GENETICALLY MODIFIED MESENCHYMAL STEM CELLS EXPRESSING ALPHA-1 ANTITRYPSIN (AAT)

Abstract

A method for treating a subject having a medical condition associated with inflammation and/or an unwanted immune response without an alpha1-antitrypsin (AAT) deficiency, wherein the method comprises administering genetically modified mesenchymal stem cells to the subject, wherein said genetically modified mesenchymal stem cells comprise an exogenous nucleic acid comprising (i) an Alpha-1 antitrypsin (AAT) encoding region operably linked to (ii) a promoter or promoter/enhancer combination.

Claims

1. A method for treating a subject having a medical condition associated with inflammation and/or an unwanted immune response without an alpha1-antitrypsin (AAT) deficiency, wherein the method comprises administering genetically modified mesenchymal stem cells to the subject, wherein said genetically modified mesenchymal stem cells comprise an exogenous nucleic acid comprising (i) an Alpha-1 antitrypsin (AAT) encoding region operably linked to (ii) a promoter or promoter/enhancer combination.

2. The method according to claim 1, wherein the exogenous nucleic acid comprises a viral vector.

3. The method according to claim 2, wherein the viral vector is a retroviral vector.

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

5. The method according to claim 4, wherein the constitutive promoter is the EFS, PGK, or EF1alpha promoter.

6. The method according to claim 1, wherein said promoter or promoter/enhancer combination is an inducible promoter.

7. The method according to claim 6, wherein the promoter is an inflammation-specific promoter.

8. The method according to claim 1, wherein a therapeutically effective number of genetically modified mesenchymal stem cells according to claim 1 are introduced into the bloodstream of the subject.

9. The method according to claim 8, wherein the therapeutically effective number of genetically modified mesenchymal stem cells is introduced into the bloodstream of the subject via intravenous injection.

10. The method according to claim 1, wherein the medical condition associated with inflammation and/or an unwanted immune response is a lung disease.

11. The method according to claim 10, wherein the lung disease is an inflammatory disease of the lung.

12. The method according to claim 10, wherein the lung disease is acute 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.

13. The method according to claim 10, wherein said therapeutically effective number of genetically modified cells are introduced to the lung of the subject by inhalation, optionally in combination with introduction of said cells into the bloodstream of the subject.

14. The method according to claim 1, wherein the medical condition associated with inflammation and/or an unwanted immune response is gout.

15. The method according to claim 1, wherein the medical condition associated with inflammation and/or an unwanted immune response is chronic fibrosis.

16. The method according to claim 15, wherein the chronic fibrosis is of the kidney, liver and/or colon of the subject.

17. The method according to claim 1, wherein the medical condition associated with inflammation and/or an unwanted immune response is of the kidney, liver and/or colon of the subject.

18. The method according to claim 1, wherein the medical condition associated with inflammation and/or an unwanted immune response is an inflammatory disease selected from the group consisting of vasculitis, nephritis, inflammatory bowel disease, rheumatoid arthritis and Graft versus Host disease.

19. The method according to claim 1, wherein the medical condition associated with inflammation and/or an unwanted immune response is an autoimmune disease.

20. The method according to claim 19, wherein the autoimmune disease is Type 1 diabetes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The following figures are presented in order to describe particular embodiments of the invention, by demonstrating a practical implementation of the invention, without being limiting to the scope of the invention or the concepts described herein.

[0091] FIG. 1: Preferred expression cassettes of the present invention. Schematic representation of the preferred expression cassettes of the present invention. Numbers depicted in the figure represent apceth's internal plasmid denomination and will be used herein for reasons of simplification. The promoters are shown by a lower case p. LTR elements relate to long terminal repeats of the gamma retroviral vector employed. An internal ribosome entry site is abbreviated as IRES. A Posttranscriptional Regulatory Element is abbreviated as oPRE. A puromycin resistance gene (pac) is employed for selection.

[0092] FIG. 2: Titration of retroviral supernatants on HT1080 cells. Larger constructs (e.g. 161 and 164 in which the SERPINA1 cDNA is driven by the full length EF1a promoter) yield reduced although sufficient titers as compared to smaller constructs such as ones comprising the EFS promoter (e.g. 159 or 194). In the present experiment, the highest titer is obtained with the lentiviral construct 215.

[0093] FIG. 3: Intracellular flow cytometry analysis of cells transduced with viral expression constructs. (A) Intracellular flow cytometry analysis of primary human MSCs transduced with viral expression constructs—% ic AAT positive cells. (A) depicts percentage of MSCs that stained positive for intracellular AAT after transduction with different gamma-retroviral and lentiviral expression constructs. (B) Intracellular flow cytometry analysis of primary human MSCs transduced with viral expression constructs—mean fluorescence intensity (MFI). (B) depicts mean fluorescence intensity (MFI) values of MSCs transduced with different gamma-retroviral and lentiviral expression constructs. All tested vector constructs are able to transduce primary human MSCs, and transgenic AAT is detected by intracellular flow cytometry in all transduced samples. Differences in transduction efficiency as measured by % is AAT positive cells are most likely due to different starting titers of the viral supernatants used for transduction. Different expression levels as analyzed by MFI are the result of distinct promoters utilized as well as varied gene cassette constellations.

[0094] FIG. 4: Assessment of transgenic AAT expression by ELISA. Transgenic AAT expression in primary human MSCs is confirmed in all samples by ELISA. Variances in the amounts expressed are due to distinct promoters utilized in the vectors as well as varied gene cassette constellations.

[0095] FIG. 5: Inhibition of Neutrophil Elastase by AAT expressed from transduced MSCs. AAT expressed from transduced primary human MSCs is functional and inhibits Neutrophil Elastase at levels comparable to medium containing 10% serum or ˜1.5 μM SPCK.

[0096] FIG. 6: Experimental design BLM-induced lung fibrosis model (adapted from Tashiro et al., 2015).

DETAILED DESCRIPTION

[0097] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

[0098] The “mesenchymal cells” disclosed herein (also referred to in some embodiments as “mesenchymal stem cells” or “MSCs”) can give rise to connective tissue, bone, cartilage, and cells in the circulatory and lymphatic systems. Mesenchymal stem cells are found in the mesenchyme, the part of the embryonic mesoderm that consists of loosely packed, fusiform or stellate unspecialized cells. As used herein, mesenchymal stem cells include, without limitation, CD34-negative stem cells.

[0099] In one embodiment of the invention, the mesenchymal cells are fibroblast-like plastic adherent cells, defined in some embodiments as multipotent mesenchymal stromal cells and also include CD34-negative cells.

[0100] For the avoidance of any doubt, the term mesenchymal cell encompasses multipotent mesenchymal stromal cells that also includes a subpopulation of mesenchymal cells, MSCs and their precursors, which subpopulation is made up of multipotent or pluripotent self-renewing cells capable of differentiation into multiple cell types in vivo.

[0101] As used herein, CD34-negative cell shall mean a cell lacking CD34, or expressing only negligible levels of CD34, on its surface. CD34-negative cells, and methods for isolating such cells, are described, for example, in Lange C. et al., “Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine”. J. Cell Physiol. 2007, Apr. 25.

[0102] Mesenchymal cells can be differentiated from hematopoietic stem cells (HSCs) by a number of indicators. For example, HSCs are known to float in culture and to not adhere to plastic surfaces. In contrast, mesenchymal cells adhere to plastic surfaces. The CD34-negative mesenchymal cells of the present invention are adherent in culture.

[0103] The genetically modified cell(s) described herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0104] The present invention encompasses treatment of a patient by introducing a therapeutically effective number of cells into a subject's bloodstream. As used herein, “introducing” cells “into the subject's bloodstream” shall include, without limitation, introducing such cells into one of the subject's veins or arteries via injection. Such administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. A single injection is preferred, but repeated injections over time (e.g., quarterly, half-yearly or yearly) may be necessary in some instances. Such administering is also preferably performed using an admixture 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, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline, as well as commonly used proprietary cryopreservation media.

[0105] Administration may also occur locally, for example by injection into an area of the subject's body in proximity to a site of inflammation. MSCs have been shown to migrate towards inflammation. Mesenchymal stem cells (MSC) exhibit tropism for sites of tissue damage as well as the tumor microenvironment. Many of the same inflammatory mediators that are secreted by wounds are found in the tumor microenvironment and are thought to be involved in attracting MSC to these sites. Cell migration is dependent on a multitude of signals ranging from growth factors to chemokines secreted by injured cells and/or respondent immune cells. MSC are likely to have chemotactic properties similar to other immune cells that respond to injury and sites of inflammation. Regardless, the local administration of the cells as described herein may lead to high levels of the cells at their site of action.

[0106] Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions, most preferably aqueous solutions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, those based on Ringer's dextrose, and the like. Fluids used commonly for i.v. administration are found, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed., p. 808, Lippincott Williams S-Wilkins (2000). Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

[0107] As used herein, a “therapeutically effective number of cells” includes, without limitation, the following amounts and ranges of amounts: (i) from about 1×10.sup.2 to about 1×10.sup.8 cells/kg body weight; (ii) from about 1×10.sup.3 to about 1×10.sup.7 cells/kg body weight; (iii) from about 1×10.sup.4 to about 1×10.sup.6 cells/kg body weight; (iv) from about 1×10.sup.4 to about 1×10.sup.5 cells/kg body weight; (v) from about 1×10.sup.5 to about 1×10.sup.6 cells/kg body weight; (vi) from about 5×10.sup.4 to about 0.5×10.sup.5 cells/kg body weight; (vii) about 1×10.sup.3 cells/kg body weight; (viii) about 1×10.sup.4 cells/kg body weight; (ix) about 5×10.sup.4 cells/kg body weight; (x) about 1×10.sup.5 cells/kg body weight; (xi) about 5×10.sup.5 cells/kg body weight; (xii) about 1×10.sup.6 cells/kg body weight; and (xiii) about 1×10.sup.7 cells/kg body weight. Human body weights envisioned include, without limitation, about 5 kg, 10 kg, 15 kg, 30 kg, 50 kg, about 60 kg; about 70 kg; about 80 kg, about 90 kg; about 100 kg, about 120 kg and about 150 kg. These numbers are based on pre-clinical animal experiments and human trials and standard protocols from the transplantation of CD34+ hematopoietic stem cells. Mononuclear cells (including CD34+ cells) usually contain between 1:23000 to 1:300000 CD34-negative cells.

[0108] As used herein, “treating” a subject afflicted with a disorder, such as inflammation, shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a subject afflicted with a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself. As used herein, ameliorating a disorder and treating a disorder are equivalent. The treatment of the present invention may also, or alternatively, relate to a prophylactic administration of said cells. Such a prophylactic administration may relate to the prevention of any given medical disorder, such as the prevention of inflammation, or the prevention of development of said disorder, whereby prevention or prophylaxis is not to be construed narrowly under all conditions as absolute prevention. Prevention or prophylaxis may also relate to a reduction of the risk of a subject developing any given medical condition, preferably in a subject at risk of said condition.

[0109] Typically, the term “inflammation” as used in its art-recognized sense relates to a localized or systemic protective response elicited by injury, infection or destruction of tissues which serves to protect the subject from an injurious agent and the injured tissue. Inflammation is preferably characterized by fenestration of the microvasculature, leakage of the elements of blood into the interstitial spaces, and migration of leukocytes into the inflamed tissue, which may lead to an uncontrolled sequence of pain, heat, redness, swelling, and loss of function.

[0110] Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

[0111] The term “unwanted inflammation” preferably refers to an inflammation in a subject that exceeds the level of a physiological beneficial inflammatory reaction and leads to damages of the cells, tissues and/or organs at the site of the inflammation.

[0112] The term “unwanted immune response” preferably refers an alteration of the reactivity of the immune system in a subject that has deleterious effect on its health and may involve the stimulation and/or production of cytokines as well as the recruitment of immune cells. Unwanted immune responses occur for instance in autoimmune diseases, transplant rejections, allergies or inflammatory diseases.

[0113] Medical conditions that are defined by unwanted inflammation and/or an immune response refer therefore to a number of diseases and/or disorders including in particular but not being limited to vasculitis, nephritis, inflammatory bowel disease, rheumatoid arthritis, Graft versus Host disease, gouty arthritis, chronic fibrosis, inflammatory diseases of the lung or autoimmune diseases.

[0114] Examples of inflammatory diseases of the lung 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 as described herein migrate towards physiological niches affected by a disease condition, such as areas of inflammation, in order to impart their therapeutic effect, for example in a local manner.

[0115] The invention is further directed towards the treatment of autoimmune disorders, in particular those with an inflammatory component. These disorders may also be referred to as rheumatic disorders. Such conditions are preferably selected from Takayasu 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, Vasculitis of connective tissue diseases, Hennoch-Schonlein purpura, Cryoglobulinemic vasculitis, Cutaneous leukocytoclastic angiitis, Tropical aortitis, Sarcoidosis, Cogan's syndrome, Wiskott-Aldrich Syndrome, Lepromatous arteritis, Primary angiitis of the CNS, Thromboangiitis obliterans, Paraneoplastic ateritis, Urticaria, Dego's disease, Myelodysplastic syndrome, Eythema elevatum diutinum, Hyperimmunoglobulin D, Allergic Rhinitis, Asthma bronchiale, chronic obstructive pulmonary disease, periodontitis, Rheumatoid Arthritis, atherosclerosis, Amyloidosis, Morbus Chron, Colitis ulcerosa, Autoimmune Myositis, Diabetes mellitus, Multiple sclerosis, Guillain-Barre Syndrome, histiocytosis, Osteoarthritis, atopic dermatitis, periodontitis, chronic rhinosinusitis, Psoriasis, psoriatic arthritis, Microscopic colitis, Pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis, cryoglobulinemia, Sjogren's syndrome, Lupus erythematosus, aplastic anemia, Osteomyelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, Idiopathic achalasia, bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, Autoimmunthrombocytopenia, Autoimmunneutropenia, Autoimmunhemolytic anemia, Autoimmunlymphocytopenia, Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's Thyroiditis, atropic thyroiditis, Graves disease, Autoimmune polyglandular syndrome, Autoimmune Addison Syndrome, Pemphigus vulgaris, Pemphigus foliaceus, Dermatitis herpetiformis, Autoimmune alopecia, Vitiligo, Antiphospholipid syndrome, Myasthenia gravis, Stiff-man syndrome, Goodpasture's syndrome, Sympathetic ophthalmia, Folliculitis, Sharp syndrome and/or Evans syndrome.

[0116] As used herein “cell migration” is intended to mean movement of a cell towards a particular chemical or physical signal. Cells often migrate in response to specific external signals, including chemical signals and mechanical signals. Chemotaxis is one example of cell migration regarding response to a chemical stimulus. In vitro chemotaxis assays such as Boyden chamber assays may be used to determine whether cell migration occurs in any given cell. For example, the cells of interest may be purified and analysed. Chemotaxis assays (for example according to Falk et al., 1980 J. Immuno. Methods 33:239-247) can be performed using plates where a particular chemical signal is positioned with respect to the cells of interest and the transmigrated cells then collected and analyzed. For example, Boyden chamber assays entail the use of chambers isolated by filters, used as tools for accurate determination of chemotactic behavior. The pioneer 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). The motile cells are placed into the upper chamber, while fluid containing the test substance is filled into the lower one. The size of the motile cells to be investigated determines the pore size of the filter; it is essential to choose a diameter which allows active transmigration. For modelling in vivo conditions, several protocols prefer coverage of filter with molecules of extracellular matrix (collagen, elastin etc.) Efficiency of the measurements can be increased by development of multiwell chambers (e.g. NeuroProbe), where 24, 96, 384 samples are evaluated in parallel. Advantage of this variant is that several parallels are assayed in identical conditions.

[0117] Alternatively, tissue samples may be obtained from subjects (for example rodent models) after cell transplantation and assayed for the presence of the cells of interest in particular tissue types. Such assays may be of molecular nature, identifying cells based on nucleic acid sequence, or of histological nature, assessing cells on the basis of fluorescent markings after antibody labeling. Such assays are also particularly useful for assessing engraftment of transplanted cells. Assays for engraftment may also provide information on cell migration, as to some extent the engraftment is dependent on cell localization prior to engraftment.

[0118] In some embodiments of the invention the MSCs as described herein engraft in physiological niches affected by a disease condition, such as areas of inflammation, in order to impart their therapeutic effect, for example in a local manner.

[0119] As used herein “engraftment” relates to the process of incorporation of grafted or transplanted tissue or cells into the body of the host. Engraftment may also relate to the integration of transplanted cells into host tissue and their survival and under some conditions differentiation into non-stem cell states.

[0120] Techniques for assessing engraftment, and thereby to some extent both migration and the bio-distribution of MSCs, can encompass either in vivo or ex vivo methods. Examples of in vivo methods include bioluminescence, whereby cells are transduced to express luciferase and can then be imaged through their metabolism of luciferin resulting in light emission; fluorescence, whereby cells are either loaded with a fluorescent dye or transduced to express a fluorescent reporter which can then be imaged; radionuclide labeling, where cells are loaded with radionuclides and localized with scintigraphy, positron emission tomography (PET) or single photon emission computed tomography (SPECT); and magnetic resonance imaging (MRI), wherein cells loaded with paramagnetic compounds (e.g., iron oxide nanoparticles) are traced with an MRI scanner. Ex vivo methods to assess biodistribution include quantitative PCR, flow cytometry, and histological methods. Histological methods include tracking fluorescently labeled cells; in situ hybridization, for example, for Y-chromosomes and for human-specific ALU sequences; and histochemical staining for species-specific or genetically introduced proteins such as bacterial β-galactosidase. These immunohistochemical methods are useful for discerning engraftment location but necessitate the excision of tissue. For further review of these methods and their application see Kean et al., MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation, Stem Cells International, Volume 2013 (2013).

[0121] Progenitor or multipotent cells, such as the mesenchymal cells of the present invention, may therefore be described as protein delivery vehicles, essentially enabling the localization and expression of therapeutic gene products in particular tissues or regions of the subject's body. Such therapeutic cells offer the potential to provide cellular therapies for diseases that are refractory to other treatments. For each type of therapeutic cell the ultimate goal is the same: the cell should express a specific repertoire of genes, preferably exogenous nucleic acids that code for therapeutic gene products, thereby modifying cell identity to express said gene product and provide a therapeutic effect, such as an anti-inflammatory effect. The cells of the invention, when expanded in vitro, represent heterogeneous populations that include multiple generations of mesenchymal (stromal) cell progeny, which lack the expression of most differentiation markers like CD34. These populations may have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages.

[0122] As used herein “inducible expression” or “conditional expression” relates to a state, multiple states or system of gene expression, wherein the gene of interest, such as the therapeutic transgene, is preferably not expressed, or in some embodiments expressed at negligible or relatively low levels, unless there is the presence of one or more molecules (an inducer) or other set of conditions in the cell that allows for gene expression. Inducible promoters may relate to either naturally occurring promoters that are expressed at a relatively higher level under particular biological conditions, or to other synthetic promoters comprising any given inducible element. Inducible promoters may refer to those induced by particular tissue- or micro-environments or combinations of biological signals present in particular tissue- or micro-environments, or to promoters induced by external factors, for example by administration of a small drug molecule or other externally applied signal.

[0123] As used herein, in “proximity with” a tissue includes, for example, within 5 mm, within 1 mm of the tissue, within 0.5 mm of the tissue and within 0.25 mm of the tissue.

[0124] Given that stem cells can show a selective migration to different tissue microenvironments in normal as well as diseased settings, the use of tissue-specific promoters linked to the differentiation pathway initiated in the recruited stem cell is encompassed in the present invention and could in theory be used to drive the selective expression of therapeutic genes only within a defined biologic context. Stem cells that are recruited to other tissue niches, but do not undergo the same program of differentiation, should not express the therapeutic gene. This approach allows a significant degree of potential control for the selective expression of the therapeutic gene within a defined microenvironment and has been successfully applied to regulate therapeutic gene expression during neovascularization. Potential approaches to such gene modifications are disclosed in WO 2008/150368 and WO 2010/119039, which are hereby incorporated in their entirety.

[0125] As used herein, “nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids or modified variants thereof. An “exogenous nucleic acid” or “exogenous genetic element” relates to any nucleic acid introduced into the cell, which is not a component of the cells “original” or “natural” genome. Exogenous nucleic acids may be integrated or non-integrated, or relate to stably transfected nucleic acids.

[0126] Any given gene delivery method is encompassed by the invention and preferably relates to viral or non-viral vectors, as well as biological or chemical methods of transfection. The methods can yield either stable or transient gene expression in the system used.

[0127] Genetically modified viruses have been widely applied for the delivery of genes into stem cells. Preferred viral vectors for genetic modification of the MSCs described herein relate to retroviral vectors, in particular to gamma retroviral vectors. The gamma retrovirus (sometimes referred to as mammalian type C retroviruses) is a sister genus to the lentivirus clade, and is a member of the Orthoretrovirinae subfamily of the retrovirus family. The Murine leukemia virus (MLV or MuLV), the Feline leukemia virus (FeLV), the Xenotropic murine leukemia virus-related virus (XMRV) and the Gibbon ape leukemia virus (GALV) are members of the gamma retrovirus genus.

[0128] A skilled person is aware of the techniques required for utilization of gamma retroviruses in genetic modification of MSCs. For example, the vectors described by Maetzig et al (Gammaretroviral vectors: biology, technology and application, 2001, Viruses June; 3(6):677-713) or similar vectors may be employed. For example, the Murine Leukemia Virus (MLV), a simple gammaretrovirus, can be converted into an efficient vehicle of genetic therapeutics in the context of creating gamma retrovirus-modified MSCs and expression of a therapeutic transgene from said MSCs after delivery to a subject.

[0129] Adenoviruses may be applied, or RNA viruses such as Lentiviruses, or other retroviruses. Adenoviruses have been used to generate a series of vectors for gene transfer cellular engineering. The initial generation of adenovirus vectors were produced by deleting the EI gene (required for viral replication) generating a vector with a 4 kb cloning capacity. An additional deletion of E3 (responsible for host immune response) allowed an 8 kb cloning capacity. Further generations have been produced encompassing E2 and/or E4 deletions. Lentiviruses are members of Retroviridae family of viruses (M. Scherr et al., Gene transfer into hematopoietic stem cells using lentiviral vectors. Curr Gene Ther. 2002 February; 2(1):45-55). Lentivirus vectors are generated by deletion of the entire viral sequence with the exception of the LTRs and cis acting packaging signals. The resultant vectors have a cloning capacity of about 8 kb. One distinguishing feature of these vectors from retroviral vectors is their ability to transduce dividing and non-dividing cells as well as terminally differentiated cells.

[0130] Non-viral methods may also be employed, such as alternative strategies that include conventional plasmid transfer and the application of targeted gene integration through the use of integrase or transposase technologies. These represent approaches for vector transformation that have the advantage of being both efficient, and often site-specific in their integration. Physical methods to introduce vectors into cells are known to a skilled person. One example relates to electroporation, which relies on the use of brief, high voltage electric pulses which create transient pores in the membrane by overcoming its capacitance. One advantage of this method is that it can be utilized for both stable and transient gene expression in most cell types. Alternative methods relate to the use of liposomes or protein transduction domains. Appropriate methods are known to a skilled person and are not intended as limiting embodiments of the present invention.

Examples

[0131] The invention is further described by the following examples. These are not intended to limit the scope of the invention. The experimental examples relate to the development of technology that enables Alpha-1 antitrypsin (AAT) expression from genetically modified MSCs. The examples further relate to therapeutic trials encompassing the treatment of conditions of the lung.

[0132] In preferred embodiments the examples relate to the preclinical development of a novel gene therapy product that combines the anti-inflammatory effect of Alpha-1 antitrypsin (AAT) with the immunomodulatory properties of primary human mesenchymal stem cells (MSCs) for the treatment of inflammatory lung diseases.

[0133] 1. Design and Cloning of Retroviral Vector Constructs:

[0134] The transgene expression cassettes are constructed using standard cloning techniques as described in Julia Lodge, Peter Lund, Steve Minchin (2007) Gene Cloning, New York: Tylor and Francis Group. The gene expressed by these constructs is the human SERPINA1 cDNA {Homo sapiens serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 1, mRNA; NCBI Reference Sequence: NM_000295.4, encoding Alpha-1 antitrypsin (AAT)}. A codon optimized cDNA as described above according to SEQ ID NO 2 has also been assessed.

[0135] The SERPINA1 gene as described herein is expressed by activation of different constitutive promoters, such as the human EEF1A1 eukaryotic translation elongation factor 1 alpha 1 promoter (pEF1a), the short form of the human EEF1A1 eukaryotic translation elongation factor 1 alpha 1 promoter (pEFS), or the human phosphoglycerate kinase promoter (pPGK). The promoters may also be inducible promoters like Tie2, RANTES or the HSP70 promoter.

[0136] The gene may or may not be fused with tag-sequences (e.g. marker proteins/peptides such as the hemagglutinin tag or the HIS tag) to allow for easy detection of expression later on (Hinrik Garoff, 1985, Annual Review of Cell Biology, Vol. 1: 403-445).

[0137] The expression cassette may or may not include a second transgene cassette consisting of a selectable marker gene, such as a cell surface marker or a resistance gene (for example the pac gene to confer puromycin resistance) to allow for enrichment of genetically modified cells later in the process (David P. Clark, Nanette J. Pazdernik, 2009, Biotechnology: Applying the Genetic Revolution, London: Elsevier). The gene is either driven by a separate promoter or located 3′ of an IRES sequence within the SERPINA1 expression cassette.

[0138] To assess potential positional effects, SERPINA1 and pac cassettes are cloned in different constellations (SERPINA1 cassette 5′ of pac cassette and vice versa). Refer to FIG. 1.

[0139] The expression cassettes disclosed in FIG. 1 are then inserted into a suitable vector system, e.g. a gamma-retroviral (e.g. pSERS11, EP2019134A1) or lentiviral backbone (e.g. U.S. Pat. No. 8,846,385, herein incorporated by reference in its entirety) by standard cloning techniques.

[0140] In the gamma-retroviral constructs, there is an oPRE sequence 3′ of the expression cassette(s). The retroviral backbone contains long terminal repeats (LTRs) which are located at the 5′- and 3′-end of the cassette. The 5′-LTR contains an SV40 enhancer, RSV promoter, an SFFVp R and U5 region. The 3′-LTR contains an SFFVp U3 region that has a deletion and thus renders the vector self-inactivating (SIN), an SFFV R and U5 region as well as a PolyA signal.

[0141] In the lentiviral construct, there also is an oPRE sequence 3′ of the expression cassette(s). The lentiviral backbone contains long terminal repeats (LTRs) which are located at the 5′- and 3′-end of the cassette. The 5′-LTR contains a CMV promoter and an HIV-1 R and U5 region. The 3′-LTR contains an HIV-1 U3 region that has a deletion and thus renders the vector self-inactivating (SIN), an HIV-1 R and U5 region as well as a PolyA signal.

[0142] 2. Titration of Retroviral Supernatants:

[0143] Viral particles encoding the designated vectors are generated by transient transfection of 293T cells (Soneoka et al., Nucleic Acids Research, 1995). In order to determine viral titers, HT1080 fibrosacroma cells are seeded in 12-well plates on day 1, viral supernatants at different dilutions are added to cells on day 2, and three control wells are used to determine cell numbers per well. Three days after transduction, transduction efficiency is analyzed by an intracellular flow cytometry assay detecting AAT protein. To enhance the detection of the protein, cells are treated with GolgiPlug Protein Transport Inhibitor (BD, 555029) for 16 to 24 hours before the staining to prevent the secretion of cytosolic proteins. Cells are permeabilized using BD Cytofix/Cytoperm Fixation and Permeabilization Solution (BD, 554722) according to manufacturer's instructions, and AAT-expressing cells are stained with a FITC-conjugated Anti-alpha 1 Antitrypsin antibody (abcam, ab19170; 1 μL antibody per 100 μL staining reaction and up to 1×10.sup.6 cells, incubation for 20-30 minutes at 4° C. in the dark). Cells are analyzed on a Beckman Coulter FC500 flow cytometer. Only values <25% is AAT positive cells are included in titer calculations.

[0144] Refer FIG. 2 for results.

[0145] 3. Preparation of Human Mesenchymal Stem Cells (MSCs):

[0146] Human MSCs are isolated from bone marrow by plastic adherence and are cultured in growth medium e.g. FBS containing DMEM as described by Pittinger, M. F. (2008) Mesenchymal stem cells from adult bone marrow, In D. J. Prockop, D. G. Phinney, B. A. Bunnell, Methods in Molecular Biology 449, Mesenchymal stem cells, Totowa: Humana Press.

[0147] 4. Genetic Modification of MSCs:

[0148] The transduction of primary MSCs is performed with modifications as described by Murray et al., 1999 Human Gene Therapy. 10(11): 1743-1752 and Davis et al., 2004 Biophysical Journal Volume 86 1234-1242. In detail: 6-well or 12-well cell culture plates (e.g. Corning) are coated with Poly-L-Lysine (PLL) (e.g. Sigma-Aldrich, P4707-50 mL); the PLL solution (0.01%) is diluted to a final concentration of 0.001% with PBS. 1 to 2 mL of diluted PLL are used for each well. The plate is incubated for at least 2 hours at room temperature. After incubation, the plates are washed once with PBS. (Diluted) viral supernatant is added to each PLL-coated well in a final volume of 0.8 to 2 mL. The loaded plate is centrifuged for 2000×g for 30 min at 4° C. The supernatant is then discarded and 1×10.sup.5 MSCs are seeded in one well of a 6-well plate in a volume of 2 mL or 4×10.sup.4 cells are seeded in a well of a 12-well plate in a volume of 1 mL. The plates are incubated at 37° C. with 5% CO.sub.2 for further use.

[0149] 5. Assessment of Transgenic AAT Expression by Intracellular Flow Cytometry:

[0150] To assess transduction efficiency and AAT expression in primary human MSCs, cells are prepared and transduced as described above with multiplicities of infection (MOIs) of 0.25 to 10. Transduced cells are selected with puromycin (Sigma Aldrich, P9620-10 mL, [10 mg/mL], final concentration: 1-5 μg/mL) for 5 to 8 days. Selected cells are analyzed by intracellular flow cytometry as described above.

[0151] All tested vector constructs are able to transduce primary human MSCs, and transgenic AAT is detected by intracellular flow cytometry in all transduced samples. Differences in transduction efficiency as measured by % is AAT positive cells are most likely due to different starting titers of the viral supernatants used for transduction, such that any of the provided cell population upon further isolation and culturing may provide suitable AAT expression. Different expression levels as analyzed by MFI are the result of distinct promoters utilized as well as varied gene cassette constellations.

[0152] Refer to FIG. 3 for results.

[0153] 6. Assessment of Transgenic AAT Expression by ELISA:

[0154] Human MSCs are transduced with the indicated retroviral constructs expressing AAT and the pac gene. The cells are selected with puromycin as described above and 1×10.sup.5 cells are seeded onto 6- or 12-well plates. Supernatants (1-2 mL) are collected after 48 hours and analyzed by ELISA (alpha 1 Antitrypsin (SERPINA1) Human ELISA Kit, abcam, ab108799) according to manufacturer's instructions. Data generated are normalized to 1×10.sup.5 cells and vector copy number (VCN).

[0155] Transgenic AAT expression in primary human MSCs is confirmed in all samples by ELISA. Variances in the amounts expressed are due to distinct promoters utilized in the vectors as well as varied gene cassette constellations, such that each of the examples provided enables sufficient AAT expression at the protein level to obtain a desired effect.

[0156] Refer to FIG. 4 for results.

[0157] 7. Inhibition of Neutrophil Elastase by AAT Expressed from Transduced MSCs:

[0158] Human MSCs are transduced with the indicated retroviral constructs expressing AAT and the pac gene. The cells are selected with puromycin and selected cells are seeded onto 6- or 12-well plates in DMEM without serum. Supernatants (1-2 mL) are collected after 48 hours and analyzed by a Neutrophil Elastase Inhibitor Screening Kit (abcam, ab118971) according to manufacturer's instructions. Supernatant of transduced MSCs is analyzed in different dilutions ranging from non-diluted (1:1) to 1:16 in DMEM without serum. SPCK in different concentrations and medium containing serum (Bio-M and Bio-1) are included as positive controls, DMEM is used as a negative control.

[0159] Inhibition of neutrophil elastase is a functional assay for detecting AAT activity in in vitro. The constructs provided herein show effective inhibition of neutrophil elastase thereby indicating expression of functional AAT from the modified MSCs of the examples.

[0160] Refer to FIG. 5 for results.

[0161] 8. Assessment of Immunomodulatory Effect of AAT Expressed from MSCs on Monocytes:

[0162] Peripheral blood mononuclear cells (PBMCs) are isolated from human blood using ficoll density gradient centrifugation as described by Ivan J. Fuss, Marjorie E. Kanof, Phillip D. Smith, Heddy Zola, 2009 Curr. Protoc. Immunol. 85: 7.1.1-7.1.8. To assess the immunomodulatory effect of AAT-MSCs in vitro, an assay of LPS-induced human monocyte activation is performed as described in Janciauskiene et al., Biochemical and Biophysical Research Communications, 2004. In brief, monocytes are stimulated with lipopolysaccharide (LPS) and the effect of AAT expressed from MSCs on the secretion of proinflammatory cytokines such as TNFalpha and IL-1beta as well as on the expression of anti-inflammatory cytokines such as IL-10 from human monocytes is assessed in supernatants by ELISA.

[0163] When human primary monocytes are cultured in the presence of AAT secreted from genetically modified MSCs (supernatants from MSCs transduced to express AAT), the expression of proinflammatory cytokines (TNFalpha, IL-1beta) in supernatants harvested from monocyte cultures is markedly decreased, whereas there is an increase in the levels of anti-inflammatory cytokines such as IL-10.

[0164] 9. Assessment of Immunomodulatory Effect of AAT-MSC Administration in Animal Models:

[0165] Cells used in in vivo experiments are prepared and genetically modified as described above. Transduced cells are selected and expanded further before either cryopreservation or harvest for administration. The cells are either thawed, washed with and re-suspended in PBS or any other suitable buffer prior to injection, or detached from the culture flasks, washed with and re-suspended in PBS or any other suitable buffer, and then injected.

[0166] 10. Bleomycin (BLM)-Induced Pulmonary Fibrosis:

[0167] To test the immunomodulatory and anti-fibrotic effect of AAT-MSCs in vivo, a mouse model of bleomycin-induced pulmonary fibrosis is performed as described in Tashiro et al., Translational Science 2015.

[0168] In brief, BLM lung fibrosis is induced in C57BL/6 mice. After the administration of anesthesia, BLM sulfate (Sigma-Aldrich) dissolved in 50 μL of sterile saline at 2.5 U per kg of body weight is administered by direct intratracheal instillation via intubation. 24 to 72 hours after BLM administration, each animal receives 200 μL either PBS (control), 1×10.sup.6 non-transduced MSCs, or 1×10.sup.6 AAT-transduced MSCs in 200 μL PBS by tail vain injection or intratracheal administration. Serum AAT-levels are monitored by retro-orbital blood collection every second day and subsequent detection of AAT protein by ELISA.

[0169] Mice are killed at 14-21 days after BLM administration.

[0170] Left lung lobes are harvested from mice for protein and messenger RNA (mRNA) analysis. For morphometry and histology studies, right lung lobes are fixed by immersion in 10% neutral-buffered formalin for 24 hours and then transferred to PBS at 4° C. Samples are paraffin embedded, and sections are obtained for hematoxylin-eosin and Masson trichrome staining. Pulmonary fibrosis is assessed using the semiquantitative Ashcroft method on Masson trichrome-stained slides (Ashcroft et al., Journal of Clinical Pathology 1988).

[0171] Refer FIG. 6 for an overview of the experimental design.

[0172] At the time point of 21-day sacrifice, BLM mice without MSC treatment demonstrate lung fibrosis by Ashcroft score, whereas mice treated with either MSCs or AAT-MSCs showed decreased fibrosis. The decrease was more marked in the group receiving MSCs expressing AAT.

[0173] 11. Cyclophosphamide-Accelerated Type 1 Diabetes:

[0174] To assess the effect of AAT-MSCs on the development of diabetes in vivo, a mouse model of cyclophosphamide-accelerated type 1 diabetes is performed (adapted from Brode et al., The Journal of Immunology 2006).

[0175] NOD mice are obtained, where the incidence of diabetes in female mice is 75% by 40 weeks of age. To accelerate and synchronize diabetes, female 8-week-old NOD mice are treated with a single i.p. injection of cyclophosphamide (CY) (200 mg/kg body weight in 0.9% normal saline). Mice are then randomly divided into treatment and control groups. One to five days after cyclophosphamide treatment, each animal receives 200 μL either PBS (control), 1×10.sup.6 non-transduced MSCs, or 1×10.sup.6 AAT-transduced MSCs in 200 μL PBS by tail vain or intraperitoneal injection. Mice are monitored weekly for hyperglycemia until they become diabetic, as defined by two consecutive (>24 hr apart) nonfasting blood glucose levels >240 mg/dl.

[0176] All control mice receiving CY and PBS developed diabetes within 30 days, whereas the onset of diabetes in mice treated with either MSCs or AAT-MSCs was delayed. Interestingly, in mice treated with AAT-MSCs, the delay in development of diabetes was increased by 2 weeks compared to non-modified MSCs.

[0177] 12. MSU/C16.0-Induced Gouty Arthritis:

[0178] To assess the anti-inflammatory effect of AAT-MSCs on gouty arthritis in vivo, a mouse model of MSU/C16.0-induced gouty arthritis is performed (adapted from Joosten et al., Annals of the Rheumatic Diseases 2015).

[0179] Male C57131/6 mice are obtained from Jackson Laboratories (Bar Harbor, Me., USA) and are used at 10-12 weeks. One to three days prior to induction of gouty arthritis, each animal receives 200 μL either PBS (control), 1×10.sup.6 non-transduced MSCs, or 1×10.sup.6AAT-transduced MSCs in 200 μL PBS by intraperitoneal administration. Joint inflammation is induced by intra-articular injection of 300 pg MSU crystals mixed with 200 μM C16.0/bovine serum albumin (BSA) in 10 μL PBS into the right knee joint of naive mice. Four hours after intra-articular injection, macroscopic joint swelling is determined. Synovial tissue is isolated and either cultured for 2 hours in tissue culture medium at 37° C. or transferred directly into 200 μL Tritonx 100 (0.5% in PBS). In addition, knee joints are removed for histology.

[0180] Treatment with either non-modified or AAT-MSCs suppressed MSCU/C16.0 induced joint inflammation; however, inflammation was more markedly decreased when mice were treated with AAT-MSCs.

[0181] 13. Effect of AAT-MSCs on GvHD Prevention in an MHC Matched, Minor Antigen Disparate Murine Transplant Model:

[0182] For the assessment of a possible anti-GvHD effect of AAT-MSCs, a murine transplant model (MHC matched, minor antigen disparate) is performed (adapted from Marcondes et al., Blood 2011).

[0183] C57/BL6J mice (H-2.sup.b; The Jackson Laboratory), 10-14 weeks old with average body weight of 28 g, receive single-dose total body irradiation with 1000 cGy followed by intra-tail vein injection of T-cell depleted bone marrow (5×10.sup.6 cells), and CD8+ splenic lymphocytes (0.2×10.sup.6 cells) from C3H.Math.SW-H2b/SnJ donors (H-2bc; The Jackson Laboratory). Mice are randomly divided into treatment and control groups. Mice in the experimental group are given either 1×10.sup.6 non-transduced MSCs or 1×10.sup.6 AAT-transduced MSCs in 200 μL PBS by intraperitoneal or intra-tail vein injection before irradiation and donor cell infusion. Mice in the control group are injected, also intraperitoneally or intravenously, with 200 μL PBS. GvHD is assessed by a standard scoring system (Cooke et al., Blood 1996): Body weights were obtained and recorded on day 0 and weekly thereafter. A weekly clinical index is generated by summation of 5 criteria scores: percentage of weight change, posture (hunching), activity, fur texture, and skin integrity (maximum score=10). Blood samples are collected sequentially for cytokine assays.

[0184] Treatment with either non-modified MSCs or AAT-MSCs results in attenuation or prevention of GvHD and superior survival compared to control mice. Interestingly, the beneficial effect was more prominent in AAT-MSCs compared to native MSCs.