New Law

Abstract

The present invention relates to a method for expanding a population of regulatory T cells in a tissue or organ of a subject, comprising administration of IL-2 and a targeting moiety specific for said tissue or organ, wherein said tissue or organ is the lung.The invention further relates to a population of regulatory T cells generated according to the method and the generation of said population in vivo.Also provided is a pharmaceutical composition comprising IL-2 and a targeting moiety as defined herein, and a method for treating a disease or disorder mediated by inflammation or for reducing inflammation comprising administration of a method as defined herein or a pharmaceutical composition as defined herein.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for expanding a population of regulatory T cells in a tissue or organ of a subject, comprising administration of IL-2 and a targeting moiety specific for said tissue or organ, wherein said tissue or organ is the lung.The invention further relates to a population of regulatory T cells generated according to the method and the generation of said population in vivo.Also provided is a pharmaceutical composition comprising IL-2 and a targeting moiety as defined herein, and a method for treating a disease or disorder mediated by inflammation or for reducing inflammation comprising administration of a method as defined herein or a pharmaceutical composition as defined herein. [Background technology]

[0002] BACKGROUND OF THEINVENTION Acute respiratory distress syndrome (ARDS) can be triggered by a variety of infectious stimuli. Despite the large number of possible contributing factors, in each case the pathology converges on several conserved inflammatory pathways that lead to inappropriate inflammation. For example, severe type 1 inflammation in the lungs can cause loss of lung function and is potentially fatal. Type 1 inflammation is associated with multiple non-infectious modalities such as chronic obstructive pulmonary disease (COPD). However, severe respiratory infections are also immune pathologies driven by type 1 inflammation. Such infections include seasonal influenza, H5N1 (avian influenza), H1N1 (swine flu), SARS (severe acute respiratory syndrome), MERS (Middle East respiratory syndrome), and COVID-19. Responses to pandemics involving novel respiratory viruses typically include the development of new vaccines to prevent infection or the use of immunosuppression to prevent immune pathologies in patients. Vaccines are highly effective and specific, but their development time entails significant delays. In contrast, immunosuppressive therapies are available sooner due to the use of generic / known therapeutics, e.g., dexamethasone, but the high level of genericity makes it difficult to achieve optimal treatment, as systemic immunosuppression prevents the development of a healthy immune response against the infecting virus.

[0003] Thus, there is a great need for therapeutics that provide a virus-agnostic approach that allows for rapid deployment and prevents pathology while still allowing for the generation of antibodies. Summary of the Invention

[0004] (Summary of the invention) According to a first aspect of the present invention there is provided a method of expanding a population of regulatory T cells in a tissue or organ of a subject in need thereof, the method comprising administration of IL-2 and a targeting moiety specific for said tissue or organ, wherein said tissue or organ is the lung.

[0005] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising IL-2 and a targeting moiety specific for a tissue or organ of a subject, said targeting moiety being specific for the lung.

[0006] According to a further aspect of the present invention there is provided a method for treating a disease or disorder mediated by inflammation and / or for reducing inflammation comprising administering to a subject in need thereof a method as defined herein or a pharmaceutical composition as defined herein. [Brief description of the drawings]

[0007] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Parabiotic analysis of pulmonary regulatory T cell dynamics. A) Parabiotic surgery was performed in Foxp3Thy1.1CD45.1 and Foxp3Thy1.1CD45.2 mice and brain and blood CD4 T cells were analyzed at 1, 2, 4, 8 and 12 weeks (n=12, 12, 18, 16, 14). B) Best fit curves of pulmonary Tregs showing CD69- and CD69+ populations. [Diagram 2]Figure 2: Local expression of IL-2 promotes lung-specific expansion of regulatory T cells. A) Wild-type littermates and Scgb1a1-ERT2Cre RosaIL2 mice were treated with tamoxifen on day -14 to induce IL-2 production by non-ciliated club cells in bronchioles. Flow cytometry was used to assess the frequency of Foxp3+ cells among CD4 T cells in blood, lung, spleen, lymph nodes, and lung-draining mediastinal lymph nodes. n=5 mice / group. B) Male Scgb1a1-ERT2Cre RosaIL2 mice aged 19–21 weeks were administered 0.2 mg / gram tamoxifen three times by oral gavage. Organs were harvested for flow analysis on days 0 and 12 and after. Quantitative data for percentage of Tregs were gated in FlowJo and plotted using GraphPad prism. All data were analyzed using two-way Anova. *P<0.05, **P<0.01, and ***P<0.001 were considered significant. n=5 animals / group. C) Male Scgb1a1-ERT2Cre RosaIL2 mice aged 19-21 weeks were administered 0.2 mg / gram tamoxifen three times by oral gavage. Lungs were processed for qPCR Taqman analysis of IL-2 after days 0, 1, 4, 7, 15, 21, 29, and 35. Values ​​were first estimated by generating a standard curve using Thermofisher ProQuantum software and plotted using GraphPad prism. All data were analyzed using two-way Anova and T-tests on day 7. *P<0.05, **P<0.01, and ***P<0.001 were considered significant. n=5 animals / group. D) Lymph nodes from animals in C were also processed for qPCR Taqman analysis of IL-2. All data were analyzed using two-way Anova. *P<0.05, **P<0.01, and ***P<0.001 were considered significant. n=5 animals / group. E) Male Scgb1a1-ERT2Cre RosaIL2 mice aged 19-21 weeks were administered 0.2 mg / gram tamoxifen three times by oral gavage. Lungs were processed for flow cytometry analysis after days 0, 1, 4, 7, 15, 21, 29, and 35.Data were analyzed using FlowJo and percentage values ​​were obtained after gating on Foxp3+, CD4+, CD3+ cells and plotted using GraphPad prism. All data were analyzed using two-way Anova. *P<0.05, **P<0.01, and ***P<0.001 were considered significant. n=5 animals / group. F) Lymph nodes from animals in E were also processed for flow cytometry analysis and analyzed similarly. [Diagram 3] Figure 3: Local expression of IL-2 prevents influenza-induced neutrophil infiltration. Wild-type littermates and Scgb1a1-ERT2Cre RosaIL2 mice were treated with tamoxifen on day -14 to induce IL-2 production by non-ciliated club cells in the bronchioles. Mice were then infected intranasally with murine influenza and cohorts were dissected at days 0, 12 and 21 post-infection. Flow cytometry was used to assess the frequency of neutrophils in the lungs of infected mice. n=5–6 mice / group. [Figure 4] Figure 4: Intranasal administration of AAV6.2-mCC10-IL2 leads to local expansion of regulatory T cells (Tregs) in the lung. 12-week-old male mice were administered 1011 (vg / ml) AAV6.2-mCC10-IL2 or PBS, and on day 14, organs were harvested for flow cytometry analysis. Data were analyzed using FlowJo, percentage values ​​were obtained, and plotted using GraphPad prism. All data were analyzed using two-way Anova between groups. *P<0.05, **P<0.01, and ***P<0.001 were considered significant. n=5 animals / group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Invention According to a first aspect of the present invention there is provided a method of expanding a population of regulatory T cells in a tissue or organ of a subject in need thereof, the method comprising administration of IL-2 and a targeting moiety specific for said tissue or organ, wherein said tissue or organ is the lung.

[0009] In one embodiment, the methods defined herein comprise expanding a population of cells, such as a population of regulatory T cells, hi a further embodiment, said expansion of a population of cells, such as a population of regulatory T cells, is in a tissue or organ of a subject in need thereof, such as a particular tissue or organ of interest.

[0010] Reference herein to the terms "expanding", "expansion" and "expanded" or the phrases "expanded regulatory T cell population" and "expanded population of regulatory T cells" includes reference to a population of cells that is larger than a non-expanded population or that contains more cells than a non-expanded population. Thus, it will be understood that such "expanded" populations generated according to the methods defined herein contain more cells than a population that has not been subjected to IL-2. Thus, in certain embodiments, an expanded population of cells generated according to the methods defined herein, such as an expanded population of regulatory T cells, contains more cells compared to a reference population of cells. In one embodiment, the reference population of cells can be a population of cells that has not been subjected to or administered IL-2. In one embodiment, the expanded population of cells contains more cells than the population prior to administration of IL-2. In further embodiments, the reference population of cells can be located in a different tissue or organ than the expanded population of cells generated according to the methods defined herein. In further embodiments, the expanded population of cells is an expanded population within a tissue or organ of interest that contains more cells compared to a population of cells that is not located in said tissue or organ of interest. For example, an expanded population of cells, particularly a expanded population of regulatory T cells, expands in the lung relative to populations of cells, particularly regulatory T cells, in the blood, spleen, liver and / or lymph nodes.

[0011] In one embodiment, an expanded population of cells generated according to the methods defined herein, such as an expanded population of regulatory T cells, comprises at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold or more than a population of cells that have not been subjected to or administered IL-2. In a further embodiment, the expanded population of cells comprises at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold or more than a population of cells that are not located in a tissue or organ of interest. In certain embodiments, the expanded population of cells is at least 2-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than a reference population, such as a population of cells in a tissue or organ of interest that has not been subjected to or administered IL-2, or a population of cells not located in a tissue or organ of interest. In some embodiments, the expanded population of cells comprises a greater proportion of cells that constitute a subset of the population (e.g., a greater proportion of regulatory T cells within the total population of T cells in the tissue or organ). In further embodiments, the expanded population of cells is maximal at 21 days after administration, i.e., the population of cells maximally expands at 21 days after administration. In some embodiments, the expanded population of cells can be detected 4-35 days after administration, i.e., the expansion of the population of cells can be detected 4-35 days after administration. Thus, in one embodiment, the expanded population of cells / expansion of the population of cells can be detected 4 days after administration. In a further embodiment, the expansion of the expanded population of cells / population of cells can be detected 35 days after administration, hi a further embodiment, maximum expansion of the cell population can be detected 21 days after administration.

[0012] It will therefore be appreciated that the expanded population of regulatory T cells defined herein may expand depending on the dose of IL-2 administered, and thus in one embodiment, the expanded population of regulatory T cells defined herein comprises a population that is IL-2 dose-dependent fold greater than a reference population.

[0013] In a further embodiment, the expanded population of regulatory T cells generated according to the methods defined herein comprises a population of cells with increased viability. Thus, in one embodiment, the expanded population of regulatory T cells generated according to the methods defined herein comprises increased viability. In a further embodiment, the expanded population of regulatory T cells comprises reduced or decreased cell death. In a further embodiment, the expanded population of regulatory T cells comprises increased proliferation. Thus, in one embodiment, the expanded population of regulatory T cells is greater than a reference population (e.g., a population of regulatory T cells that have not been subjected to or administered IL-2 or a population of cells that are not located in a tissue or organ of interest) due to increased viability of the expanded population of regulatory T cells. In a further embodiment, the expanded population of regulatory T cells is greater than a reference population due to reduced or decreased cell death in the expanded population of regulatory T cells. In a further embodiment, the expanded population of regulatory T cells is greater than a reference population due to increased proliferation. In a further embodiment, the expanded population of regulatory T cells is greater than a reference population due to a combination of one or more of increased viability, reduced / reduced cell death and increased proliferation.

[0014] It will be understood that reference herein to an "expanded population" generated according to the methods defined herein, such as an "expanded population of regulatory T cells", may also include a population of activated cells. Reference herein to "expansion" may include activation of a population of cells generated according to the methods defined herein, such as a population of regulatory T cells. Similarly, "expansion" also includes the expansion of an activated population of regulatory T cells, e.g., a population that is already activated prior to administration of IL-2. Such activation of a population of cells generated according to the methods defined herein, such as a population of regulatory T cells, may be independent of the expansion or may be associated with the expansion of said population. Thus, in one embodiment, the expanded population of regulatory T cells includes activated regulatory T cells. In a further embodiment, the expanded population of regulatory T cells is an activated population of regulatory T cells.

[0015] In an alternative embodiment, reference to "expanded" or "expanded population" generated according to the methods defined herein does not include activation of said population or activated population of cells. Thus, according to this embodiment, an expanded population of cells generated according to the methods defined herein, such as an expanded population of regulatory T cells, does not include an activated phenotype. In a further embodiment, an expanded population of regulatory T cells does not include activated regulatory T cells. Thus, in a further embodiment, an expanded population of regulatory T cells includes a phenotype, such as a surface phenotype, of a population of regulatory T cells that have not been subjected to or administered IL-2.

[0016] Regulatory T cells (also called Tregs) are a subpopulation of T cells that regulate the immune system, maintain tolerance, and prevent autoimmune diseases. They generally suppress or downregulate the activation and / or proliferation of effector T cells, and have been shown to have utility in immunosuppression. Regulatory T cells are therefore very promising cells that combine multiple immunosuppressive and regenerative capabilities, and there is great interest in using exogenous regulatory T cells as cell therapy or exogenous factors to stimulate, activate or expand endogenous regulatory T cells. We have demonstrated that pulmonary regulatory T cells recirculate approximately every 6 weeks (Figure 1), and thus altering the dynamics of homeostasis in this tissue provides a window for expanding these cell populations in the lung for immunosuppressive treatments, such as anti-inflammatory treatments.

[0017] Thus, in one embodiment, the expanded population of regulatory T cells generated according to the methods defined herein comprises an increased anti-inflammatory potential. Such increased anti-inflammatory potential may be compared to a non-expanded population of regulatory T cells, such as a non-expanded population of regulatory T cells present in a tissue or organ, or a population of regulatory T cells present elsewhere than the tissue or organ of interest. In one embodiment, the expanded population of regulatory T cells comprises a phenotype similar to the non-expanded regulatory T cells in the tissue or organ of interest, or to regulatory T cells from elsewhere than the tissue or organ of interest. Such phenotypes may include surface marker phenotypes, transcriptome phenotypes / signatures (e.g., gene expression signatures), gene and / or protein expression profiles, and cytokine expression profiles. Thus, in certain embodiments, the expanded population of regulatory T cells comprises or retains the anti-inflammatory potential of the non-expanded population of regulatory T cells or the expanded population of regulatory T cells prior to expansion. In further embodiments, the expanded population of regulatory T cells comprises or retains the anti-inflammatory potential of a population of regulatory T cells from elsewhere than the tissue or organ of interest.

[0018] Reference herein to the phrase "in a tissue or organ" refers to a discrete location in a subject, such as within a particular tissue or organ. It will be understood that such terms do not relate to cases where the effect occurs systemically or outside the tissue or organ of interest, or where a cell type or cell population that is not located in the tissue or organ of interest is affected (e.g., proliferates or is activated). Thus, in one embodiment, the population of regulatory T cells generated according to the methods defined herein is affected (e.g., proliferates) in a particular tissue or organ, i.e., locally. In a further embodiment, the population of regulatory T cells is affected (e.g., proliferates) only in a particular tissue or organ. In a further embodiment, the population of regulatory T cells that is located outside the tissue or organ of interest, i.e., not in the tissue or organ, is not affected (e.g., does not proliferate). Thus, in certain embodiments, the systemic or peripheral population of regulatory T cells is not affected (e.g., does not proliferate). In a further embodiment, the population of regulatory T cells in the lymph nodes and / or spleen is not affected. In a further embodiment, the population of regulatory T cells in the blood is not affected. In a further embodiment, the population of regulatory T cells in the liver is not affected.

[0019] A tissue or organ as defined herein includes a discrete location of a body or organism. For example, a tissue or organ may include a lung. Thus, in a particular embodiment, the tissue or organ is a lung.

[0020] IL-2 is a key population regulator of regulatory T cells. Regulatory T cells have a naturally high turnover frequency compared to other T cells with rapid proliferation and high apoptosis rates. IL-2 can increase the frequency of regulatory T cells through induction of the anti-apoptotic protein Mcl1, which reduces the rate of Bim-dependent apoptosis (Pierson et al., 2013, doi: https: / / doi.org / 10.1038 / ni.2649). Thus, increasing IL-2 levels can expand the size of the regulatory T cell population (Liston and Gray et al., 2014, doi: https: / / doi.org / 10.1038 / nri3605). IL-2 delivery has been shown to be a promising anti-inflammatory measure through the expansion of this regulatory T cell population in multiple preclinical studies, and optimization of IL-2 delivery is being investigated clinically. Thus, in the context of the lung, local delivery of IL-2 would provide a very promising treatment for inflammation by increasing the number of lung-specific proliferation or regulatory T cells without the deleterious effects of systemic immunosuppression. In the context of treating respiratory infections, this ability would be understood to be important, since systemic immunosuppression would inhibit the body's ability to make antibodies, allowing viruses to spread. Thus, the present invention provides the ability to block harmful inflammation in the lungs while allowing systemic antibody production.

[0021] Thus, according to one embodiment of the invention, a method is provided for expanding a population of regulatory T cells in a tissue or organ of a subject in need thereof, thereby avoiding extensive peripheral or systemic immunosuppression that would be intolerable to the patient, e.g., due to reduced production of antibodies against infectious agents of pulmonary immune pathology or increased risk of secondary infection.

[0022] It will be understood that references herein to "administration" refer to providing or making available IL-2 at a discrete location or site of an organism, such as a specific tissue or organ. Such administration is therefore similar to the definition of "in a tissue or organ" as set out herein above. Thus, in one embodiment, administration of IL-2 includes administration to or within a specific tissue or organ. In certain embodiments, administration of IL-2 includes expression of IL-2 in a specific tissue or organ (e.g., lung), i.e., tissue- or organ-specific expression of IL-2 in said tissue or organ of said subject. In one embodiment, administration includes expression of a gene encoding IL-2 in a specific tissue or organ (e.g., lung). Thus, in one embodiment, administration of IL-2 includes tissue- or organ-specific expression of IL-2 in said tissue or organ of said subject. In certain embodiments, the tissue- or organ-specific expression of IL-2 is lung. In some embodiments, the tissue- or organ-specific expression of IL-2 is maximal 15 days after administration. In some embodiments, tissue or organ specific expression of IL-2 can be detected 4-35 days after administration. Thus, in one embodiment, tissue or organ specific expression of IL-2 can be detected 4 days after administration. In a further embodiment, tissue or organ specific expression of IL-2 can be detected 35 days after administration. In a further embodiment, maximum tissue or organ specific expression of IL-2 can be detected 15 days after administration. In a further embodiment, expression of IL-2 is not detectable outside of the tissue or organ of interest, such as in the periphery (e.g., in the blood). In a further embodiment, expression of IL-2 is not detectable in lymph nodes and / or spleen. In a further embodiment, expression of IL-2 is expression that is restricted to a particular tissue or organ of interest. In a further embodiment, expression of IL-2 is tissue or organ specific expression. Thus, in certain embodiments, tissue or organ specific expression of IL-2 is driven by a tissue or organ specific promoter. In some embodiments, administration or expression of IL-2 can be in two or more tissues or organs of interest. In one embodiment, administration or expression of IL-2 is in one, two, or more relevant tissues or organs (eg, lung and airway tissues).In another embodiment, administration or expression of IL-2 is in one, two, or more tissues or organs that are not considered to be involved.

[0023] Furthermore, references herein to "administration" and "expression" also refer to the case where IL-2 is provided to a population of cells of a tissue or organ. Such provision of IL-2, in one embodiment, includes administration of IL-2 in protein or peptide form to or within the tissue or organ of interest, i.e., locally. In a further embodiment, provision of IL-2 includes expression of IL-2 in cells of the tissue or organ of interest. Thus, in certain embodiments, expression of IL-2 includes cells of the tissue or organ of interest that express IL-2, such as cells that compose said tissue or organ. In some embodiments, expression of IL-2 includes epithelial cells; airway epithelial cells (e.g., goblet cells, ciliated cells, club cells, neuroendocrine cells (neuroendocrine), basal cells, intermediate (parabasal) cells, serous cells, brush cells, specialized type cells with numerous intracytoplasmic membrane-bound inclusions, non-ciliated columnar cells, and metaplastic cells); and alveolar cells (e.g., type 1 and type 2 pneumocytes, transitional type 1 and type 2 pneumocytes, and cuboidal non-ciliated cells). In one embodiment, expression of IL-2 includes airway epithelial cells, e.g., club cells, and / or alveolar cells, e.g., type 2 alveolar cells. In a particular embodiment, expression of IL-2 includes club cells. In a further embodiment, expression of IL-2 includes expression in cells other than regulatory T cells that constitute the expanded population of regulatory T cells generated according to the methods defined herein. Thus, in a further embodiment, expression of IL-2 is not within the regulatory T cell population generated according to the methods defined herein.

[0024] In an alternative embodiment, the administration or expression of IL-2 comprises introducing an exogenous sequence encoding IL-2 into cells of the tissue or organ. Thus, in one embodiment, the administration or expression of IL-2 comprises expression from an exogenous sequence. In a further embodiment, the administration or expression of IL-2 comprises expression from a transgene. In a further embodiment, the transgene comprises a gene or element encoding IL-2. In a particular embodiment, the exogenous sequence is an IL-2 coding sequence. In a further embodiment, the transgene comprises an IL-2 coding sequence or gene.

[0025] In one embodiment, the exogenous sequence encoding IL-2 is in the form of a transgene comprising a tissue or organ specific promoter. Such tissue or organ specific promoters are known in the art and include promoters that drive expression of tissue or organ specific genes. In one embodiment, the transgene comprises a tissue or organ specific promoter that drives expression specifically in a tissue or organ of interest. In a further embodiment, the transgene comprises a tissue or organ specific promoter that does not result in expression in a tissue or organ other than the tissue or organ of interest. Thus, in one embodiment, the tissue or organ specific promoter is a lung specific promoter. In a further embodiment, the transgene comprises a promoter that drives expression specifically in cells of the lung. In a further embodiment, the transgene comprises a promoter that drives expression specifically in epithelial cells, airway epithelial cells and / or alveolar cells as defined herein above. In one embodiment, the transgene comprises a promoter that drives expression specifically in the lung. In a particular embodiment, the transgene comprises a promoter that drives expression specifically in airway epithelial cells, e.g., club cells, and / or alveolar cells, e.g., type 2 alveolar cells. In a further embodiment, the transgene comprises a promoter that drives expression specifically in club cells. In a further embodiment, the transgene comprises the promoter for surfactant protein B (SFTPB). In another embodiment, the transgene comprises the promoter for club cell specific protein (CC10).

[0026] Thus, in one embodiment, the tissue or organ specific promoter is the promoter of surfactant protein B (SFTPB). In another embodiment, the tissue or organ specific promoter is the promoter of club cell specific protein (CC10). In a particular embodiment, the tissue or organ specific promoter is the SFTPB promoter and drives expression specifically in club cells and / or type 2 alveolar cells. In a further embodiment, the tissue or organ specific promoter is the CC10 promoter and drives expression specifically in club cells.

[0027] In a further embodiment, the administration or expression of IL-2 includes a transgene that includes an element that promotes or induces expression of IL-2 in the presence of an exogenous compound. Such elements that promote or induce expression are known in the art and include, for example, the tetracycline (Tet) inducible system. Tet inducible systems provide reversible control of transcription and utilize a tetracycline-controlled transactivator (tTA) that binds to a tetracycline operator (TetO) sequence contained in a tetracycline response element (TRE) that is placed upstream of the gene / coding region of interest (and its promoter, such as a tissue-specific promoter). These can be either TetOff or TetOn systems. The TetOff system of inducible expression (also called the tTA-dependent system) uses the tTA protein, created by fusing the tetracycline repressor (TetR) found in E. coli to the activation domain of another protein, VP16, found in herpes simplex virus. The resulting tTA can bind to the TetO sequence in the TRE in the absence of tetracycline and promote expression of the downstream gene / coding region. In the presence of tetracycline, tTA binding to the TetO sequence is prevented, resulting in reduced gene expression. Conversely, the TetOn system (also called the rtTA-dependent system) uses a reverse Tet repressor (rTetR) to create a reverse tetracycline-controlled transactivator (rtTA) protein, which depends on the presence of tetracycline to promote expression. Thus, rtTA only binds to the TetO sequence in the TRE and promotes expression in the presence of tetracycline. Specific examples of the TetOn system include, but are not limited to, Life Technologies' TetOn Advanced, TetOn3G, and T-REx systems. Derivatives and analogs of tetracycline can be used with either the TetOff or TetOn systems, including, but not limited to, doxycycline. It will be appreciated that such derivatives / analogs offer significant advantages over tetracycline, such as increased stability in the case of doxycycline.Thus, in one embodiment, the exogenous sequence encoding IL-2, such as a transgene comprising a tissue or organ specific promoter, further comprises a tetracycline response element (TRE). Thus, in one embodiment, administration or expression of IL-2 is tetracycline dependent or tetracycline inducible. In a further embodiment, administration or expression of IL-2 comprises introducing an exogenous sequence encoding a reverse tetracycline controlled transactivator (rtTA) into cells of the tissue or organ. In one embodiment, the exogenous sequence encoding rtTA comprises a tissue or organ specific promoter, i.e., expression of the rtTA coding sequence is under the control of a tissue or organ specific promoter disclosed herein. Thus, in a further embodiment, the exogenous sequence encoding rtTA comprises a lung specific promoter. In a further embodiment, expression of the rtTA coding sequence is under the control of a lung specific promoter. In a particular embodiment, the exogenous sequence encoding rtTA comprises a promoter that specifically promotes expression in airway epithelial cells, e.g., club cells, and / or alveolar cells, e.g., type 2 alveolar cells. In a further embodiment, the exogenous sequence encoding rtTA comprises a promoter of surfactant protein B (SFTPB). In a further embodiment, the exogenous sequence encoding rtTA comprises a promoter of club cell specific protein (CC10). Such an exogenous sequence encoding rtTA may be separate from the exogenous sequence encoding IL-2, e.g., separate from an IL-2 transgene that includes a tissue-specific or organ-specific promoter. Alternatively, such an exogenous sequence encoding rtTA may be included with an IL-2 coding sequence, e.g., within the same transgene. Thus, in some embodiments, administration or expression of IL-2 comprises the TetOn system. Thus, it will be appreciated that in one embodiment, administration or expression of IL-2 comprises administration of tetracycline or a derivative / analog of tetracycline, such as doxycycline.

[0028] The use of tetracycline-dependent or tetracycline-inducible administration or expression of IL-2 provides another level of control, allowing the administration or expression of IL-2 to be "switched" on or off. It will be appreciated that such switching is advantageous in the methods described herein by allowing the expansion of regulatory T cell populations in a tissue or organ to be controlled in time. For example, expression of IL-2 can be switched "on" by administering tetracycline or a derivative / analog thereof when pulmonary inflammation, such as type 1 inflammation, is detected / diagnosed. Alternatively, expression of IL-2 can be switched "on" after infection, such as infection with a respiratory virus. Expression of IL-2 can then be switched "off" by removing tetracycline or a derivative / analog thereof when inflammation, such as type 1 inflammation, is no longer detectable or is reduced. Expression can also be switched "off" after the subject is deemed no longer at risk of infection, such as infection with a respiratory virus. Said use of tetracycline-dependent or tetracycline-induced administration or expression of IL-2 further provides for dose-dependent administration or expression of IL-2. For example, the level and / or amount of IL-2 administration or expression can be modified and / or titrated in a tissue or organ so as to be dependent on the level and / or amount of inflammation, e.g., type 1 inflammation, in the tissue or organ. Thus, IL-2 expression can be "switched on" by administering a certain dose of tetracycline or a derivative / analogue thereof when pulmonary inflammation, such as type 1 inflammation, is detected / diagnosed, and said dose can be increased if inflammation persists. Similarly, said dose can be decreased if inflammation decreases after an initial administration of tetracycline or a derivative / analogue thereof.

[0029] It will be appreciated that according to embodiments in which administration or expression of IL-2 comprises the TetOn system, the presence of a tissue or organ specific promoter to control the expression of IL-2 may not be required. For example, expression of the rtTA-coding sequence may be under the control of a tissue or organ specific promoter, but expression of IL-2 is under the control of a tetracycline response element (TRE) rather than a tissue or organ specific promoter. Thus, in one embodiment, administration or expression of IL-2 is tetracycline-dependent or tetracycline-inducible, and the exogenous sequence encoding rtTA comprises a lung specific promoter (e.g., SFTPB promoter or CC10 promoter), but the exogenous sequence encoding IL-2 does not comprise a lung specific promoter. In a further embodiment, administration or expression of IL-2 is tetracycline-dependent or tetracycline-inducible, and expression of the rtTA-coding sequence is under the control of a lung specific promoter (e.g., SFTPB promoter or CC10 promoter), but expression of the exogenous sequence encoding IL-2 is not under the control of a lung specific promoter.

[0030] In another embodiment, administration or expression of IL-2 is tetracycline-dependent or tetracycline-inducible, and both the exogenous sequence encoding rtTA and the exogenous sequence encoding IL-2 comprise a lung-specific promoter (e.g., the SFTPB promoter or the CC10 promoter). In a further embodiment, administration or expression of IL-2 is tetracycline-dependent or tetracycline-inducible, and expression of both the rtTA-coding sequence and the exogenous sequence encoding IL-2 are under the control of a lung-specific promoter (e.g., the SFTPB promoter or the CC10 promoter). In such an embodiment, expression of IL-2 is subject to an additional level of control to further ensure tissue- or organ-specific administration or expression.

[0031] In one embodiment, the transgene as defined herein is introduced into the cells of the tissue or organ of interest by transduction, such as transduction with a virus or viral vector. In a particular embodiment, the transduction uses an adeno-associated virus. Thus, in one embodiment, the administration of IL-2 comprises transduction, such as viral transduction. In a further embodiment, the administration of IL-2 comprises adeno-associated viral transduction.

[0032] In one embodiment, transduction of the transgene as defined herein utilizes a viral vector that specifically targets or infects the cells of the tissue or organ of interest. Thus, in one embodiment, transduction of the transgene as defined herein specifically targets or infects the cells of the tissue or organ of interest. According to this embodiment, it will be understood that transduction of the transgene as defined herein with a viral vector does not target or infect a population of regulatory T cells. In a further embodiment, transduction of the transgene as defined herein comprises a viral vector that is able to access the tissue or organ of interest and cross a barrier that separates the tissue or organ of interest from other tissues, organs or the rest of the organism. Thus, in one embodiment, transduction comprises a viral vector that is able to specifically target or infect the lung. In a further embodiment, transduction comprises a viral vector that is able to target or infect the cells of the lung.

[0033] In one embodiment, the transduction comprises a lung-targeting virus or viral vector or a virus or viral vector that infects the lung. Examples of lung-targeting viruses / viral vectors include, but are not limited to, AAV6 and its variants and derivatives (e.g., AAV6.2 and AAV6.2FF). In an embodiment, the transgene as defined herein is comprised in a viral vector, such as an adeno-associated viral vector (e.g., AAV6.2). In a further embodiment, the transduction comprises the adeno-associated viral variant AAV6 and its derivatives, such as AAV6.2 and AAV6.2FF. In a further embodiment, the transduction comprises an AAV6.2 viral vector. In a further embodiment, the transduction comprises an AAV6.2FF viral vector. In another embodiment, the transgene as defined herein is comprised in an AAV6.2 viral vector. In a further embodiment, the transgene as defined herein is comprised in an AAV6.2FF viral vector. Thus, in one embodiment, the transducing and / or viral vector comprises AAV6.2-SFTPB-IL2, an AAV6.2 derivative of AAV6 comprising a transgene comprising an IL-2 coding sequence and the lung-specific promoter SFTPB. In another embodiment, the transducing and / or viral vector comprises AAV6.2FF-SFTPB-IL2, an AAV6.2FF derivative of AAV6 comprising an IL-2 coding sequence and a transgene comprising an SFTPB lung-specific promoter. In a further embodiment, the transducing and / or viral vector comprises AAV6.2-CC10-IL2, an AAV6.2 derivative of AAV6 comprising an IL-2 coding sequence and a transgene comprising a CC10 lung-specific promoter. In a further embodiment, the transducing and / or viral vector comprises AAV6.2FF-CC10-IL2, an AAV6.2FF derivative of AAV6 comprising an IL-2 coding sequence and a transgene comprising a CC10 lung-specific promoter. Viral vectors can be used to integrate a target sequence, such as a transgene, into a host cell genome, such as the genome of the cells of a tissue or organ of interest.Thus, in one embodiment, transduction involves the integration of a transgene as defined herein into the genome of cells of a tissue or organ of interest, such that long-term expression of the transgene in the tissue or organ is achieved. Viral vectors, such as adeno-associated viral vectors, may also be used to allow stable or long-term expression without integrating the target sequence into the host cell genome. Thus, in one embodiment, the transgene and / or target sequence are stably maintained outside the host cell genome.

[0034] Reference herein to "virus" and / or "viral vector" includes viruses that are non-lytic or lysogenic. It will be understood that such viruses achieve the introduction of a transgene into cells without infection of, or death or destruction of, cells, such as cells of a tissue or organ of interest.

[0035] It will be appreciated from the disclosure presented herein that the combination of a virus or viral vector that specifically targets or infects cells of a tissue or organ of interest (e.g., a lung-specific virus or viral vector) with a promoter that drives expression specifically in cells of the tissue or organ of interest provides exceptional specificity. Such specificity provides a so-called "double lock" that restricts both the cells that the transgene targets or infects and the cells in which the transgene is expressed. Thus, in one embodiment, the combination of a tissue or organ specific viral vector as defined herein with a tissue or organ specific promoter provides that only cells of the tissue or organ of interest contain a transgene as defined herein and are capable of expressing said transgene. In a further embodiment, the combination of a tissue or organ specific viral vector as defined herein with a tissue or organ specific promoter provides that only cells of the tissue or organ of interest contain an IL-2 encoding gene and are capable of expressing said gene.

[0036] In a further embodiment, the combination of a tissue or organ specific viral vector and a tissue or organ specific promoter as defined herein, together with an inducible element, such as a tetracycline inducible element, provides that only cells of the tissue or organ of interest contain a transgene as defined herein, and that only cells of the tissue or organ of interest are capable of expressing said transgene when an activator of the inducible element is administered (e.g., tetracycline or doxycycline). In one embodiment, the combination of a tissue or organ specific viral vector and a tissue or organ specific promoter as defined herein, together with an inducible element, such as a tetracycline inducible element, provides that only cells of the tissue or organ of interest contain an IL-2 encoding gene, and that only cells of the tissue or organ of interest are capable of expressing said gene when an activator of the inducible element is administered (e.g., tetracycline or doxycycline). In a further embodiment, the combination provides that only cells of the tissue or organ of interest contain an inducible IL-2 encoding gene and that only cells of the tissue or organ of interest are capable of expressing a reverse tetracycline-controlled transactivator (rtTA) gene that results in expression of IL-2 when an activator of the inducible element is administered (e.g., tetracycline or doxycycline).

[0037] Administration of IL-2 as defined herein may further include direct administration of IL-2 to the tissue or organ of interest. Examples of direct administration include direct injection into the tissue or organ of interest or utilizing a suitable delivery device. Such delivery devices are known in the art and, according to the present disclosure, allow for controlled and / or sustained administration of IL-2 during the treatment period (e.g., chronically or during the treatment of an acute inflammatory disease or disorder). For administration / delivery to the lungs, inhalation and / or intranasal delivery may be utilized, for example, using an inhaler or nebulizer, or by spraying an atomized solution containing IL-2 and / or a viral vector as described herein.

[0038] The duration of IL-2 administration as defined herein can vary depending on the treatment and characteristics of the particular inflammatory condition or disease being treated by the methods described herein. For example, administration of IL-2 can be chronic. Alternatively, administration of IL-2 can be within the duration of treatment of the disease or disorder, such as in the treatment of an acute inflammatory condition or infection. Thus, in certain embodiments, the duration of administration or expression of IL-2 depends on the disease or disorder being treated or the duration of treatment. In one embodiment, administration or expression of IL-2 is acute. In another embodiment, administration or expression of IL-2 is chronic.

[0039] It will be understood that IL-2 may be combined or co-administered with a tissue or organ specific targeting moiety. Thus, administration of IL-2 may include expression of IL-2 in a tissue or organ of interest as defined herein (e.g., tissue-specific or organ-specific expression) and may be combined with a targeting moiety specific to the tissue or organ of interest. Furthermore, administration of IL-2 may include administration of IL-2 in a protein or peptide form and may be combined with a targeting moiety specific to the tissue or organ of interest.

[0040] Reference herein to the term "targeting moiety" refers to any moiety that provides tissue- or organ-specific administration or expression of IL-2 as defined herein. It will be further appreciated that said targeting moiety provides localized administration or expression of IL-2 as defined herein.

[0041] Therefore, in one embodiment of the invention, the method defined herein comprises the administration of a targeting moiety specific for a tissue or organ of a subject. In a further embodiment, the targeting moiety specific for a tissue or organ of a subject localizes IL-2 at or to the tissue or organ of interest. Thus, in one embodiment, the targeting moiety specific for a tissue or organ of a subject localizes only IL-2 at or to the tissue or organ of interest. In a further embodiment, the targeting moiety specific for a tissue or organ of a subject prevents localization of IL-2 to other tissues or organs other than the tissue or organ of interest, or localizes IL-2 away from tissues or organs other than the tissue or organ of interest. In another embodiment, the targeting moiety provides expression of IL-2 in the tissue or organ of interest. Thus, in one embodiment, the targeting moiety specific for a tissue or organ of a subject provides expression of only IL-2 in the tissue or organ of interest. Such references herein to "in a tissue or organ of interest" further include cases where the effect is within cells that make up said tissue or organ (e.g., epithelial cells, airway epithelial cells and / or alveolar cells).

[0042] In one embodiment, the targeting moiety specific for a tissue or organ of interest is a virus or viral vector as defined herein. In a further embodiment, said virus or viral vector specifically targets or infects the tissue or organ of interest, or specifically targets or infects the cells of the tissue or organ of interest. Thus, according to this embodiment, said targeting moiety specific for a tissue or organ of interest is a virus or viral vector that does not target or infect cells of other tissues or organs other than the tissue or organ of interest, or does not target or infect cells that constitute tissues or organs other than the tissue or organ of interest. It will also be understood that according to this embodiment, said targeting moiety specific for a tissue or organ as defined herein does not target or infect a population of regulatory T cells. In a further embodiment, the targeting moiety specific for a tissue or organ of interest as defined herein comprises a virus or viral vector that can access the tissue or organ of interest and can cross a barrier that separates the tissue or organ of interest from other tissues, organs or the rest of the subject. Therefore, in one embodiment, the tissue or organ specific targeting moiety comprises a virus or viral vector that can specifically target or infect lungs. In a further embodiment, the tissue or organ specific targeting moiety comprises a virus or viral vector that can target or infect epithelial cells, airway epithelial cells and / or alveolar cells. In a particular embodiment, the tissue or organ specific targeting moiety comprises a virus or viral vector that can target or infect club cells. In another embodiment, the tissue or organ specific targeting moiety comprises a virus or viral vector that can target or infect type 2 alveolar cells.

[0043] In one embodiment, the tissue or organ specific targeting moiety comprises an adeno-associated virus. In a further embodiment, the targeting moiety is an adeno-associated virus selected from AAV6 and its variants / derivatives, such as AAV6.2 and AAV6.2FF. In a particular embodiment, the tissue or organ specific targeting moiety comprises the adeno-associated virus variant AAV6.2. In another embodiment, the tissue or organ specific targeting moiety comprises the adeno-associated virus variant AAV6.2FF. In an embodiment, the transgene as defined herein is comprised in a tissue or organ specific targeting moiety, such as an adeno-associated virus vector contained within an adeno-associated virus as defined herein. In one embodiment, the transgene as defined herein is comprised in an adeno-associated virus selected from AAV6 and its variants / derivatives, such as AAV6.2 and AAV6.2FF. Thus, in one embodiment, the transgene comprising the IL-2 coding sequence and the lung-specific promoter SFTPB is comprised within an AAV6.2 virus / viral vector of an AAV6 derivative, and the virus / viral vector is AAV6.2-SFTPB-IL2. In another embodiment, the transgene comprising the IL-2 coding sequence and the lung-specific promoter CC10 is comprised within an AAV6.2 virus / viral vector, and the virus / viral vector is AAV6.2-CC10-IL2. In a further embodiment, the transgene comprising the IL-2 coding sequence and the lung-specific promoter SFTPB is comprised within an AAV6.2FF virus / viral vector of an AAV6 derivative, and the virus / viral vector is AAV6.2FF-SFTPB-IL2. In a further embodiment, the transgene comprising the IL-2 coding sequence and the lung-specific promoter CC10 is comprised within an AAV6.2FF virus / viral vector, and the virus / viral vector is AAV6.2FF-CC10-IL2.

[0044] According to a further aspect of the present invention, there is provided a method for expanding a regulatory T cell population in a tissue or organ in vivo. It will be understood that embodiments of this aspect are equivalent and comparable to all embodiments previously described herein. Thus, in one embodiment, the term "subject" as used herein is synonymous with "in vivo."

[0045] In one embodiment, the method of expanding a regulatory T cell population in vivo in a tissue or organ comprises administration of IL-2 as described herein. In a further embodiment, the method of expanding a regulatory T cell population in vivo in a tissue or organ comprises administration of a targeting moiety specific for the tissue or organ of interest in vivo. In one embodiment, administration of IL-2, which may include expression of IL-2, is combined in vivo with a targeting moiety specific for the tissue or organ. In a further embodiment, the method of expanding a regulatory T cell population in vivo in a tissue or organ comprises a virus or viral vector comprising an IL-2 coding gene. In one embodiment, the virus or viral vector is capable of targeting or infecting a tissue or organ of interest. In a particular embodiment, the virus or viral vector capable of targeting or infecting a tissue or organ of interest specifically targets or infects cells of the tissue or organ of interest. In a further embodiment, the method of expanding a regulatory T cell population in vivo in a tissue or organ comprises a virus or viral vector comprising a tissue or organ specific promoter. Thus, in certain embodiments, the method of expanding a regulatory T cell population in a tissue or organ in vivo comprises administering a targeting moiety specific for the tissue or organ of interest, said targeting moiety being a virus or viral vector that specifically targets or infects the lung. In further embodiments, the method of expanding a regulatory T cell population in a tissue or organ in vivo comprises administering a targeting moiety specific for the tissue or organ of interest, said targeting moiety being specific for epithelial cells, airway epithelial cells, and / or alveolar cells. In further embodiments, the targeting moiety specific for the tissue or organ of interest is specific for club cells and / or type 2 alveolar cells. In another embodiment, a method of expanding a regulatory T cell population in a tissue or organ in vivo comprises administration of a lung-specific virus or viral vector comprising a transgene defined herein, e.g., administration of AAV6.2-SFTPB-IL2, AAV6.2FF-SFTPB-IL2, AAV6.2-CC10-IL2 or AAV6.2FF-CC10-IL2 defined herein.

[0046] According to one aspect of the invention there is provided a population of regulatory T cells expanded according to or obtained by the methods described herein. Thus, in one embodiment there is provided an expanded population of regulatory T cells expanded in a tissue or organ of a subject by administration of IL-2 and a targeting moiety specific for said tissue or organ.

[0047] Pharmaceutical Compositions According to one aspect of the present invention there is provided a pharmaceutical composition comprising IL-2 and a targeting moiety specific for a tissue or organ of a subject, said targeting moiety being specific for the lung.

[0048] In one embodiment, the pharmaceutical composition comprises IL-2 to promote the expansion of a population of regulatory T cells. In a further embodiment, the pharmaceutical composition comprises a targeting moiety specific for a tissue or organ of a subject. In one embodiment, the targeting moiety specific for a tissue or organ of a subject is a virus or viral vector that specifically targets or infects the cells of the tissue or organ and promotes tissue- or organ-specific expression of IL-2 as described herein. Thus, according to this aspect of the invention, there is provided a pharmaceutical composition comprising a tissue- or organ-specific viral vector that expands a population of regulatory T cells in said tissue or organ of a subject. In a particular embodiment, the pharmaceutical composition expands a population of regulatory T cells specifically or locally in a tissue or organ of interest of a subject.

[0049] In one embodiment, the pharmaceutical composition defined herein comprises a targeting moiety capable of passing through a barrier separating a tissue or organ of interest from other tissues or organs or the remainder of an organism. Thus, in one embodiment, the pharmaceutical composition defined herein comprises an adeno-associated virus selected from AAV6 and variants / derivatives thereof, such as AAV6.2 and AAV6.2FF. In a further embodiment, the pharmaceutical composition defined herein comprises the adeno-associated virus variant AAV6.2. In a further embodiment, the pharmaceutical composition defined herein comprises the adeno-associated virus variant AAV6.2FF. In a further embodiment, the viral vector comprised in the pharmaceutical composition defined herein comprises a gene, such as a transgene encoding IL-2. In a further embodiment, the transgene comprised in the viral vector of the pharmaceutical composition further comprises a tissue or organ specific promoter as defined herein.

[0050] Thus, in one embodiment, the pharmaceutical composition defined herein comprises a tissue or organ specific virus or viral vector capable of targeting or infecting cells of a tissue or organ of interest, comprising an IL-2 encoding gene whose expression is driven by a tissue or organ specific promoter. In one particular embodiment, the pharmaceutical composition defined herein comprises a viral vector, such as an adeno-associated virus (e.g., AAV6 and its variants / derivatives, e.g., AAV6.2 and AAV6.2FF), that specifically targets or infects lung, such as lung cells (e.g., epithelial cells, airway epithelial cells and / or alveolar cells), and comprises an IL-2 encoding gene whose expression is driven by a tissue or organ specific promoter. In a further embodiment, the pharmaceutical composition defined herein comprises an adeno-associated virus AAV6.2, comprising an IL-2 encoding gene whose expression is driven locally in the lung or lung cells by the SFTPB promoter. In another embodiment, the pharmaceutical composition comprises an adeno-associated virus AAV6.2FF, comprising an IL-2 encoding gene whose expression is driven locally in the lung or lung cells by the SFTPB promoter. In a further embodiment, the pharmaceutical composition comprises an adeno-associated virus AAV6.2 comprising an IL-2 coding gene, the expression of which is driven locally in the lung or in lung cells by the CC10 promoter. In a further embodiment, the pharmaceutical composition comprises an adeno-associated virus AAV6.2FF comprising an IL-2 coding gene, the expression of which is driven locally in the lung or in lung cells by the CC10 promoter. Thus, in one embodiment, the pharmaceutical composition comprises AAV6.2-SFTPB-IL2. In another embodiment, the pharmaceutical composition comprises AAV6.2FF-SFTPB-IL2. In a further embodiment, the pharmaceutical composition comprises AAV6.2-CC10-IL2. In a further embodiment, the pharmaceutical composition comprises AAV6.2FF-CC10-IL2.

[0051] In some embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable excipients in addition to a tissue- or organ-specific virus or viral vector as defined herein.

[0052] Generally, the pharmaceutical compositions are utilized with pharmacologically suitable excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's. Suitable physiologically acceptable adjuvants may be selected from viscosity enhancing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates, if necessary, to hold the composition comprising the tissue or organ-specific targeting moiety defined herein in a separate location (e.g., within the tissue or organ of interest). Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Excipients, carriers, and vehicles suitable for delivery of pharmaceutical compositions by intranasal administration, inhalation, or use of a nebulizer are also known, and will be understood to be particularly useful with the pharmaceutical compositions defined herein. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases. (Mack, (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0053] Therapeutic Uses and Methods It will be appreciated from the disclosure provided herein that the methods of expanding a population of regulatory T cells, the pharmaceutical compositions and treatment methods of the invention are particularly useful for treating and / or ameliorating a disease or disorder mediated by inflammation and / or reducing inflammation. It will further be appreciated that the population of regulatory T cells expanded according to the methods and disclosure provided herein is also useful for treating and / or ameliorating a disease or disorder mediated by inflammation and / or reducing inflammation.

[0054] Thus, according to one aspect of the invention there is provided a method of expanding a population of regulatory T cells in a tissue or organ of a subject for use in the treatment and / or amelioration of a disease or disorder mediated by inflammation, wherein the tissue or organ is the lung. In another aspect of the invention there is provided a method of expanding a population of regulatory T cells in a tissue or organ of a subject for use in reducing inflammation, wherein the tissue or organ is the lung. In a further aspect of the invention there is provided a method of expanding a population of regulatory T cells in a tissue or organ of a subject for use in the treatment and / or amelioration of an autoimmune disease, wherein the tissue or organ is the lung.

[0055] In another aspect of the invention, there is provided a regulatory T cell population expanded in a tissue or organ of a subject, generated according to the methods defined herein, for use in the treatment and / or amelioration of a disease or disorder mediated by inflammation or for use in reducing inflammation. Such diseases or disorders may include inflammatory conditions, autoimmune diseases and / or diseases associated with transplantation, such as transplant rejection or graft-versus-host disease. In one embodiment, the regulatory T cell population expanded in a tissue or organ of a subject, generated according to the methods defined herein, has been expanded by administration of IL-2 and a targeting moiety specific for said tissue or organ. In a further embodiment, the regulatory T cell population expanded in a tissue or organ of a subject, generated according to the methods defined herein, has been expanded by tissue- or organ-specific expression of IL-2, as defined herein. In another embodiment, the regulatory T cell population expanded in a tissue or organ of a subject, has been expanded by tissue- or organ-specific expression of IL-2, which is promoted or induced by an inducible element, such as a tetracycline-inducible element. In a further embodiment, the regulatory T cell population expanded in a tissue or organ of a subject generated according to the methods defined herein is for use in the treatment and / or amelioration of a pulmonary disease or disorder. In one embodiment, the regulatory T cell population expanded in a tissue or organ of a subject generated according to the methods defined herein is for use in the treatment and / or amelioration of a pulmonary disease or disorder. In a further embodiment, the regulatory T cell population expanded in a tissue or organ of a subject generated according to the methods defined herein is for use in the treatment and / or amelioration of type 1 inflammation. In an embodiment, the regulatory T cell population expanded in a tissue or organ of a subject generated according to the methods defined herein is for use in the treatment and / or amelioration of inflammation in the lungs. Thus, according to one embodiment, the inflammation as defined herein is pulmonary inflammation. In a further embodiment, the pulmonary inflammation is due to a respiratory disease or disorder. Thus, according to one embodiment, the inflammation in the lungs is due to a respiratory disease or disorder.In a further embodiment, the regulatory T cell population expanded in a tissue or organ of a subject generated according to the methods defined herein is for use in the treatment and / or amelioration of pulmonary inflammation, wherein the inflammation is caused by a respiratory infection or the respiratory disease or disorder is a respiratory infection such as an infection with influenza, coronavirus or an emerging virus. In an alternative embodiment, the inflammation is caused by a non-infectious disease or disorder or the respiratory disease or disorder is non-infectious, such as chronic obstructive pulmonary disease (COPD). Another example is an autoimmune disease or disorder and / or the inflammation is due to an autoimmune disease or disorder.

[0056] According to a further aspect of the present invention, there is provided a method for treating a disease or disorder mediated by inflammation and / or a method for reducing inflammation, said method comprising administering to a subject in need thereof a pharmaceutical composition comprising IL-2 and a targeting moiety specific for a tissue or organ of the subject as defined herein, either as defined herein. In one embodiment, said method of treatment comprises administering to a subject in need thereof a virus or viral vector comprising a gene encoding IL-2 as defined herein. In one embodiment, the method of treatment defined herein comprises administering to a subject in need thereof a virus or viral vector that specifically targets or infects a tissue or organ affected by or affected by a disease or disorder mediated by inflammation. In an embodiment, the method of treatment defined herein further comprises administering to a subject in need thereof a virus or viral vector comprising a gene encoding IL-2, the expression of which is driven by a tissue or organ specific promoter. In a further embodiment, the method of treatment defined herein comprises administering to a subject in need thereof a virus or viral vector comprising a gene encoding IL-2, the expression of which is driven by a tissue or organ specific promoter and an inducible element, such as a tetracycline inducible element. In an alternative embodiment, the method of treatment comprises administering to a subject a virus or viral vector comprising a gene encoding IL-2, the expression of which is driven by an inducible element, such as a tetracycline inducible element, under the control of a tissue or organ specific promoter. In a further embodiment, the method of treatment defined herein comprises administering to a subject in need thereof a virus or viral vector comprising a gene encoding IL-2, the expression of which is driven by a tissue or organ specific promoter, such as administration of AAV6.2-SFTPB-IL2, AAV6.2FF-SFTPB-IL2, AAV6.2-CC10-IL2 or AAV6.2FF-CC10-IL2.

[0057] In one embodiment, the subject in need thereof suffers from a disease or disorder mediated by inflammation. In a further embodiment, the subject in need thereof suffers from inflammation. In a further embodiment, the subject in need thereof suffers from type 1 inflammation. In another embodiment, the subject in need thereof suffers from an autoimmune disease or disorder. In one embodiment, the disease or disorder is a pulmonary disease or disorder. In a further embodiment, the disease or disorder is a respiratory disease or disorder. In a further embodiment, the inflammation or respiratory disease or disorder comprises type 1 inflammation. In another embodiment, the inflammation is caused by a respiratory infection or the respiratory disease or disorder is a respiratory infection such as influenza or coronavirus infection, or an emerging respiratory virus. In an alternative embodiment, the inflammation is caused by a non-infectious disease or disorder or the respiratory disease or disorder is non-infectious, such as chronic obstructive pulmonary disease (COPD). EXAMPLES

[0058] (Example) Example 1: Parabiosis studies to calculate steady-state population exchange Parabiosis studies allow the calculation of steady-state population exchange. Foxp3 Thy1.1 CD45.1 mice and Foxp3 Thy1.1 Parabiosis of CD45.2 mice and high-dimensional flow cytometry analysis at five consecutive time points (Figure 1A) allowed monitoring the kinetics of normalization for Tregs in blood and lung tissue. Unlike blood, where normalization occurred within days, exchange rate calculations showed that CD69 - The median lung residence time of CD4 T cells is approximately 2–3 weeks, whereas CD69 + The median pulmonary residence time of their counterparts was determined to be approximately 7 weeks (Figure 1B).

[0059] Example 2: Proof-of-principle transgenic mouse model for lung-specific regulatory T cell expansion Based on the lung residence time of approximately 7 weeks, it was reasoned that the lung-resident regulatory T cell population could be expanded by local delivery of IL-2. To test this possibility, we developed a genetic switch for IL-2 expression in which IL-2 could be switched on only in lung tissue. Using the constitutive Rosa26 promoter and a floxed-stop expression system, we created a system in which Cre expression induces lineage-specific IL-2 expression. A weak endogenous Rosa26 promoter was used to ensure that IL-2 expression remained at physiological levels. Indeed, transgenic IL-2 expression in this system is only approximately 5% of the expression level of natural IL-2 production by conventional CD4 T cells (data not shown). To induce lung-specific expression, we replaced this transgene with Scgb1a1-ERT2 Cre Crossing with a transgenic mouse line allowed tamoxifen-induced recombination in bronchiolar nonciliated club cells. Scgb1a1-ERT2 Cre RosaIL2 mice were treated with tamoxifen, and the number of regulatory T cells was assessed, and lung-specific expansion of this population was observed (Figures 2A and 2B). No major changes were observed in regulatory T cell populations in blood, secondary lymphoid organs, or other non-pulmonary tissues (Figures 2A and 2B).

[0060] To determine the dynamics of IL-2 expression in the lungs, Scgb1a1-ERT2 mice treated with tamoxifen were Cre Lymph node tissues were collected from RosaIL2 mice 0, 1, 4, 7, 15, 21, 29, and 35 days after treatment and analyzed by quantitative PCR (qPCR). Data are shown in Figures 2C and 2D, and show that Scgb1a1-ERT2 Cre Lung-specific expression of IL-2 in RosaIL2 mice (Figure 2C). Expression was detected from day 4 after administration and was still detectable on day 35, the final day of analysis. Expression peaked on day 15 after tamoxifen administration. Compared to littermate controls, Scgb1a1-ERT2 Cre There was no significant difference in IL-2 expression in lymph nodes of RosaIL2 mice ( Fig. 2D ).

[0061] Figure 2E shows that the maximum increase in regulatory T cells is seen in the lungs at day 21 after lung-specific IL-2 expression. The percentage of Treg proliferation in the lungs is detected from day 4 after tamoxifen administration and remains elevated compared to littermate controls at day 35, the final day of analysis. This correlates with the kinetics of IL-2 expression seen in Figure 2C, where a slight delay in cell numbers is expected. No significant changes were seen over time in the percentage of Tregs in the lymph nodes of mice treated with tamoxifen (Figure 2F).

[0062] These data indicate that local provision of IL-2 can specifically expand regulatory T cell populations in the lung without expanding their numbers in the periphery, and that IL-2 can be expressed locally in the lung but not in peripheral tissues such as lymph nodes.

[0063] Example 3: Expansion of pulmonary regulatory T cells reduces inflammatory infiltrates during influenza infection To evaluate the protective effect of lung-specific regulatory T cell proliferation against respiratory infection, we investigated Scgb1a1-ERT2 Cre RosaIL2 mice were infected with murine influenza via the intranasal route. Unlike wild-type mice, which showed a chronic increase in inflammatory neutrophil infiltration in the lungs after infection, Scgb1a1-ERT2 mice Cre RosaIL2 mice showed a reduction in both the magnitude and duration of disease (Figure 3).

[0064] These results suggest that lung-targeted IL-2 administration / delivery has the potential to reduce the immunopathology of respiratory infections, which, together with the data above showing lung-specific expansion of regulatory T cells, indicates that this potential can be achieved without increasing the systemic regulatory T cell load.

[0065] Example 4: Intranasal administration of AAV6.2-mCC10-IL2 results in local expansion of regulatory T cells in the lungs To test the ability of the viral vector to specifically and locally deliver / express IL-2, mice were intranasally administered AAV6.2-mCC10-IL2 or control PBS as described herein above, and immune cells in the lungs were analyzed 14 days after administration. Compared to mice administered control PBS, mice administered AAV6.2-mCC10-IL2 intranasally showed a significant increase in the proportion of Tregs in the lungs (Figure 4). This increase was not seen in other tissues such as lymph nodes, or in the periphery / blood.

[0066] Thus, this data indicates that IL-2 can be delivered locally to the lung using a viral vector such as AAV6.2 to expand Treg populations specifically in the lung, consistent with the use of both a lung-targeting moiety and the lung-specific promoter CC10.

Claims

1. 1. A pharmaceutical composition comprising an exogenous IL-2 coding sequence and a targeting moiety specific for a tissue or organ of a subject, the tissue or organ being the lung; the targeting moiety is a lung-specific viral vector; The pharmaceutical composition, wherein expression of the exogenous IL-2 coding sequence is tissue- or organ-specific in the tissue or organ of the subject, and wherein the tissue- or organ-specific expression of IL-2 is driven by a lung-specific promoter.

2. 2. The pharmaceutical composition according to claim 1, wherein the viral vector is an adeno-associated virus selected from AAV6 and its mutants and derivatives, in particular AAV6.2 or AAV6.2FF.

3. 3. The pharmaceutical composition according to claim 1 or 2, wherein the lung-specific promoter is the promoter of surfactant protein B (SFTPB) or the promoter of club cell specific protein (CC10).

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the tissue- or organ-specific expression of IL-2 comprises an inducible element that promotes or induces expression of IL-2 in the presence of an exogenous compound.

5. 5. The pharmaceutical composition of claim 4, wherein the inducible element is a tetracycline-dependent or tetracycline-inducible element, such as a tetracycline response element (TRE).

6. 6. The pharmaceutical composition of claim 5, wherein the tissue or organ specific expression of IL-2 comprises administration of tetracycline or a derivative / analogue of tetracycline, such as doxycycline.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the exogenous IL-2 coding sequence further comprises a sequence encoding a reverse tetracycline-controlled transactivator (rtTA).

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the viral vector is AAV6.2 or AAV6.2FF.

9. 9. A pharmaceutical composition according to any one of claims 1 to 8 for use in the treatment of a disease or disorder mediated by inflammation and / or in reducing inflammation.

10. 10. The pharmaceutical composition for use according to claim 9, wherein the inflammation is inflammation of the lungs.

11. 10. The pharmaceutical composition for use according to claim 9, wherein the disease or disorder is a respiratory disease or disorder.

12. 12. The pharmaceutical composition for use according to claim 10 or claim 11, wherein the inflammatory or respiratory disease or disorder comprises type 1 inflammation.

13. 13. The pharmaceutical composition for use according to any one of claims 10 to 12, wherein said inflammation is caused by a respiratory infection or said respiratory disease or disorder is a respiratory infection such as an influenza or coronavirus infection.

14. 13. The pharmaceutical composition for use according to any one of claims 10 to 12, wherein said inflammation is caused by a non-infectious disease or disorder or said respiratory disease or disorder is non-infectious, such as chronic obstructive pulmonary disease (COPD).

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