized as upper respiratory tract infections and lower respiratory tract infections. The most common upper respiratory tract infection is the common cold. However, specific organ infections of the upper respiratory tract, such as sinusitis, tonsillitis, otitis media, pharyngitis, and laryngitis, are also considered upper respiratory tract infections. The most common lower respiratory tract infection is pneumonia, which in Western countries is usually caused by bacteria, particularly Streptococcus pneumoniae, in the lungs. Worldwide, tuberculosis is a major cause of pneumonia. Other pathogens such as viruses and fungi can cause pneumonia, such as severe acute respiratory syndrome and Pneumocystis pneumonia. Pneumonia can lead to complications such as lung abscess (a round cavity in the lung caused by infection) or may spread to the pleural cavity.

[0076] One aspect of this invention relates to the treatment of vascular diseases using genetically modified AAT-modified MSCs as described herein. Pulmonary vascular diseases are conditions affecting pulmonary circulation. An example is pulmonary embolism, which is a blood clot (thromboembolism) that forms in a vein, detaches, passes through the heart, and remains in the lungs. Large pulmonary embolisms are fatal, leading to sudden death. Many other substances can also cause pulmonary embolisms (through blood flow), but these are rare, such as fat embolisms (especially after bone injury), amniotic fluid embolisms (with childbirth complications), or air embolisms (iatrogenic – caused by invasive medical procedures). According to the invention, the following lung diseases can also be treated: pulmonary hypertension, i.e., elevated blood pressure in the pulmonary arteries; pulmonary edema, fluid leakage from pulmonary capillaries into the alveoli (or air chambers); pulmonary hemorrhage, inflammation, and pulmonary capillary damage leading to blood leakage into the alveoli.

[0077] The genetically modified mesenchymal stem cells described in this article can also be used as a medicine to treat medical conditions related to α1-antitrypsin deficiency (A1AD) in patients with or without α1-antitrypsin deficiency.

[0078] Examples of medical conditions associated with α1-antitrypsin deficiency include cirrhosis, COPD, pneumothorax, asthma, Wegener's granulomatosis, pancreatitis, gallstones, bronchiectasis, pelvic organ prolapse, primary sclerosing cholangitis, autoimmune hepatitis, emphysema, α1-antitrypsin deficiency primarily involving the lower lobes and causing bullae, and secondary membranoproliferative glomerulonephritis.

[0079] In a further aspect, the present invention relates to genetically modified mesenchymal stem cells used as medicaments for treating inflammatory diseases as described herein.

[0080] In a further embodiment, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the inflammatory disease is vasculitis, nephritis, inflammatory bowel disease, rheumatoid arthritis, and / or graft-versus-host disease.

[0081] In one embodiment, the genetically modified mesenchymal stem cells described herein as a drug are characterized by treating an inflammatory disease called gout. Gout is a medical condition characterized by recurrent acute inflammatory arthritis, typically presenting as swelling and pain in a specific joint. The metatarsophalangeal joint at the base of the big toe is a commonly affected area. Gout is generally recognized as being caused by elevated levels of uric acid in the blood, leading to uric acid crystals that deposit in the joints, tendons, and surrounding tissues of the patient's body. In some cases, gout may manifest as tophi, kidney stones, or uric acid nephropathy.

[0082] Surprisingly, the AAT-modified MSCs of the present invention can be an effective treatment option for gout, resulting in a reduction of swelling and pain in the affected area. Successful treatment can be observed through reduction of inflammation and uric acid crystals in the synovial fluid. In a further aspect, the present invention relates to genetically modified mesenchymal stem cells used herein as a medicament for treating chronic fibrosis.

[0083] In one embodiment, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the inflammatory fibrotic disease and / or chronic fibrotic disease is a fibrotic disease of the subject's kidneys, liver, and / or colon.

[0084] Fibrosis is generally considered to be the formation of excessive fibrous connective tissue in an organ or tissue. Fibrosis can be a reactive, benign, or pathological condition. Unwanted deposits of connective tissue can strip away the structure and function of the underlying organ or tissue, leading to a pathological condition. Fibrosis can occur in many tissues throughout the body, often due to inflammation or damage potentially associated with unwanted inflammatory and / or immune responses. Examples of fibrosis include pulmonary fibrosis (pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis), liver fibrosis (cirrhosis), cardiac fibrosis (endocardial fibrosis, myocardial infarction in the elderly, atrial fibrosis) or fibrosis from other causes (mediastinal (mediastinal soft tissue) fibrosis), myelofibrosis (bone marrow), retroperitoneal fibrosis (retroperitoneal soft tissue), progressive massive fibrosis (lung); complications of pneumoconiosis in coal miners, mephrogenic systemic fibrosis (skin), Crohn's disease (intestine), keloids (skin), scleroderma / systemic sclerosis (skin, lung), joint fibrosis (knee, shoulder, other joints), Dupuytren's contracture (hand, fingers) or adhesive capsulitis (shoulder)).

[0085] Surprisingly, local expression of AAT in fibrotic areas may lead to enhanced therapeutic effects. MSCs can exhibit unexpected migration properties toward fibrotic tissue and result in a reduction in fibrotic tissue formation through AAT expression. In this approach, AAT-MSCs are specifically used as anti-inflammatory and / or immunomodulatory therapeutic agents.

[0086] The use of AAT-modified MSCs for the treatment of type 1 diabetes as described herein represents a further aspect of the invention. Therefore, the invention also covers the treatment of complications of type 1 diabetes. Potential complications associated with type 1 diabetes include cardiovascular disease, particularly the accelerated progression of atherosclerosis and an increased risk of heart attack and / or stroke, nephropathy, neuropathy, and retinopathy.

[0087] In one implementation, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the subject is a human.

[0088] In one embodiment, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the genetically modified cells are allogeneic relative to the subject.

[0089] In one embodiment, the genetically modified mesenchymal stem cells used as a drug as described herein are characterized in that the genetically modified cells are relative to the subject's own cells.

[0090] A further aspect of the invention relates to AAT-modified MSCs described herein for the treatment of inflammatory or autoimmune diseases such as those described herein, wherein cancer is excluded from the group of diseases to be treated.

[0091] In a further aspect of the invention, genetically modified or unmodified MSCs are typically provided as medicaments for treating lung diseases. The characteristics of the cells mentioned herein, relative to AAT-modified MSCs, also apply to further embodiments of the invention. According to this embodiment, the MSCs do not contain AAT-encoding nucleic acids. Genetic modification may be absent, or the MSCs may contain exogenous nucleic acids encoding therapeutic proteins (such as AAT). Therefore, the invention relates to the use of MSCs for treating lung diseases, depending on whether MSC modification or specific genetic modification has occurred. As described herein, the anti-inflammatory properties of MSCs lead to surprisingly good efficacy in treating lung diseases. The potential lung diseases to be treated mentioned herein, relative to treatment with AAT-modified MSCs, also apply to this specific embodiment of the invention.

[0092] In a further aspect of the invention, MSCs as described herein may comprise a combination of a foreign nucleic acid encoding the protein CXCR4 and a further nucleic acid sequence suitable for expressing said protein. CXCR4 is a cell surface chemokine receptor involved in MSC migration and is expressed on the surface of a small fraction of MSCs. CXCR4 expression has been proposed to play a role in the efficiency of MSC homing to tissue damage. Recent results have shown that CXCR4 expression in MSCs enhances the chemotactic and paracrine characteristics of cells in vitro and improves MSC homing and colonization in damaged lung tissue in vivo. The CXCR4 coding sequence may be present in the same foreign nucleic acid molecule encoding AAT or in a separate foreign nucleic acid. Multiple integrated nucleic acid constructs or cassettes may be present in the MSCs of the invention, each of which carries one or more genes of interest, such as therapeutic genes (e.g., AAT) or other genes involved in cell migration (e.g., CXCR4)). Detailed Implementation

[0093] All cited patent and non-patent documents are incorporated into this paper in their entirety by reference.

[0094] The “mesenchymal cells” disclosed herein (also referred to in some embodiments as “mesenchymal stem cells” or “MSCs”) can generate cells in connective tissue, bone, cartilage, and the circulatory and lymphatic systems. Mesenchymal stem cells are found in the mesenchyme, a part of the embryonic mesoderm composed of loosely packed spindle-shaped or star-shaped unspecialized cells. As used herein, mesenchymal stem cells include, but are not limited to, CD34-negative stem cells.

[0095] In one embodiment of the invention, the mesenchymal cells are fibroblast-like plastic-adherent cells, defined in some embodiments as pluripotent mesenchymal matrix cells, and also include CD34-negative cells.

[0096] To avoid any confusion, the term mesenchymal cells encompass pluripotent mesenchymal stromal cells, which also include a subpopulation of mesenchymal cells, MSCs, and their precursors, consisting of pluripotent or pluripotent self-renewing cells capable of differentiating into multiple cell types in vivo.

[0097] As used herein, CD34-negative cells should refer to cells that lack CD34 on their surface or express only negligible levels of CD34. Methods for isolating CD34-negative cells are described, for example, in Lange C. et al., “Accelerated and safe expansion of human mesenchymal stromal cells in animalserum-free medium for transplantation and regenerative medicine”. J. Cell Physiol. 2007, Apr. 25.

[0098] Mesenchymal cells can be distinguished from hematopoietic stem cells (HSCs) using many indicators. For example, HSCs are known to float in cultures and not adhere to plastic surfaces. In contrast, mesenchymal cells adhere to plastic surfaces. The CD34-negative mesenchymal cells of this invention adhere in cultures.

[0099] The genetically modified cells described in this article can be administered in solid, liquid, or spray form, and for such administration routes, as well as whether sterility is required when used as an injectable agent, including different types of carriers. This invention can be administered via intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrathoracic, intratracheal, intranasal, intravitreal, intravaginal, intravaginal, vaginal, intrarectal, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularly, mucosal, intraperitoneal, intraumbilical, intraocular, oral, topically, locally, inhalation (e.g., nebulized inhalation), injection, infusion, continuous infusion, local perfusion, direct washing of target cells, via catheter, via irrigation, in paste, in lipid composition (e.g., liposomes), or by other methods known to those skilled in the art or any combination thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0100] This invention covers treating a patient by introducing a therapeutically effective number of cells into the bloodstream of a subject. As used herein, introducing cells “into the bloodstream of a subject” includes, but is not limited to, introducing such cells into one of the subject’s veins or arteries by injection. Such administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods. A single injection is preferred, but in some cases, repeated injections over a period of time (e.g., quarterly, semi-annually, or annually) are necessary. Such administration is also preferably performed using a mixture of CD34-negative cells and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate-buffered saline or 0.8% saline, and commonly used specialized cryopreservation media.

[0101] It can be administered locally, for example by injection into an area of ​​the subject's body near the site of inflammation. MSCs have been shown to migrate toward inflammation. Mesenchymal stem cells (MSCs) exhibit tropism toward sites of tissue injury and the tumor microenvironment. Many of the same inflammatory mediators secreted by wounds are found in the tumor microenvironment, and these inflammatory mediators are thought to be involved in attracting MSCs to these sites. Cell migration depends on a wide range of signals, from growth factors to chemokines, secreted by damaged cells and / or responding immune cells. MSCs likely possess chemotactic properties similar to other immune cells that respond to sites of injury and inflammation. In any case, local application of the cells described herein results in high levels of cells at their site of action.

[0102] Furthermore, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions, with aqueous solutions being the most preferred. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, and suspensions, including saline and buffer media. Non-gastrointestinal carriers include sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solution, and fixed oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements such as Ringer's glucose, Ringer's glucose-based carriers, etc. Fluids commonly used for intravenous administration can usually be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808, Lippincott Williams S-Wilkins (2000). Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc., may also be present.

[0103] As used herein, “therapeutic effective number of cells” includes, but is not limited to, the following amounts and ranges: (i) approximately 1 × 10 2 To approximately 1×10 8(ii) Approximately 1 × 10⁻⁶ cells / kg body weight; 3 To approximately 1×10 7 (iii) Approximately 1 × 10⁻⁶ cells / kg body weight; 4 To approximately 1×10 6 (iv) Approximately 1 × 10⁻⁶ cells / kg body weight; 4 To approximately 1×10 5 Cells / kg body weight; (v) approximately 1×10 5 To approximately 1×10 6 Cells / kg body weight; (vi) Approximately 5 × 10 4 To approximately 0.5 × 10 5 Cells / kg body weight; (vii) Approximately 1×10 3 (viii) Approximately 1 × 10⁻⁶ cells / kg body weight; 4 Cells / kg body weight; (ix) approximately 5 × 10⁻⁶ 4 Cells / kg body weight; (x) approximately 1×10 5 Cells / kg body weight; (xi) approximately 5 × 10 5 Cells / kg body weight; (xii) approximately 1×10 6 Cells / kg body weight; and (xiii) approximately 1×10 7 Cells / kg body weight. Expected body weight includes, but is not limited to, approximately 5 kg, 10 kg, 15 kg, 30 kg, 50 kg, approximately 60 kg; approximately 70 kg; approximately 80 kg, approximately 90 kg; approximately 100 kg, approximately 120 kg, and approximately 150 kg. These numbers are based on preclinical animal and human trials and standard protocols from CD34+ hematopoietic stem cell transplantation. Monocytes (including CD34+ cells) typically contain 1:23,000 to 1:300,000 CD34-negative cells.

[0104] As used herein, “treating” a subject suffering from a disease (such as inflammation) should mean slowing, stopping, or reversing the progression of the disease. In a preferred embodiment, treating a subject suffering from a disease means ideally reversing the progression of the disease to a point where the disease itself is eliminated. As used herein, improving a disease and treating a disease are equivalent. The treatment of the present invention may also, or alternatively, involve the prophylactic application of the cells. Such prophylactic application may involve preventing any given medical condition (such as preventing inflammation) or preventing the development of said disease, thereby preventing or avoiding, in all conditions, not to be interpreted narrowly as absolute prevention. Prevention or avoidance may also involve reducing the risk of a subject developing any given medical condition, preferably for subjects at risk of developing said condition.

[0105] Generally, the term "inflammation," used in its recognized sense in the art, refers to a local or systemic protective response resulting from tissue damage, infection, or destruction, which protects the subject from harmful substances and damaged tissue. Inflammation is preferably characterized by microvascular perforation, the infiltration of blood elements into the intercellular space, and the migration of leukocytes to inflamed tissue, leading to uncontrolled pain, fever, redness, swelling, and loss of function.

[0106] Inflammation can be classified as acute or chronic. Acute inflammation is the body's initial response to a harmful stimulus and is achieved through an increased movement of plasma and white blood cells (especially granulocytes) from the blood to the damaged tissue. A series of biochemical events enable the inflammatory response to spread and mature, involving the local vascular system, immune system, and various cells within the damaged tissue. Long-term inflammation, known as chronic inflammation, leads to progressive changes in the cell types present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissue during the inflammatory process.

[0107] The term “unwanted inflammation” preferably refers to inflammation in a subject that exceeds the level of a physiologically beneficial inflammatory response and results in damage to cells, tissues and / or organs at the site of inflammation.

[0108] The term "unwanted immune response" preferably refers to a reactive alteration of a subject's immune system that has a detrimental effect on their health and may involve the stimulation and / or production of cytokines and the recruitment of immune cells. Unwanted immune responses can occur in, for example, autoimmune diseases, transplant rejection, allergies, or inflammatory diseases.

[0109] Therefore, medical conditions defined by unwanted inflammation and / or immune responses refer to a wide range of diseases and / or conditions, specifically including but not limited to vasculitis, nephritis, inflammatory bowel disease, rheumatoid arthritis, graft-versus-host disease, gouty arthritis, chronic fibrosis, inflammatory lung diseases, or autoimmune diseases.

[0110] Examples of inflammatory lung diseases include, but are not limited to, lung injury, chronic obstructive pulmonary disease (COPD) (including chronic bronchitis, emphysema, bronchiectasis, and bronchiolitis), acute respiratory distress syndrome, asthma, sarcoidosis, hypersensitivity pneumonitis, and / or pulmonary fibrosis. In some embodiments of the invention, the MSCs described herein migrate, for example locally, toward physiological locations affected by the disease condition (such as areas of inflammation) in order to provide the therapeutic effect of the MSCs.

[0111] This invention also relates to the treatment of autoimmune diseases, particularly those with inflammatory components. These diseases may also refer to rheumatic diseases.Such diseases are preferably selected from Gauden's arteritis, giant cell arteritis, familial Mediterranean fever, Kawasaki disease, polyarteritis nodosa, cutanous polyarteritis nodosa, hepatitis-associated arteritis, Behcet's syndrome, Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, connective tissue disease vasculitis, Hennoch-Schonlein purpura, cryoglobulin vasculitis, cutaneous leukocyte mitotic vasculitis, tropical aortitis, sarcoidosis, Kogan's syndrome, Wiskott-Aldrich syndrome, leprosy nodular arteritis, primary CNS vasculitis, thromboangiitis obliterans, peritumoral arteritis, urticaria, and Dego's disease. Diseases including myelodysplastic syndrome, persistent erythema raisedae, hyperimmune globulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Morbus Chronosis, ulcerative colitis, autoimmune myositis, diabetes, multiple sclerosis, Guillain-Barré syndrome, histiocytosis, osteoarthritis, atopic dermatitis, periodontitis, chronic sinusitis, psoriasis, psoriatic arthritis, microscopic colitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis media, cryoglobulinemia, Sjogren's syndrome, lupus erythematosus, aplastic anemia, myelofibroma, chronic inflammatory demyelinating polyneuropathy, and Kimura's disease. Diseases including: systemic sclerosis, chronic peri-aortic inflammation, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, idiopathic achalasia, bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, autoimmune thrombocytopenic purpura, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atrophic thyroiditis, Graves' disease, autoimmune polyglandular syndrome, autoimmune Addison's syndrome, pemphigus vulgaris, pemphigus foliaceus, herpetic dermatitis, autoimmune alopecia, vitiligo, antiphospholipid syndrome, myasthenia gravis, stiff-person syndrome, Goodpasser syndrome, sympathetic ophthalmia, folliculitis, Sharp syndrome and / or Evans syndrome.

[0112] As used herein, “cell migration” is intended to refer to the movement of cells toward a specific chemical or physical signal. Cells typically migrate in response to specific external signals, including chemical and mechanical signals. Chemotaxis is one example of cell migration in response to chemical stimuli. In vitro chemotaxis assays, such as Boyden chamber assays, can be used to determine whether cell migration is occurring in any given cell. For example, cells of interest can be purified and analyzed. Chemotaxis assays can be performed using plates (e.g., according to Falk et al., 1980 J. Immuno. Methods 33:239-247), in which specific chemical signals are placed relative to the cells of interest and migrating cells, and then collected and analyzed. For example, Boyden chamber assays require the use of chambers separated by filters, which serve as tools for accurately determining chemotactic behavior. The pioneering type of these chambers was constructed by Boyden (Boyden (1962), "The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes". J Exp Med 115(3):453). Motile cells are placed in the upper chamber while a fluid containing the test substance is overfilled into the lower chamber. The size of the motile cells to be studied determines the pore size of the filter; choosing a diameter that allows for active migration is necessary. For modeling under in vivo conditions, several protocols preferably use filters covered with extracellular matrix molecules (collagen, elastin, etc.). The validity of measurements is increased by developing porous chambers (e.g., NeuroProbe), where 24, 96, and 384 samples can be evaluated in parallel. The advantage of this variant is that it allows for the determination of several parallel samples under identical conditions.

[0113] Alternatively, tissue samples can be obtained from the subject (e.g., a rodent model) after cell transplantation, and the presence of cells of interest in a specific tissue type within said tissue sample can be determined. Such assays may be molecular in nature, i.e., based on nucleic acid sequence recognition of cells, or histological in nature, i.e., based on fluorescent labeling followed by antibody labeling to assess cells. This assay is particularly useful for evaluating the transplantation of transplanted cells. Assays of the graft can also provide information about cell migration, as transplantation depends to some extent on pre-transplant cell localization.

[0114] In some embodiments of the invention, as described herein, MSCs are transplanted into physiological locations affected by disease conditions (such as areas of inflammation) in order to impart therapeutic effects to the MSCs, for example, in a local manner.

[0115] As used in this article, “infiltration” refers to the process of incorporating grafted or transplanted tissues or cells into a host body. Infiltration also involves the integration of transplanted cells into host tissues and their survival and, under certain conditions, differentiation into a non-stem cell state.

[0116] Techniques used to evaluate MSC inoculation and thus the migration and biodistribution of MSCs to some extent can encompass in vivo or in vitro methods. Examples of in vivo methods include bioluminescence, whereby cells are transduced to express luciferase, which can then be imaged by their luciferin metabolism, resulting in light emission; fluorescence, whereby cells are loaded with fluorescent dyes or transduced to express fluorescent reporter molecules that can then be imaged; and radiolabeling, where cells are loaded with radionuclides and located using scintigraphy, positron emission tomography (PET) or single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI), where cells loaded with paramagnetic compounds (e.g., iron oxide nanoparticles) are tracked using an MRI scanner. In vitro methods for evaluating biodistribution include quantitative PCR, flow cytometry, and histological methods. Histological methods include tracking fluorescently labeled cells; in situ hybridization of, for example, Y chromosome and human-specific ALU sequences; and histochemical staining for species-specific or genetically introduced proteins (such as bacterial β-galactosidase). These immunohistochemical methods are used to identify inoculation sites, but tissue resection is necessary. For a further review of these methods and their applications, see Kean et al., MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation, StemCells International, Volume 2013 (2013).

[0117] Therefore, progenitor cells or pluripotent cells, such as the mesenchymal cells of the present invention, can be described as protein delivery carriers, essentially capable of localizing and expressing therapeutic gene products in specific tissues or regions within a subject's body. Such therapeutic cells offer the ability to provide cell therapy for other therapeutically refractory diseases. For each type of therapeutic cell, the ultimate goal is the same: the cells should express a full range of specific genes, preferably exogenous nucleic acids encoding therapeutic gene products, thereby modifying cell consistency to express said gene products, and providing therapeutic effects, such as anti-inflammatory effects. When the cells of the present invention are expanded in vitro, they exhibit a heterologous population comprising multiple generations of mesenchymal (stromal) cell progeny lacking expression of most differentiation markers such as CD34. These populations may retain limited proliferative potential and responsiveness along both mesenchymal and non-mesenchymal lineages of terminal differentiation and maturation.

[0118] As used herein, "inducible expression" or "conditional expression" refers to a state, multiple state, or system of gene expression in which the gene of interest, such as a therapeutic transgene, is preferably not expressed, or in some embodiments, expressed at negligible or relatively low levels, unless one or more molecules (inducers) or other set of conditions that allow gene expression are present in the cell. An inducible promoter can refer to a naturally occurring promoter that is expressed at a relatively high level under specific biological conditions, or to other synthetic promoters that include any given inducible element. An inducible promoter can refer to a promoter induced by a specific tissue or microenvironment or a combination of biological signals present in a specific tissue or microenvironment, or to a promoter induced by external factors, such as by the administration of small drug molecules or other externally applied signals.

[0119] As used herein, “close to” tissue includes, for example, within 5 mm, 1 mm, 0.5 mm, and 0.25 mm of tissue.

[0120] Given that stem cells can exhibit selective migration to different tissue microenvironments under both normal and pathological conditions, this invention covers the use of tissue-specific promoters associated with differentiation pathways initiated in recruited stem cells, and these tissue-specific promoters can theoretically be used to drive selective expression of therapeutic genes only within a defined biological environment. Stem cells recruited to other tissue sites but not undergoing the same differentiation process should not express therapeutic genes. This method enables potentially significant control over the selective expression of therapeutic genes within a defined microenvironment, and has been successfully applied to regulate therapeutic gene expression during angiogenesis. Possible methods of such gene modification are disclosed in WO 2008 / 150368 and WO 2010 / 119039, the entire contents of which are incorporated herein by reference.

[0121] As used herein, “nucleic acid” should refer to any nucleic acid molecule, including but not limited to DNA, RNA, and their hybrids or modified variants. “Exogenous nucleic acid” or “exogenous genetic element” refers to any nucleic acid introduced into the cell that is not a component of the cell’s “original” or “natural” genome. Exogenous nucleic acids can be integrated or non-integrated, or involve nucleic acids that are stably transduced.

[0122] This invention covers any given gene delivery method, preferably involving viral or non-viral vectors, and biological or chemical methods of transfection. This method can produce stable or transient gene expression in the system used.

[0123] Genetically modified viruses have been widely used to deliver genes to stem cells. Preferred viral vectors for the genetic modification of MSCs described herein involve retroviral vectors, particularly gamma retroviral vectors. Gamma retroviruses (sometimes called mammalian C-retroviruses) are sister genera of the lentiviral clade and members of the subfamily Ortho- and Retrovirinae of the family Retroviridae. Murine leukemia virus (MLV or MuLV), feline leukemia virus (FeLV), heterophilic murine leukemia virus-associated virus (XMRV), and gibberish leukemia virus (GALV) are members of the genus Gamma retrovirus. Those skilled in the art are familiar with the techniques required for the use of gamma retroviruses in the genetic modification of MSCs. Vectors or similar vectors, as described by Maetzig et al. (Gamma retroviral vectors: biology, technology and application), 2001, Viruses Jun; 3(6):677-713, can be used. For example, murine leukemia virus (MLV) is a simple gamma retrovirus that can be converted into an effective genetic therapy vector by creating gamma retrovirus-modified MSCs and having the MSCs express therapeutic transgenes after delivery to a subject.

[0124] Adenoviruses, or RNA viruses such as lentiviruses, or other retroviruses can be used. Adenoviruses have been used to generate a series of vectors for gene transfer in cell engineering. Primary adenovirus vectors are produced by deleting the E1 gene (required for viral replication) to generate vectors with 4kb cloning capacity. Deleting the E3 gene (responsible for the host immune response) allows for 8kb cloning capacity. Further generations are generated by including deletions of E2 and / or E4. Lentivirals are members of the retroviral family of viruses (M. Scherr et al., Gene transfer into hematopoietic stem cells using lentiviral vectors. Curr Gene Ther. 2002 Feb; 2(1):45-55). Lentiviral vectors are generated by deleting the entire viral sequence except for the LTR and cis-acting packaging signals. The resulting vectors have approximately 8kb cloning capacity. A distinguishing feature of these vectors from retroviral vectors is their ability to transduce dividing and non-dividing cells as well as terminally differentiated cells.

[0125] Non-viral methods can also be employed, such as the following alternative strategies, which include: conventional plasmid transfer and application of target gene integration using integrase or transposase technology. These represent methods for vector transformation that have the advantage of being efficient and generally site-specific in their integration. Physical methods for introducing vectors into cells are known to those skilled in the art. One example involves electroporation, which relies on short, high-voltage electrical pulses that create transient pores on the membrane by overcoming its capacitance. One advantage of this method is that it can be used for stable and transient gene expression in most cell types. Alternative methods involve the use of liposomes or protein transduction domains. Suitable methods are known to those skilled in the art and are not intended as limiting embodiments of the invention. Attached Figure Description

[0126] The following drawings are provided to illustrate specific embodiments of the invention, without limiting the scope of the invention or the concepts described herein. Brief description of the attached diagram:

[0128] Figure 1 The preferred expression box of the present invention.

[0129] Figure 2 Titration of retroviral supernatant on HT1080 cells.

[0130] Figure 3 Intracellular flow cytometry analysis of primary human MSCs transduced using viral expression constructs.

[0131] Figure 4 The expression of transgenic AAT was assessed by ELISA.

[0132] Figure 5 : It inhibits neutrophil elastase by AAT expressed by transduced MSCs.

[0133] Figure 6 Experimental design: BLM-induced pulmonary fibrosis model.

[0134] Detailed description of the attached diagram:

[0135] Figure 1 The preferred expression box of the present invention.

[0136] A schematic diagram of a preferred expression cassette of the present invention. The numbers shown in the diagram denote the names of the internal plasmids of the apceth and will be used herein for simplicity. Promoters are denoted by lowercase p. LTR elements relate to the long terminal repeat sequences of the γ-retroviral vector used. The internal ribosome entry site is abbreviated as IRES. Posttranscriptional regulatory elements are abbreviated as oPRE. Selection is performed using a puromycin resistance gene (pac).

[0137] Figure 2 Titration of retroviral supernatant on HT1080 cells.

[0138] Larger constructs (e.g., 161 and 164, where the SERPINA1 cDNA is driven by the full-length EF1a promoter) produced reduced titers, although they yielded adequate titers, compared to smaller constructs such as those containing the EFS promoter (e.g., 159 or 194). In this experiment, the highest titers were obtained with lentiviral construct 215.

[0139] Figure 3 Intracellular flow cytometry analysis of cells transduced by viral expression constructs.

[0140] Figure 3 a: Intracellular flow cytometry analysis of primary human MSCs transduced with viral expression constructs - ic AAT-positive cells. Figure 3 a depicts the percentage of intracellular AAT-positive MSCs after transduction with different γ-retroviral and lentiviral expression constructs. Figure 3 b: Intracellular flow cytometry analysis of primary human MSCs transduced with viral expression constructs - mean fluorescence intensity (MFI). Figure 3b depicts the mean fluorescence intensity (MFI) values ​​of MSCs transduced with different γ-retroviral and lentiviral expression constructs. All tested vector constructs were capable of transducing primary human MSCs, and transgenic AAT was detected by intracellular flow cytometry in all transduced samples. The difference in transduction efficiency, measured by the percentage of AAT-positive cells per ic cell, is most likely due to the different starting titers of the viral supernatant used for transduction. The different expression levels analyzed by MFI are a result of using different promoters and varying gene cassette contingencies.

[0141] Figure 4 The expression of transgenic AAT was assessed by ELISA.

[0142] The expression of transgenic AAT in primary human MSCs was confirmed by ELISA in all samples. The differences in expression levels were due to the use of different promoters in the vectors and the varying distribution of the gene cassette.

[0143] Figure 5 : It inhibits neutrophil elastase by AAT expressed by transduced MSCs.

[0144] AAT expressed by transduced primary human MSCs is functional and inhibits neutrophil elastase at levels comparable to those in a medium containing 10% serum or approximately 1.5 μM PCK.

[0145] Figure 6 Experimental design of a BLM-induced pulmonary fibrosis model (adapted from Tashiro et al., 2015).

[0146] Example

[0147] The invention is further described through the following examples. These examples are not intended to limit the scope of the invention. Experimental examples relate to the development of a technique capable of expressing α-1 antitrypsin (AAT) by genetically modified MSCs. Examples further relate to therapeutic trials covering the treatment of lung diseases.

[0148] In a preferred embodiment, the examples relate to the preclinical development of a novel gene therapy product that combines the anti-inflammatory effects of α-1 antitrypsin (AAT) with the immunomodulatory properties of primary human mesenchymal stem cells (MSCs) for the treatment of inflammatory lung diseases.

[0149] 1. Design and cloning of retroviral vector constructs:

[0150] Transgenic expression cassettes were constructed using standard cloning techniques as described in *GeneCloning*, New York: Tylor and Francis Group, Julia Lodge, Peter Lund, and Steve Minchin (2007). These constructs expressed the human SERPINA1 cDNA {Homo sapiens serine protease peptidase inhibitor, branch 1 (α-1 antiprotease, antitrypsin), member 1 (SERPINA1), transcript variant 1, mRNA; NCBI reference sequence: NM_000295.4, encoding α-1 antitrypsin (AAT)}. Codon-optimized cDNAs according to SEQ ID NO 2 described above were also evaluated.

[0151] The SERPINA1 gene described in this article is expressed by activating different constitutive promoters, such as the human EEF1A1 eukaryotic translation elongation factor 1α1 promoter (pEF1a), the short form of the human EEF1A1 eukaryotic translation elongation factor 1α1 promoter (pEFS), or the human phosphoglycerate kinase promoter (pPGK). The promoter can also be an inducible promoter, such as the Tie2, RANTES, or HSP70 promoter.

[0152] The gene may or may not be fused with a tag sequence (e.g., a tag protein / peptide, such as a hemagglutinin tag or a HIS tag) so that expression can be easily detected subsequently (Hinrik Garoff, 1985, Annual Review of Cell Biology, Vol 1: 403-445).

[0153] The expression cassette may or may not include a second transgenic cassette consisting of selective marker genes (such as cell surface markers or resistance genes, e.g., the pac gene for conferring puromycin resistance) to allow for the enrichment of genetically modified cells in subsequent processes (David P. Clark, Nanette J. Pazdernik, 2009, Biotechnology: Applying the Genetic Revolution, London: Elsevier). The gene is driven by a separate promoter or a promoter located at the 3' end of the IRES sequence within the SERPINA1 expression cassette.

[0154] To assess potential positional effects, SERPINA1 and pac boxes were cloned with different contellations (pac box cloned in SERPINA1 box 5', and SERPINA1 box cloned in pac box 5'), see [link to documentation]. Figure 1 .

[0155] Then Figure 1 The expression cassettes disclosed herein are inserted into suitable vector systems, such as γ-retroviral backbones (e.g., pSERS11, EP2019134A1) or lentiviral backbones (e.g., U.S. Patent 8,846,385, the entire contents of which are incorporated herein by reference).

[0156] In the γ-retroviral construct, the oPRE sequence is present at the 3' end of the expression cassette. The retroviral backbone contains long terminal repeats (LTRs) located at the 5' and 3' ends of the cassette. The 5'-LTR contains the SV40 enhancer, RSV promoter, SFFVp R, and U5 regions. The 3'-LTR contains the SFFVp U3 region, SFFV R, and U5 regions, as well as the PolyA signal, wherein the SFFVp U3 region is deleted, thus providing self-inactivation (SIN) for the vector.

[0157] In the lentiviral construct, the oPRE sequence is present at the 3' end of the expression cassette. The lentiviral backbone contains long terminal repeats (LTRs) located at the 5' and 3' ends of the cassette. The 5'-LTR contains the CMV promoter and the HIV-1R and U5 regions. The 3'-LTR contains the HIV-1 U3 region, the HIV-1R and U5 regions, and the PolyA signal, wherein the HIV-1 U3 region is deleted, thus providing self-inactivation (SIN) for the vector.

[0158] 2. Titration of retroviral supernatant:

[0159] Viral particles encoding a specified vector were generated by transient transfection of 293T cells (Soneoka et al., Nucleic Acids Research, 1995). To determine viral titers, HT1080 fibrosarcoma cells were seeded in 12-well plates on day 1, and viral supernatant was added to the cells at different dilutions on day 2, with three control wells used to determine the cell count per well. Transduction efficiency was analyzed by intracellular flow cytometry to detect AAT protein 3 days post-transduction. To enhance protein detection, cells were treated with a GolgiPlug protein transport inhibitor (BD, 555029) for 16 to 24 hours prior to staining to prevent cytosolic protein secretion. Cells were permeabilized using BD Cytofix / Cytoperm fixation and permeabilization solution (BD, 554722) according to the manufacturer's instructions, and FITC-conjugated anti-α1 antitrypsin antibody (abcam, ab19170; 1 μL antibody per 100 μL staining reaction, up to 1 × 10⁻⁶) was used. 6 Cells expressing AAT were stained (incubated in the dark at 4°C for 20 to 30 minutes). Cells were analyzed using a Beckman Coulter FC500 flow cytometer. Values ​​for only <25% of icAAT-positive cells were included in the titer calculation.

[0160] See results Figure 2 .

[0161] 3. Preparation of human mesenchymal stem cells (MSCs):

[0162] As described in DJProckop, DGPhinney, BABunnell, Methods in Molecular Biology 449, Mesenchymal stem cells, in Pittinger, MF (2008) of Totowa: Humana Press, human MSCs are isolated from bone marrow by plastic attachment and cultured in growth medium (e.g., DMEM containing FBS).

[0163] 4. Genetic modifications of MSCs:

[0164] Primary MSC transduction was performed using methods described and modified as described by Murray et al., 1999, Human Gene Therapy, 10(11):1743-1752 and Davis et al., 2004, Biophysical Journal, Vol. 86, 1234–1242. Specifically: 6-well or 12-well plates (e.g., Corning) were coated with poly-L-lysine (PLL) (e.g., Sigma-Aldrich, P4707-50 mL); the PLL solution (0.01%) was diluted to a final concentration of 0.001% with PBS. 1 to 2 mL of diluted PLL was used per well. The plates were incubated at room temperature for at least 2 hours. After incubation, the plates were washed once with PBS. The (diluted) viral supernatant was added to each PLL-coated well in a final volume of 0.8 to 2 mL. The loaded plates were centrifuged at 2000 × g for 30 minutes at 4 °C. Then discard the supernatant and use 1×10 5 One MSC is seeded in one well of a 6-well plate at a volume of 2 mL, or 4 × 10⁶ MSCs are seeded in one well of a 6-well plate. 4 One cell was seeded at a volume of 1 mL into the wells of a 12-well plate. The plate was incubated at 37°C in 5% CO2 for further use.

[0165] 5. Assess transgenic AAT expression using intracellular flow cytometry:

[0166] To assess transduction efficiency and AAT expression in primary human MSCs, cells were prepared and transduced with multiples of infection (MOI) ranging from 0.25 to 10 as described above. Transduced cells were selected for 5 to 8 days with puromycin (Sigma Aldrich, P9620-10 mL, [10 mg / mL], final concentration: 1 to 5 μg / mL). Selected cells were analyzed by intracellular flow cytometry as described above.

[0167] All tested vector constructs were capable of transducing primary human MSCs, and transgenic AAT was detected in all transduced samples by intracellular flow cytometry. Differences in transduction efficiency, measured by the percentage of AAT-positive cells (icAAT), are most likely due to different initial titers of the viral supernatant used for transduction, meaning that any cell population provided during further isolation and culture may offer suitable AAT expression. Different expression levels, as analyzed by MFI, are a result of the different promoters used and varying contellations.

[0168] See results Figure 3 .

[0169] 6. Assess transgenic AAT expression using ELISA:

[0170] Human MSCs were transduced using a specified retroviral construct expressing the AAT and pac genes. Cells were selected with puromycin as described above, and 1 × 10⁶ cells were added. 5 Cells were seeded into 6-well or 12-well plates. After 48 hours, the supernatant (1 to 2 mL) was collected and analyzed by ELISA (α-1 antitrypsin (SERPINA1) human ELISA kit, abcam, ab108799) according to the manufacturer's instructions. The resulting data were normalized to 1 × 10⁻⁶. 5 Cell and vector copy number (VCN).

[0171] Transgenic AAT expression in primary human MSCs was confirmed in all samples by ELISA. The differences in expression levels were due to the different promoters used in the vectors and the varying gene cassette distributions, which made it possible to achieve adequate AAT expression at the protein level in each sample to obtain the desired results.

[0172] See results Figure 4 .

[0173] 7. AAT expressed by transduced MSCs inhibits neutrophil elastase:

[0174] Human MSCs were transduced using a specified retroviral construct expressing the AAT and pac genes. Cells were selected with puromycin and seeded in serum-free DMEM in 6-well or 12-well plates. After 48 hours, the supernatant (1 to 2 mL) was collected and analyzed according to the manufacturer's instructions using a neutrophil elastase inhibitor screening kit (abcam, ab118971). The supernatant of transduced MSCs was analyzed in serum-free DMEM at various dilutions from undiluted (1:1) to 1:16. Different concentrations of SPCK and serum-containing media (Bio-M and Bio-1) were included as positive controls, and DMEM was used as a negative control.

[0175] Inhibition of neutrophil elastase is a functional assay used to detect AAT activity in vitro. The constructs provided herein demonstrate effective inhibition of neutrophil elastase, thereby indicating that the modified MSCs of the examples express functional AAT.

[0176] See results Figure 5 .

[0177] 8. Evaluation of the immunomodulatory effect of AAT expressed by MSCs on monocytes:

[0178] Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using sucrose density gradient centrifugation as described in Ivan J. Fuss, Marjorie E. Kanof, Phillip D. Smith, Heddy Zola, 2009 Curr. Protoc. Immunol. 85:7.1.1-7.1.8. To evaluate the in vitro immunomodulatory effects of AAT-MSCs, LPS-induced activation of human monocytes was performed as described in Janciauskiene et al., Biochemical and Biophysical Research Communications, 2004. In short, monocytes were stimulated with lipopolysaccharide (LPS), and the effects of AAT expressed by MSCs on the secretion of pro-inflammatory cytokines (such as TNFα and IL-1β) and the expression of anti-inflammatory cytokines (such as IL-10) by human monocytes were assessed by ELISA in the supernatant.

[0179] When human primary monocytes were cultured in the presence of AAT secreted by genetically modified MSCs (from the supernatant of MSCs transduced to express AAT), the expression of pro-inflammatory cytokines (TNFα, IL-1α) in the supernatant harvested from the monocyte culture was significantly reduced, while the level of anti-inflammatory cytokines such as IL-10 was increased.

[0180] 9. Evaluation of the immunomodulatory effects of AAT-MSC administration in animal models:

[0181] Prepare cells for in vivo experiments and genetically modify the cells as described above. Further select and amplify the transduced cells before cryopreservation or harvesting for administration. Thaw the cells prior to injection, wash and resuspend them with PBS or any other suitable buffer, or separate them from the culture flask, wash and resuspend them with PBS or any other suitable buffer, and then inject.

[0182] 10. Bleomycin (BLM)-induced pulmonary fibrosis:

[0183] To test the immunomodulatory and antifibrotic effects of AAT-MSCs in vivo, a mouse model of bleomycin-induced pulmonary fibrosis was developed, as described by Tashiro et al. in Translational Science 2015.

[0184] In short, BLM pulmonary fibrosis was induced in C57BL / 6 mice. After anesthesia, BLM sulfate (Sigma-Aldrich) dissolved in 50 μL sterile saline was administered via direct intratracheal instillation at a dose of 2.5 U / kg body weight. 24 to 72 hours after BLM administration, each animal received 200 μL PBS (control) via tail vein injection or intratracheal administration, or 1 × 10⁻⁶ BLM in 200 μL PBS. 6 1×10 untransduced MSCs or 200 μL PBS 6 AAT-transduced MSCs were collected. Serum AAT levels were monitored every other day via retroorbital blood sampling, and AAT protein was subsequently detected by ELISA.

[0185] Mice were sacrificed 14 to 21 days after BLM administration.

[0186] Left lung lobes were harvested from mice for protein and messenger RNA (mRNA) analysis. For morphometric and pathological studies, right lung lobes were fixed by immersion in 10% neutral buffered formalin for 24 hours, followed by transfer to PBS at 4°C. Samples were paraffin-embedded to obtain sections for hematoxylin-eosin and Masson trichrome staining. Pulmonary fibrosis was assessed using the semi-quantitative Ashcroft method on Masson trichrome stained slides (Ashcroft et al., Journal of Clinical Pathology, 1988).

[0187] See the overview of experimental design. Figure 6 .

[0188] At day 21, when mice were sacrificed, BLM mice untreated with MSCs showed pulmonary fibrosis according to Ashkov scores, while mice treated with MSCs or AAT-MSCs showed reduced fibrosis. This reduction was more pronounced in the group receiving MSCs expressing AAT.

[0189] 11. Cyclophosphamide-induced type 1 diabetes:

[0190] To assess the effects of AAT-MSCs on the development of diabetes in vivo, a mouse model of cyclophosphamide-induced type 1 diabetes was established (adapted from Brode et al., The Journal of Immunology, 2006).

[0191] NOD mice were obtained, with a 75% incidence of diabetes in female mice by 40 weeks of age. To promote and synchronize diabetes, 8-week-old female NOD mice were treated with a single intraperitoneal injection of cyclophosphamide (CY) (200 mg / kg body weight in 0.9% saline). Mice were then randomly assigned to treatment and control groups. One to five days after cyclophosphamide treatment, each animal received 200 μL of PBS (control) or 1 × 10⁻⁶ mg / kg body weight in 200 μL of PBS via tail vein or intraperitoneal injection. 6 1×10 untransduced MSCs or 200 μL PBS 6 AAT-transduced MSCs were used. Mice were monitored for hyperglycemia weekly until they were classified as diabetic mice according to the definition of two consecutive (interval >24 hours) non-fasting blood glucose levels >240 mg / dL.

[0192] All control mice receiving CY and PBS developed diabetes within 30 days, while the onset of diabetes was delayed in mice treated with MSCs or AAT-MSCs. Interestingly, in mice treated with AAT-MSCs, the delay in diabetes development was increased by 2 weeks compared to mice with unmodified MSCs.

[0193] 12. MSU / C16.0-induced gouty arthritis:

[0194] To evaluate the anti-inflammatory effect of AAT-MSCs on gouty arthritis in vivo, a mouse model of gouty arthritis induced by MSU / C16.0 was established (adapted from Joosten et al., Annals of the Rheumatic Diseases 2015).

[0195] Male C57Bl / 6 mice were obtained from Jackson Laboratories (Barthon, Maine, USA) and used at 10 to 12 weeks of age. One to three days prior to inducing gouty arthritis, each animal was administered intraperitoneally 200 μL of PBS (control) or 1 × 10⁻⁶ PBS. 6 1×10 untransduced MSCs or 200 μL PBS 6AAT-transduced MSCs were used. Joint inflammation was induced in the right knee joint of naive mice by intra-articular injection of 300 μg MSU crystals mixed with 200 μM C16.0 / bovine serum albumin (BSA) in 10 μL PBS. Gross joint swelling was observed 4 hours after intra-articular injection. Synovial tissue was isolated and cultured in tissue culture medium at 37°C for 2 hours, or directly transferred to 200 μL Triton × 100 (0.5% in PBS). Additionally, the knee joint was excised for pathological examination.

[0196] Treatment with unmodified MSCs or AAT-MSCs suppressed MSCU / C16.0-induced joint inflammation; however, when mice were treated with AAT-MSCs, inflammation was reduced more significantly.

[0197] 13. The role of AAT-MSCs in the prevention of GvHD in a few MHC-matched mouse transplantation models with different antigens:

[0198] To assess the potential anti-GvHD effect of AAT-MSCs, a mouse transplantation model (MHC-matched, with a few antigens different) was established (adapted from Marcondes et al., Blood 2011).

[0199] C57 / BL6J mice (H-2) aged 10 to 14 weeks with an average weight of 28g b Jackson Laboratory) received a single dose of 1000 cGy whole-body irradiation, followed by intravenous injection of T-cells to deplete bone marrow (5 × 10⁻⁶ cells). 6 (0.2 × 10⁻⁶ cells), and CD8+ spleen lymphocytes (0.2 × 10⁻⁶ cells) from C3H.SW-H2b / SnJ donor (H-2bc; Jackson Laboratory). 6 Mice were randomly assigned to treatment and control groups. Prior to irradiation and donor cell fusion, mice in the experimental group were administered 1×10⁻⁶ cells in 200 μL of PBS via intraperitoneal or tail artery injection. 6 1×10 untransduced MSCs or 200 μL PBS 6 AAT-transduced MSCs were administered. Mice in the control group were also given 200 μL of PBS via intraperitoneal or intravenous injection. GvHD was assessed using a standardized scoring system (Cooke et al., Blood 1996): body weight was acquired and recorded on day 0 and then weekly thereafter. Weekly clinical indices were generated by summing scores from five criteria: (percentage of weight change, posture (arched back), activity, coat texture, and skin integrity) (maximum score = 10). Blood samples were continuously collected for cytokine assays.

[0200] Compared to control mice, treatment with unmodified MSCs or AAT-MSCs resulted in attenuation or prevention of GvHD and excellent survival rates. Interestingly, the beneficial effects of AAT-MSCs were more pronounced compared to native MSCs.

Claims

1. The use of genetically modified mesenchymal stem cells in the preparation of drugs for the treatment of inflammatory lung diseases, gout, graft-versus-host disease, and type 1 diabetes in subjects without α-1 antitrypsin deficiency, wherein, The mesenchymal stem cells comprise exogenous nucleic acids, the exogenous nucleic acids comprising (i) a region encoding an α-1 antitrypsin protein according to SEQ ID NO 3, the region encoding the α-1 antitrypsin according to SEQ ID NO 3 being operatively linked to (ii) a promoter or a promoter / enhancer combination.

2. The use according to claim 1, wherein, The exogenous nucleic acid includes viral vectors.

3. The use according to claim 2, wherein, The viral vector is a gamma retrovirus vector.

4. The use according to claim 1, wherein, The promoter or promoter / enhancer combination is a constitutive promoter.

5. The use according to claim 4, wherein, The constitutive promoter is an EFS, PGK, or EF1α promoter.

6. The use according to claim 1, wherein, The promoter or promoter / enhancer combination is an inductive promoter.

7. The use according to claim 6, wherein, The promoter of the mesenchymal stem cells is inducible during differentiation after administration.

8. The use according to claim 1, wherein, The promoter is an inflammation-specific promoter.

9. The use according to claim 1, wherein, The promoter is the Tie2 promoter.

10. The use according to claim 1, wherein, The promoter is the RANTES promoter.

11. The use according to claim 1, wherein, The promoter is the HSP70 promoter.

12. The use according to claim 1, wherein, The mesenchymal stem cells are administered by introducing a therapeutically effective number of cells into the patient's bloodstream.

13. The use according to claim 1, wherein, The inflammatory lung disease mentioned is a respiratory disease.

14. The use according to claim 1, wherein, The inflammatory lung diseases mentioned are acute lung injury, chronic obstructive pulmonary disease, acute respiratory distress syndrome, asthma, sarcoidosis, allergic pneumonia, and / or pulmonary fibrosis, and the chronic obstructive pulmonary disease includes chronic bronchitis, emphysema, bronchiectasis, and bronchiolitis.

15. The use according to claim 1, wherein, The mesenchymal stem cells are administered by introducing a therapeutically effective number of cells into the patient's lungs via inhalation.

16. The use according to claim 1, wherein, The drug is used to treat gout.

17. The use according to claim 1, wherein, The drug is used to treat graft-versus-host disease.

18. The use according to claim 1, wherein, The drug is used to treat type 1 diabetes.

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