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, the method comprising administering IL-2 and a targeting moiety specific for the tissue or organ, wherein the tissue or organ is the central and / or peripheral nervous system. The invention further relates to a population of regulatory T cells generated according to the method and the generation of the population in vivo. Also provided are pharmaceutical compositions comprising IL-2 and a targeting moiety as defined herein, and methods for treating a disease or disorder mediated by inflammation or for reducing inflammation comprising administering a method as defined herein or a pharmaceutical composition as defined herein.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for expanding a population of regulatory T cells in a tissue or organ of a subject, the method comprising administering IL-2 and a targeting moiety specific for the tissue or organ, wherein the tissue or organ is the central and / or peripheral nervous system. The invention further relates to a population of regulatory T cells generated according to the method and the generation of the population in vivo. Also provided are pharmaceutical compositions comprising IL-2 and a targeting moiety as defined herein, and methods for treating a disease or disorder mediated by inflammation or for reducing inflammation comprising administering a method as defined herein or a pharmaceutical composition as defined herein. [Background technology]

[0002] BACKGROUND OF THE INVENTION Neuroinflammation is a pathogenic process in multiple neuroinflammatory diseases. As the inflammatory process is well understood, multiple anti-inflammatory immunosuppressive drugs are available, and in principle, neuroinflammation should be a manageable problem. The main issues hindering the use of immunosuppressants in neuroinflammatory diseases are 1) the blood-brain barrier and 2) off-target immunosuppression. Essentially, a dose of immunosuppressant sufficient to suppress neuroinflammation would be impossible in patients because it would need to be high enough to provide widespread peripheral immunosuppression.

[0003] Avles et al. (2017) Brain and WO 2017 / 060510 disclose reduced IL-2 levels in hippocampal biopsies from Alzheimer's disease patients and explain that systemic delivery of IL-2 in a transgenic mouse model of Alzheimer's disease promotes the proliferation and activation of regulatory T cells in the body and brain.

[0004] Dashkoff et al. (2016) Molecular Therapy describes and characterizes adeno-associated viruses that express GFP under the control of an astrocytic or neuronal promoter.

[0005] Rouse et al. (2013) in Immunobiology describe the effectiveness of systemic IL-2 treatment in ameliorating pathology in a mouse model of multiple sclerosis (MS) when delivered before disease onset.

[0006] Therefore, there is a great need for effective treatments for inflammatory diseases or disorders. Summary of the Invention [Effects of the Invention]

[0007] (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, said method comprising administration of IL-2 and a targeting moiety specific for said tissue or organ, wherein said tissue or organ is the central and / or peripheral nervous system.

[0008] 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, wherein the targeting moiety is specific for the central nervous system and / or the peripheral nervous system.

[0009] 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 the reduction of inflammation, said method comprising administering to a subject in need thereof a method as defined herein or a pharmaceutical composition as defined herein. [Brief explanation of the drawings]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1: Regulatory T cells are present in the parenchyma of healthy mouse brains. A) Representative confocal microscopy images showing regulatory T cells immunostained with CD4 (first column) and FoxP3 (a specific marker for regulatory T cells—second column) located in the parenchyma, perivascular space, and intravascular regions of mouse brains. Fluorescently labeled lectins were used to label vascular structures (third column), and cell nuclei were stained with DAPI (fourth column). Scale bar = 20 μm. B) Enlargement and 3D reconstruction of an example of a CD4+Foxp3+ T cell. Scale bar = 10 μm. C) Regulatory T cells were assessed in perfused mouse brains by high-dimensional flow cytometry. Wild-type mice were sampled during healthy aging (weeks 8, 12, 30, and 52). n = 8, 5, 6, and 5, respectively. [Figure 2]Figure 2: Brain-resident regulatory T cells acquire a resident phenotype in situ during long-term brain migration. A) Schematic of the parabiotic experiment (n=12, 12, 18, 16, 14). B) Best-fit curves for the origin of CD4+Foxp3+ regulatory T cells in blood and brain, showing the CD69+ population in the brain. C) tSNE of CD4+Foxp3+ regulatory T cells constructed on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4, and gated on CD4+Foxp3-CD3+CD8-CD45+. CD69 expression is shown in grayscale. Host and inbound cells are defined by CD45.1 vs. CD45.2 expression and are shown at 2, 4, and 8 weeks. D) CD69 histograms of CD4+Foxp3+ regulatory T cells. Host and influent cells were defined by CD45.1 vs. CD45.2 expression and are shown at 2, 4, and 8 weeks. E) Population flow diagram of CD4+Foxp3+ regulatory T cells in homeostatic conditions. The area of ​​the circle represents the population frequency calculated independently for blood and brain. Small black circles represent cell death. The size of the arrowhead is proportional to the rate of population flow as an exit (outgoing arrow) or entry (incoming arrow). All arrowhead sizes are scaled equally in each panel, so populations with the highest turnover have arrows covering the full circumference (thus, the graphical representation of this population flow is the same regardless of the units used for the migration rate). The numbers near the end of each arrow indicate the corresponding entry or exit rate in events / 1000 cells / day. Numbers with asterisks indicate rates with high estimated uncertainty. Population migration with rates less than 0.1 / 1000 cells / day at either end is not shown. [Figure 3]Figure 3: Proof-of-principle transgenic mouse model for brain-specific regulatory T cell expansion. A) The Rosafl-Stop-flIL-2 allele contains a loxP-flanked stop cassette, and IL-2 expression is activated after Cre activation. Using a CD4Cre driver, transgene-induced levels of IL-2 production were compared to reduced levels of IL-2 induced by endogenous stimuli. B) and C) Schematic diagram of tamoxifen-inducible Cre (CreERT2) under the control of the brain-specific promoters tested in this study: B) Plp1 and C) CaMKII. D) Effect of brain-specific IL-2 production on the expansion and proliferation of regulatory T cell populations. Plot comparing Treg (Foxp3+CD25+) proliferation in the blood and brain of wild-type, IL-2 Plp1Cre, and IL-2 aCaMKIICre mice. E) Histogram showing the percentage of Foxp3+ cells in the CD4+ cell population. Mean ± SEM (P value, one-way anova). F) 10X Chromium single-cell sequencing was performed on CD4+ T cells from wild-type perfused adult IL-2 aCaMKII Cre mouse brains. tSNE visualizes cell clusters formed in the combined population. Clusters of naive CD4+ T cells, activated CD4+ T cells, and CD4+Foxp3+ regulatory T cells were identified and labeled (top) based on characteristic expression of transcriptional markers (bottom). G) Fold change in all expressed genes between conventional and regulatory T cells from IL-2 aCaMKII Cre mice. H) Transcriptional profile of cytokines in CD4+ T cells purified from the brains of mouse IL-2 aCaMKII Cre mice analyzed via the 10X single-cell pipeline. I)-S) Behavioral assessment of IL-2 aCaMKII Cre (αCamKIIIL2) and control mice. I) Time spent on the rod, average of four 300-second replicates (n=23, 17). J) Open field, total distance traveled and K) time in the corner (n=23, 16). L) Nest building score (n=24, 18). M) Latency to enter the light zone in the light-dark test and N) time spent in the light zone (n=20, 17). O) Time of immobility during the forced swim test (n=24, 16).P) Sociability test trial monitoring interaction with a strange mouse (S) compared to an empty chamber (E) (n=28, 18). Q) Freezing behavior over time during context acquisition acclimation (n=28, 18). Mean ± SEM. R) Context discrimination during generalization testing. Mean ± SEM (n=28, 18). S) Spatial learning in the Morris water maze. Path length to find the hidden platform (n=16, 8), probe test after 5 and 10 days and reversal learning (n=28, 20). Mean ± SEM. [Figure 4] Figure 4: Expanded brain regulatory T cells protect against traumatic brain injury. Controlled cortical impact was administered to wild-type and IL-2 aCaMKII Cre (αCamKIIIL2) mice to induce moderate traumatic brain injury (TBI), and the mice were examined 15 days after TBI. A) Macroscopic damage to the brain surface at the injury site. B) Representative confocal images taken within the brains of IL-2 aCaMKII Cre (αCamKIIIL2) or littermate control mice 15 days after ipsilateral cortical injury. C) Immunofluorescent staining of cortical tissue after controlled cortical impact surgery. GFAP (astrocytes), NeuN (neurons), and DAPI (nuclei). Scale bar = 50 μm. D) Damage area shown as a percentage of the entire hemisphere (n = 3). [Figure 5] Figure 5: Astrocyte-specific expression using the GFAP promoter. A) The GFAP promoter restricts TdTomato expression to astrocytes in the adult mouse brain, as judged by characteristic cell morphology and immunostaining for the astrocyte-specific markers GFAP and S100β. No off-target expression was observed when sections were counterstained with NeuN (neurons), APC (oligodendrocytes), IBA1 (microglia), and PDGFRa (NG2+ cells). Scale bar = 20 µm. Data are representative images of three sections from three independent mice administered the GFAP-TdTomato construct. B) Quantified representative staining (left) and quantitative expression (right) of GFAP in the cortex and striatum of adult mouse brains 14 days after induction of traumatic brain injury (TBI; n = 5). [Figure 6]Figure 6: PHP.B-GFAP-IL2 specifically increases Tregs in the brain and controls neuroinflammation. A) Flow cytometry analysis of cells isolated from the brains of C57Bl6 mice infected with PHP.B control (PHP.B-GFP) or PHP.B-GFAP-IL2. Cells were gated on live CD45+CD11b-CD19-CD3+. B) Frequency of Tregs (CD4+Foxp3+ cells) in the brain. Data are shown as mean ± SEM (n = 3 per group). C) Flow cytometry analysis of cells isolated from the spleens of C57Bl6 mice infected with PHP.B control (PHP.B-GFP) or PHP.B-GFAP-IL2. D) Frequency of regulatory T cells in the spleen. Data are shown as mean ± SEM (n = 3 per group). E) Blood, spleen, and perfused mouse brains from PHP.B-GFAP-GFP control and PHP.B-GFAP-IL2-treated mice were compared by high-dimensional flow cytometry for regulatory T cell counts. F) Wild-type mice were administered 10, 10, or 10 vector genomes (total dose) of PHP.B-GFP control vector or PHP.B-GFAP-IL2 via tail vein injection, and the number of conventional (left) and regulatory (right) T cells in the perfused brain was assessed 14 days after treatment (n = 3–5 per group). G) C57Bl6 mice were infected with PHP.B-GFP control or PHP.B-GFAP-IL2 (10 vg / mouse). To induce EAE 14 days after infection with PHP.B, mice were immunized with MOG(35–55) in CFA. Mononuclear cells were isolated on day 30 of EAE. The mean and SEM of clinical and cumulative clinical scores were calculated (n = 15, 14; mean ± SEM; P value, Mann-Whitney U test). H) Cells were isolated from the CNS (brain and spinal cord). Top: Absolute numbers or frequencies of indicated brain-infiltrating cells are shown. Bottom: CNS-derived cells were stimulated with PMA and ionomycin, and IL-10, IL-17, GM-CSF, and amphiregulin (AREG) were analyzed by flow cytometry in CD4 or regulatory T cells. Symbols represent individual mice. Data are shown as mean ± SEM (n = 6–7 per group).I) Same as G), but mice were treated with PHP.B-GFAP-IL2 or PHP.B-GFP control on day 10 after EAE induction (indicated by arrows). Incidence, daily clinical scores (mean ± SEM), and cumulative mean clinical scores (n = 15, 14). [Figure 7] Figure 7: PHP.B-GFAP-IL2 protects against traumatic brain injury. Mice were intravenously injected with a 1x dose of 1x10 vector genomes per mouse of PHP.B-GFAP-IL2 or PHP.B control (PHP.B-GFP) 14 days before a controlled cortical impact to induce moderate traumatic brain injury (TBI). Mouse brains were examined 15 days after TBI. A) Macroscopic damage to the brain surface at the injury site. B) Representative confocal images showing NeuN, BrdU, and GFAP in the brains of control PHP.B-GFP, PHP.B-GFAP-IL2, or sham-operated mice after ipsilateral cortical injury. C) Quantification of the lesion area lost, relative Iba1 expression in the cortex and striatum, and GFAP expression in the cortex and striatum (ratio of expression in the ipsilateral vs. contralateral hemisphere). D) Representative MRIs and MRI-based quantification of lesion size in PHP.B-GFAP-GFP control or PHP.B-GFAP-IL2-treated mice at days 1, 7, 14, 35, and 150 post-TBI (control n=16, 16, 12, 11, 10; IL2 n=16, 16, 16, 12, 9). E) Percentage of total time spent in the target quadrant during the probe trial. F) Ratio of exploration time of the novel object relative to the old object on day 2 of the novel object recognition paradigm. [Figure 8]Figure 8: Normal peripheral influx after PHP.B-GFAP-IL2 treatment in mice with traumatic brain injury. Mice treated with PHP.B-GFAP-IL2 or control PHP.B-GFAP-GFP 14 days prior were administered a controlled cortical impact to induce moderate traumatic brain injury (TBI) and examined 15 days after TBI (n = 3, 4, 4). Sham-TBI mice were included in the control PHP.B-GFAP-GFP group. TBI-induced perfused brains from sham-treated, TBI-treated, and PHP.B-GFAP-IL2-treated TBI mice were compared by high-dimensional flow cytometry. Microglia were gated on A) percentage of CD45+ cells or B) absolute numbers of CD11b+ CX3CR1+ CD64+ CD45mod Ly6G- cells. C) MHCII expression on microglia. D) Percentage of CD4 and CD8 T cells as a percentage of CD45+ CD11b- TCRβ+ CD19- cells. E) Percentage of regulatory T cells (CD4+ Foxp3+) as a percentage of CD4 T cells. F) Frequency of CD25, CD44, CD69, Ki67 and PDL1 expressing cells. G) Frequency of amphiregulin-producing cells within conventional CD4 T cell populations or (H) mean fluorescence intensity (MFI). I) Frequency of CD25, CD44, CD69, Ki67 and PDL1 expressing cells. J) Frequency of amphiregulin-producing cells within conventional CD4 T cell populations or (K) mean expression level. Mean ± SEM. [Figure 9] Figure 9: Expansion of regulatory T cells in the brain reduces stroke severity. A) Wild-type mice (n = 7, 10) treated with control PHP.B-GFAP-GFP or PHP.B-GFAP-IL2 14 days prior underwent distal middle cerebral artery occlusion (dMCAO) stroke. Macroscopic damage was examined 15 days post-stroke, with B) TTC-based damage quantification. C) Wild-type mice (n = 5, 5) treated with control PHP.B-GFAP-GFP or PHP.B-GFAP-IL2 14 days prior underwent photothrombotic stroke. Macroscopic damage was examined 1 day post-stroke in representative images, with D) TTC-based quantification. [Figure 10]Figure 10: Small molecule induction system for brain-specific regulatory T cell expansion. Wild-type mice were administered 10 vector genomes (total dose) of PHP.B-GFAP-GFP control vector or PHP.B-GFAP-TetR-T2A-rtTA(V7 / V14).TetO-IL2 (PHP.GFAP / TetO-IL2) via tail vein injection. Mice were gavaged daily with 50 mg / kg minocycline or PBS control (n = 4-5 mice per group), and the proportion of Tregs was assessed in the spleen or perfused brain 14 days after treatment. DETAILED DESCRIPTION OF THE INVENTION

[0011] (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, said method comprising administration of IL-2 and a targeting moiety specific for said tissue or organ, wherein said tissue or organ is the central and / or peripheral nervous system.

[0012] 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.

[0013] 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. As such, 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. As such, 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, an expanded population of cells generated according to the methods defined herein, such as an expanded population of regulatory T 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 a further embodiment, an expanded population of cells produced according to the methods defined herein, such as an expanded population of regulatory T cells, is an expanded population within a tissue or organ of interest and comprises more cells than a population of cells that is not located in said tissue or organ of interest. In a further embodiment, an expanded population of cells produced according to the methods defined herein, such as an expanded population of regulatory T cells, is located in a tissue or organ separated from other tissues or organs by a barrier (such as the blood-brain barrier) and comprises more cells than a population of cells that is not located with said barrier-separating tissue or organ.

[0014] 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 greater population than a population of cells that have not been subjected to or administered IL-2. In a further 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 greater population than a population of cells that are not located in a tissue or organ of interest. In certain embodiments, an expanded population of cells produced according to the methods defined herein 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 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. In some embodiments, an expanded population of cells produced according to the methods defined herein, such as an expanded population of regulatory T 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 a tissue or organ).

[0015] It will therefore be understood 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 larger than a reference population.

[0016] 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 generated according to the methods defined herein 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 generated according to the methods defined herein 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 generated according to the methods defined herein 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 generated according to the methods defined herein is greater than the reference population due to a combination of one or more of increased viability, reduced / decreased cell death, and increased proliferation.

[0017] 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," can also include a population of activated cells. Reference herein to "expansion" can 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, can be independent of proliferation or can be associated with the expansion of said population. Thus, in one embodiment, an expanded population of regulatory T cells generated according to the methods defined herein comprises activated regulatory T cells. In a further embodiment, an expanded population of regulatory T cells generated according to the methods defined herein is an activated population of regulatory T cells.

[0018] In an alternative embodiment, reference to "expanded" or "expanded population" produced 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 produced according to the methods defined herein, such as an expanded population of regulatory T cells, does not comprise an activated phenotype. In a further embodiment, an expanded population of regulatory T cells produced according to the methods defined herein does not comprise activated regulatory T cells. Thus, in a further embodiment, an expanded population of regulatory T cells produced according to the methods defined herein comprises a phenotype, such as the surface phenotype, of a population of regulatory T cells that have not been subjected to or administered IL-2.

[0019] 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 be useful for immunosuppression. Therefore, regulatory T cells are highly 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. The present inventors have demonstrated that, despite the conventional view that the brain is a tissue separated from the immune system (e.g., by the blood-brain barrier), regulatory T cells are present in the healthy brain (Figure 1) and therefore may be a valid target for immunosuppressive therapies, such as anti-inflammatory treatments, in the brain.

[0020] Thus, in one embodiment, the expanded population of regulatory T cells generated according to the methods defined herein comprises 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 at another location other than the tissue or organ of interest. In one embodiment, the expanded population of regulatory T cells generated according to the methods defined herein comprises a phenotype similar to non-expanded regulatory T cells within the tissue or organ of interest, or regulatory T cells from a location other than the tissue or organ of interest. Such phenotype may include surface marker phenotype, transcriptome phenotype / signature (e.g., gene expression signature), gene and / or protein expression profile, and cytokine expression profile. Thus, in certain embodiments, the expanded population of regulatory T cells generated according to the methods defined herein 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 a further embodiment, the expanded population of regulatory T cells generated according to the methods defined herein comprise or retain the anti-inflammatory potential of a population of regulatory T cells from elsewhere than the tissue or organ of interest.

[0021] 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 pertain to cases where the effect occurs systemically or outside the tissue or organ of interest, or where cell types or populations of cells not located in the tissue or organ of interest are affected (e.g., proliferated or activated). Thus, in one embodiment, a population of regulatory T cells generated according to the methods defined herein is affected (e.g., proliferated) in a particular tissue or organ, i.e., locally. In a further embodiment, a population of regulatory T cells generated according to the methods defined herein is affected (e.g., proliferated) only in a particular tissue or organ. In a further embodiment, a population of regulatory T cells located outside the tissue or organ of interest, i.e., not within the tissue or organ, is not affected (e.g., not proliferated). Thus, in certain embodiments, a systemic or peripheral population of regulatory T cells is not affected (e.g., not proliferated).

[0022] As defined herein, a tissue or organ includes a separate location of a body or organism. For example, a tissue or organ may include a compartment of a body, such as the nervous system (e.g., the central nervous system or the peripheral nervous system or the brain). In certain embodiments, a tissue or organ is separated from other tissues or organs by a barrier, such as the blood-brain barrier. Thus, in one embodiment, the tissue or organ is the central nervous system and / or the peripheral nervous system. In a further embodiment, the tissue or organ is the brain.

[0023] IL-2 is a key population regulator of regulatory T cells. Regulatory T cells naturally have a high turnover rate compared to other T cells, with rapid proliferation and a high apoptosis rate. IL-2 can increase the frequency of regulatory T cells by inducing 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). Therefore, 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 shown promise as an anti-inflammatory agent 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 brain context, given the potential use of IL-2 as an anti-inflammatory mediator, systemic delivery of IL-2 should theoretically promote an increase in the number of regulatory T cells in the brain as this population is seeded by circulating regulatory T cells ( Figure 2 ).

[0024] However, in reality, systemic expansion of regulatory T cells via IL-2 delivery disproportionately increases the naive regulatory T cell population that disseminates to the brain at an approximately 10-fold lower efficiency level (see Figure 2E). Thus, systemic IL-2 delivery levels that produce a significant increase in anti-inflammatory potential in the periphery do not produce a significant increase in regulatory T cell numbers in the brain. The findings presented herein indicate that while IL-2 has great potential as a therapeutic agent for brain inflammation, including neuroinflammation, systemic delivery within the physiological range required to increase regulatory T cell numbers in the brain is likely to induce systemic immunosuppression. In contrast, brain-specific expansion or augmentation of regulatory T cell numbers was able to locally induce the anti-inflammatory properties of regulatory T cells without the deleterious effects of systemic immunosuppression.

[0025] Thus, one embodiment of the present invention provides a method for expanding a population of regulatory T cells in a tissue or organ of a subject in need thereof, wherein the tissue or organ is separated from other tissues or organs by a barrier, such as the blood-brain barrier. It will be appreciated, therefore, that the methods defined herein provide for the expansion of regulatory T cell populations in tissues or organs that are difficult to achieve with systemic delivery of IL-2 due to the presence of a barrier, such as the blood-brain barrier. For example, due to the presence of such a barrier, a dose of IL-2 sufficient to affect a population of cells present in the tissue or organ must be at a level high enough to have a widespread peripheral or systemic effect. If a regulatory T cell population were expanded in a tissue or organ using IL-2 as described herein, the resulting widespread peripheral or systemic immunosuppression would be unacceptable to patients due to increased risk of infection.

[0026] It will be understood that references herein to "administration" refer to providing or making available IL-2 to a discrete location or region of an organism, such as a specific tissue or organ. Accordingly, such administration is analogous to the definition of "in a tissue or organ" set forth previously herein. Thus, in one embodiment, administration of IL-2 includes administration to or within a specific tissue or organ. In a specific embodiment, administration of IL-2 includes expression of IL-2 in a specific tissue or organ (e.g., the brain or nervous system). In one embodiment, administration includes expression of a gene encoding IL-2 in a specific tissue or organ (e.g., the brain or nervous system). In a further embodiment, expression of IL-2 is undetectable outside the tissue or organ of interest, such as the periphery. In a further embodiment, expression of IL-2 is restricted to a specific tissue or organ of interest. In a further embodiment, expression of IL-2 is tissue-specific or organ-specific. In certain embodiments, administration or expression of IL-2 can be within 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 (e.g., tissues of the brain and nervous system or the intestinal tract), hi another embodiment, administration or expression of IL-2 is in one, two, or more tissues or organs that are not considered relevant.

[0027] Furthermore, references herein to "administration" and "expression" also refer to the delivery of IL-2 to a population of cells in a tissue or organ. Such delivery of IL-2, in one embodiment, includes administration of IL-2 in the form of a protein or peptide to or within a tissue or organ of interest, i.e., locally. In a further embodiment, delivery 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 (e.g., neurons) that comprise the tissue or organ. In some embodiments, expression of IL-2 includes neurons, oligodendrocytes, and / or astrocytes. In one embodiment, expression of IL-2 includes astrocytes. It will be appreciated that expression of IL-2 by / in astrocytes offers several advantages: 1) astrocytes are competent secretory cells that are widely distributed throughout the brain; and 2) astrocytes are well represented in the spinal cord, providing the possibility of administering or expressing IL-2 in the spinal cord. 3) astrocytes demonstrate increased temporal and spatial numbers during neuroinflammatory events, such as traumatic brain injury. And 4) expression of the astrocyte-specific promoter GFAP is upregulated in response to injury and disease (Figure 5B). In a further embodiment, expression of IL-2 includes expression in cells other than regulatory T cells constituting 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. In one embodiment, administration or expression of IL-2 includes expression from an endogenous IL-2-encoding gene in cells of a tissue or organ of interest. According to this embodiment, expression of IL-2 in cells of a tissue or organ does not involve transfection, transduction, or introduction of exogenous sequences. Thus, in one embodiment, expression of IL-2 in cells of a tissue or organ includes tissue- or organ-specific stimulation with a compound that upregulates or "activates" expression of the IL-2-encoding gene only in cells of the tissue or organ of interest. It will be appreciated that, according to this embodiment, stimulation of expression of the endogenous gene encoding IL-2 is specific and localized only to the tissue or organ of interest.

[0028] In an alternative embodiment, administering or expressing IL-2 comprises introducing an exogenous sequence encoding IL-2 into cells of the tissue or organ. Thus, in one embodiment, administering or expressing IL-2 comprises expression from an exogenous sequence. In a further embodiment, administering or expressing 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.

[0029] 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 specifically drives expression 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 tissues or organs other than the tissue or organ of interest. Thus, in one embodiment, the transgene comprises a promoter that specifically drives expression in neurons. In a further embodiment, the transgene comprises a promoter that specifically drives expression in cells of the central nervous system and / or peripheral nervous system. In a further embodiment, the transgene comprises a promoter that drives expression in the central nervous system but not in the peripheral nervous system. In another embodiment, the transgene comprises a promoter that drives expression in the peripheral nervous system but not in the central nervous system. In one embodiment, the transgene comprises a promoter that specifically drives expression in the brain. In a particular embodiment, the transgene comprises a promoter that specifically drives expression in astrocytes. In a further embodiment, the transgene comprises a GFAP promoter. In a further embodiment, the transgene comprises a minimal GFAP promoter.

[0030] In a further embodiment, the administration or expression of IL-2 involves a transgene containing elements that promote or induce 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) 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 Escherichia coli to the activation domain of VP16, another protein found in herpes simplex virus. The resulting tTA can bind to the TetO sequence within 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 generate the reverse tetracycline-controlled transactivator (rtTA) protein, which depends on the presence of tetracycline to promote expression. Thus, rtTA only binds to the TetO sequence within the TRE and promotes expression in the presence of tetracycline. Specific examples of TetOn systems include, but are not limited to, Life Technologies' TetOn Advanced, TetOn3G, and T-REx systems. Tetracycline derivatives and analogs can be used with either the TetOff or TetOn systems, including, but not limited to, doxycycline and minocycline (e.g., minocycline).It will be appreciated that such derivatives / analogs offer significant advantages over tetracycline, such as increased stability in the case of doxycycline and / or increased ability to cross the blood-brain barrier in the case of minocycline (Chtarto et al., 2003, doi: https: / / doi.org / 10.1016 / j.neulet.2003.08.067). 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 into cells of the tissue or organ an exogenous sequence encoding a reverse tetracycline-controlled transactivator (rtTA). 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 promoter specific to the nervous system, such as the central nervous system (e.g., the brain). In a further embodiment, expression of the rtTA coding sequence is under the control of a promoter specific to the nervous system, such as the central nervous system (e.g., the brain). In certain embodiments, the exogenous sequence encoding rtTA comprises a promoter that specifically promotes expression in astrocytes, such as the GFAP promoter or a minimal GFAP promoter. 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 comprises a tissue- or organ-specific promoter. Alternatively, such an exogenous sequence encoding rtTA may be included together with the IL-2 coding sequence, e.g., within the same transgene. Thus, in some embodiments, administration or expression of IL-2 comprises the TetOn system. It will therefore be understood that in one embodiment, administration or expression of IL-2 includes administration of tetracycline or a derivative / analogue of tetracycline such as doxycycline or minocycline.In certain embodiments, the administration or expression of IL-2 comprises administration of minocycline, such as administration of minocycline.

[0031] The use of tetracycline-dependent or tetracycline-inducible administration or expression of IL-2 provides another level of control, allowing IL-2 administration or expression to be "switched" on or off. Such switching will be understood to be advantageous in the methods described herein by allowing for temporal control of the expansion of regulatory T cell populations within a tissue or organ. For example, IL-2 expression can be switched "on" by administering tetracycline or a derivative / analogue thereof when inflammation of the central and / or peripheral nervous system, such as brain neuroinflammation and / or inflammation, is detected / diagnosed. Alternatively, IL-2 expression can be switched "on" after an acute injury to the brain or head, such as a traumatic brain injury or stroke. IL-2 expression can then be switched "off" by removing tetracycline or a derivative / analogue thereof when inflammation, such as neuroinflammation, is no longer detectable or has decreased. Expression can also be switched "off" after the subject is deemed no longer at risk of acute brain injury, such as a traumatic brain injury or stroke. The use of tetracycline-dependent or tetracycline-inducible IL-2 administration or expression further provides dose-dependent IL-2 administration or expression. For example, the level and / or amount of IL-2 administration or expression can be altered and / or titrated in a tissue or organ depending on the level and / or amount of inflammation, such as neuroinflammation, in the tissue or organ. Thus, IL-2 expression can be "turned on" by administering a specific dose of tetracycline or a derivative / analogue thereof when inflammation of the central nervous system and / or peripheral nervous system, such as neuroinflammation and / or inflammation of the brain, is detected / diagnosed, and the dose can be increased if inflammation persists. Similarly, if inflammation decreases after initial administration of tetracycline or a derivative / analogue thereof, the dose can be decreased.

[0032] In another embodiment, the administration or expression of IL-2 involves a transgene containing elements that prevent expression of IL-2. Such elements that prevent expression can be removed and / or inactivated in cells of the tissue or organ of interest. In certain embodiments, there is no removal or inactivation of elements that prevent expression in cells other than cells of the tissue or organ of interest. Thus, in one embodiment, removal or inactivation of elements that prevent expression does not occur in a population of regulatory T cells generated according to the methods defined herein. In a further embodiment, the element that prevents expression is a stop cassette. In one embodiment, the stop cassette is contained within a transgene defined herein and located upstream of the gene encoding IL-2. In a further embodiment, the stop cassette is flanked by sites recognized by a recombinase enzyme. Such recombinase enzymes include Cre recombinase and Flp recombinase, which can recognize and recombine sites such as LoxP and FRT, respectively. Recombination of the sites results in the removal, deletion, and / or inactivation of the sequences contained between them. Thus, in one embodiment, the termination cassette is flanked by LoxP recombination sites. According to this embodiment, cells of a tissue or organ of interest can express Cre recombinase to recombine the recombination sites in said cells. In certain embodiments, the Cre recombinase expression is localized specifically or exclusively within cells of the tissue or organ of interest. Such localized or specific expression of Cre recombinase in cells of a tissue or organ of interest can be driven by the methods defined herein using a tissue- or organ-specific promoter, or by any other method known in the art. Such methods can include tissue- or organ-specific delivery of the Cre recombinase enzyme, as well as tissue- or organ-specific delivery of a Cre recombinase-encoding sequence, such as tissue- or organ-specific delivery of an mRNA encoding Cre recombinase or a transgene encoding Cre recombinase. Thus, in certain embodiments, localized or specific expression of Cre recombinase is driven by a tissue- or organ-specific promoter.In one embodiment, the local or specific expression of Cre recombinase is driven by a promoter that drives specific expression in neurons. In a further embodiment, the local or specific expression of Cre recombinase is driven by a promoter that drives specific expression in cells of the central nervous system and / or peripheral nervous system. In a further embodiment, the local or specific expression of Cre recombinase is driven by a promoter that drives expression in the central nervous system but not the peripheral nervous system. In another embodiment, the local or specific expression of Cre recombinase is driven by a promoter that drives expression in the peripheral nervous system but not the central nervous system. In one embodiment, the local or specific expression of Cre recombinase is driven by a promoter that drives expression specifically in the brain. In a particular embodiment, the local or specific expression of Cre recombinase is driven by a promoter that drives expression specifically in astrocytes. In a further embodiment, the local or specific expression of Cre recombinase is driven by a PLP promoter. In another embodiment, the local or specific expression of Cre recombinase is driven by the CaMKIIa promoter.

[0033] It will be appreciated that in embodiments utilizing elements that prevent expression in cells other than cells of the tissue or organ of interest, the presence of a tissue- or organ-specific promoter controlling expression of IL-2 may not be necessary. Thus, in one embodiment, a transgene comprising elements that prevent expression in cells other than cells of the tissue or organ of interest does not comprise a tissue- or organ-specific promoter. In another embodiment, a transgene comprising elements that prevent expression in cells other than cells of the tissue or organ of interest further comprises a tissue- or organ-specific promoter. In such embodiments, expression of IL-2 is subject to an additional level of control to further ensure tissue- or organ-specific administration or expression.

[0034] In one embodiment, a transgene as defined herein is introduced into cells of a tissue or organ of interest by transduction, such as transduction using a virus or viral vector. In a particular embodiment, transduction uses an adeno-associated virus. Thus, in one embodiment, administration of IL-2 comprises transduction, such as viral transduction. In a further embodiment, administration of IL-2 comprises adeno-associated virus transduction.

[0035] In one embodiment, transduction of a transgene defined herein utilizes a viral vector that specifically targets or infects cells of a tissue or organ of interest. Thus, in one embodiment, transduction of a transgene defined herein specifically targets or infects cells of a tissue or organ of interest. It will be understood that, according to this embodiment, transduction of a transgene defined herein using a viral vector does not target or infect a population of regulatory T cells. In a further embodiment, transduction of a transgene defined herein involves a viral vector that is accessible to the tissue or organ of interest and can cross barriers that separate the tissue or organ of interest from other tissues, organs, or the rest of the organism. Thus, in one embodiment, transduction involves a viral vector that can specifically target or infect the nervous system. In a further embodiment, transduction involves a viral vector that can target or infect the central nervous system. In an alternative embodiment, transduction involves a viral vector that can target or infect the peripheral nervous system. In a further embodiment, the transduction involves a viral vector capable of targeting or infecting the brain.

[0036] In certain embodiments, transduction involves a viral vector capable of crossing the blood-brain barrier. In one embodiment, transduction involves an adeno-associated virus, which crosses the blood-brain barrier. Thus, in one embodiment, transduction involves a neurotropic virus or viral vector. In another embodiment, the viral vector is a neurotropic virus or viral vector. Examples of neurotropic viruses and viral vectors capable of crossing the blood-brain barrier include, but are not limited to, AAVrh.8, AAVrh10, and AAV9, as well as variants and derivatives thereof (e.g., AAVhu68 and PHP.B). In certain embodiments, the transgene defined herein is comprised in a viral vector, such as a neurotropic virus or viral vector and / or an adeno-associated viral vector. In a further embodiment, transduction involves the adeno-associated virus variant AAV9 and its derivatives, such as PHP.B. In a further embodiment, transduction involves a PHP.B viral vector. In another embodiment, the transgene defined herein is comprised in a PHP.B viral vector. Thus, in one embodiment, the transduction and / or viral vector comprises PHP.B-GFAP-IL2, a PHP.B derivative of AAV9 that contains a transgene comprising an IL-2 coding sequence and the astrocyte-specific promoter GFAP. Viral vectors can be used to integrate a target sequence, such as a transgene, into a host cell genome, such as the genome of cells of a tissue or organ of interest. Thus, in one embodiment, transduction involves 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 neurotropic viruses or viral vectors and adeno-associated viral vectors, can also be used to enable 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.

[0037] 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 infecting, killing, or destroying cells, such as cells of a tissue or organ of interest.

[0038] It will be appreciated from the disclosure provided herein that the combination of a virus or viral vector (e.g., a neurotropic virus or viral vector) that specifically targets or infects cells of a tissue or organ of interest with a promoter that drives expression specifically in the 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 a 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 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 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.

[0039] In a further embodiment, the combination of a tissue- or organ-specific viral vector defined herein and a tissue- or organ-specific promoter, 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 defined herein, and that only cells of the tissue or organ of interest are capable of expressing said transgene upon administration of an activator of the inducible element (e.g., tetracycline, doxycycline, or minocycline / minomycin). In one embodiment, the combination of a tissue- or organ-specific viral vector defined herein and a tissue- or organ-specific promoter, 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 upon administration of an activator of the inducible element (e.g., tetracycline, doxycycline, or minocycline / minomycin). 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) that results in expression of IL-2 when an activator of the inducible element is administered (e.g., tetracycline, doxycycline, or minocycline / minomycin).

[0040] Administration of IL-2, as defined herein, may further include direct administration of IL-2 to a tissue or organ of interest. Examples of direct administration include direct injection into a tissue or organ of interest, such as by intracranial injection, 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 treatment (e.g., chronically or during treatment of an acute inflammatory disease or disorder).

[0041] 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 traumatic injury. Thus, in certain embodiments, the duration of IL-2 administration or expression depends on the disease or disorder being treated or the duration of treatment. In one embodiment, administration or expression of IL-2 is acute.

[0042] 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.

[0043] 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 the targeting moiety provides for localized administration or expression of IL-2 as defined herein.

[0044] Thus, in one embodiment of the invention, the methods defined herein comprise administering 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 to or at 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 to or at 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 for expression of IL-2 in the tissue or organ of a subject. Thus, in one embodiment, the targeting moiety specific for a tissue or organ of a subject provides for expression of only IL-2 in the tissue or organ of a subject. Such references herein to "in a tissue or organ of interest" further include cases where the effect is within cells (e.g., neurons and / or astrocytes) that make up said tissue or organ.

[0045] 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, the virus or viral vector specifically targets or infects the tissue or organ of interest, or specifically targets or infects cells in the tissue or organ of interest. Thus, according to this embodiment, the targeting moiety specific for a tissue or organ of interest is a virus or viral vector that does not target or infect cells in tissues or organs other than the tissue or organ of interest, or that does not target or infect cells comprising tissues or organs other than the tissue or organ of interest. It will also be understood that according to this embodiment, the 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 cross a barrier that separates the tissue or organ of interest from other tissues, organs, or the rest of the subject. Thus, in one embodiment, the tissue- or organ-specific targeting moiety comprises a virus or viral vector capable of specifically targeting or infecting the nervous system, such as a neurotropic virus or viral vector. In a further embodiment, the tissue- or organ-specific targeting moiety comprises a virus or viral vector capable of targeting or infecting the central nervous system. In an alternative embodiment, the tissue- or organ-specific targeting moiety comprises a virus or viral vector capable of targeting or infecting the peripheral nervous system.

[0046] In certain embodiments, the tissue- or organ-specific targeting moiety comprises a virus or viral vector capable of crossing the blood-brain barrier. In one embodiment, the tissue- or organ-specific targeting moiety comprises an adeno-associated virus capable of crossing the blood-brain barrier. Thus, in certain embodiments, the tissue- or organ-specific targeting moiety comprises a neurotropic virus or viral vector. In one embodiment, the targeting moiety is selected from a neurotropic virus or viral vector, such as AAVrh.8, AAVrh10, or AAV9, and variants and derivatives (e.g., AAVhu68 and PHP.B). In a further embodiment, the tissue- or organ-specific targeting moiety comprises the adeno-associated virus variant PHP.B. In certain embodiments, a transgene as defined herein is comprised in a tissue- or organ-specific targeting moiety, such as an adeno-associated viral vector, which is comprised within an adeno-associated virus as defined herein. In one embodiment, a transgene as defined herein is comprised in a neurotropic virus or viral vector, such as a PHP.B viral vector. Therefore, in a further embodiment, a transgene comprising an IL-2 coding sequence and an astrocyte-specific promoter GFAP or minimal GFAP is contained within an AAV9 derivative PHP.B virus / viral vector, and the virus / viral vector is PHP.B-GFAP-IL2.

[0047] 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 certain embodiments, the term "in a subject" as used herein is synonymous with "in vivo."

[0048] In one embodiment, the method for expanding a regulatory T cell population in vivo in a tissue or organ comprises administering IL-2 as described herein. In a further embodiment, the method for expanding a regulatory T cell population in vivo in a tissue or organ comprises administering 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 for expanding a regulatory T cell population in vivo in a tissue or organ comprises a virus or viral vector comprising an IL-2-encoding 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 for 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, a method for expanding a regulatory T cell population in vivo in a tissue or organ comprises administering a targeting moiety specific for the tissue or organ of interest, wherein the targeting moiety is a virus or viral vector that crosses the blood-brain barrier as defined herein. In further embodiments, a method for expanding a regulatory T cell population in vivo in a tissue or organ comprises administering a targeting moiety specific for the tissue or organ of interest, wherein the targeting moiety is specific for the nervous system, such as the central nervous system and / or the peripheral nervous system. In a further embodiment, the targeting moiety specific for the tissue or organ of interest is specific for astrocytes. In another embodiment, a method for expanding a regulatory T cell population in vivo in a tissue or organ comprises administering a neurotropic virus or viral vector comprising a transgene as defined herein, such as administering PHP.B-GFAP-IL2.

[0049] According to one aspect of the present 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.

[0050] Pharmaceutical Composition 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, wherein the targeting moiety is specific for the central nervous system and / or the peripheral nervous system.

[0051] In one embodiment, the pharmaceutical composition comprises IL-2 that promotes 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 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 in a subject.

[0052] In one embodiment, the pharmaceutical composition defined herein comprises a targeting moiety capable of crossing a barrier separating a tissue or organ of interest from other tissues or organs or the rest of an organism. Thus, in one embodiment, the pharmaceutical composition defined herein comprises a virus or viral vector that crosses the blood-brain barrier, such as an adeno-associated virus and / or a neurotropic virus or viral vector. In a further embodiment, the pharmaceutical composition defined herein comprises the adeno-associated virus variant AAV9 or a derivative thereof, such as PHP.B. 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.

[0053] Thus, in certain embodiments, the pharmaceutical compositions defined herein comprise a tissue- or organ-specific virus or viral vector that contains an IL-2-encoding gene whose expression is driven by a tissue- or organ-specific promoter and that can target or infect cells of a tissue or organ of interest. In one particular embodiment, the pharmaceutical compositions defined herein comprise a viral vector, such as an adeno-associated virus (e.g., AAV9 or a derivative thereof, such as PHP.B), that specifically targets or infects neurons or the nervous system, such as the brain (i.e., a neurotropic virus or viral vector), and contains an IL-2-encoding gene whose expression is driven by a tissue- or organ-specific promoter. In a further embodiment, the pharmaceutical compositions defined herein comprise the adeno-associated virus AAV9, which contains an IL-2-encoding gene whose expression is driven locally in neurons / astrocytes or the nervous system by a GFAP promoter or a minimal GFAP promoter. In a further embodiment, the adeno-associated virus is a derivative of AAV9, such as PHP.B. Thus, in one embodiment, the pharmaceutical composition comprises PHP.B-GFAP-IL2.

[0054] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients in addition to a tissue- or organ-specific virus or viral vector as defined herein.

[0055] Generally, the pharmaceutical compositions are utilized with pharmacologically appropriate 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 solution. Suitable physiologically acceptable adjuvants can be selected from viscosity enhancing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates, if necessary, to maintain the composition containing 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. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th ed.).

[0056] Therapeutic Uses and Methods It will be appreciated from the disclosure provided herein that the methods for expanding populations of regulatory T cells, pharmaceutical compositions, and treatment methods of the present invention are particularly useful for treating and / or ameliorating diseases or disorders mediated by inflammation and / or reducing inflammation. It will further be appreciated that populations of regulatory T cells expanded according to the methods and disclosure provided herein are also useful for treating and / or ameliorating diseases or disorders mediated by inflammation and / or reducing inflammation.

[0057] Thus, according to one 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, for use in the treatment and / or amelioration of a disease or disorder mediated by inflammation, wherein the tissue or organ is the central and / or peripheral nervous system. In another 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, wherein the tissue or organ is the central and / or peripheral nervous system, for use in reducing inflammation. In a further 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, wherein the tissue or organ is the central and / or peripheral nervous system, for use in the treatment and / or amelioration of an autoimmune disease.

[0058] In another aspect of the present invention, there is provided a regulatory T cell population expanded in a tissue or organ of a subject, produced 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, produced 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, produced 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 that is driven 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 produced according to the methods defined herein is for use in the treatment and / or amelioration of a disease or disorder of the nervous system. In one embodiment, the regulatory T cell population expanded in a tissue or organ of a subject produced according to the methods defined herein is for use in the treatment and / or amelioration of a central nervous system and / or peripheral nervous system. In a further embodiment, the regulatory T cell population expanded in a tissue or organ of a subject produced according to the methods defined herein is for use in the treatment and / or amelioration of neuroinflammation. In one embodiment, the regulatory T cell population expanded in a tissue or organ of a subject produced according to the methods defined herein is for use in the treatment and / or amelioration of inflammation in the brain. Thus, according to one embodiment, inflammation as defined herein is brain inflammation. In a further embodiment, the brain inflammation is due to a brain or head injury, such as a traumatic brain injury or stroke.In another embodiment, the regulatory T cell population expanded in a tissue or organ of a subject produced according to the methods defined herein is for use in the treatment and / or amelioration of a neurological disease or disorder. Thus, in one embodiment, the inflammation in the brain is due to a neurological disease or disorder, such as a traumatic neurological disease or disorder. In another embodiment, the regulatory T cell population expanded in a tissue or organ of a subject produced according to the methods defined herein is for use in the treatment and / or amelioration of a cognitive disorder, such as a cognitive disorder caused by neuroinflammation. In one embodiment, the regulatory T cell population expanded in a tissue or organ is for use in reducing a cognitive disorder. In a further embodiment, the inflammation in the brain is due to an acute traumatic injury, disease, or disorder. Thus, in a further embodiment, the neurological disease or disorder is other than (i.e., is not) a neurodegenerative disease or disorder, such as Alzheimer's disease and / or Parkinson's disease. Another example is an autoimmune disease or disorder, and / or the inflammation is due to an autoimmune disease or disorder.

[0059] 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, the 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, either as defined herein. In one embodiment, the treatment method 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 treatment method 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 the disease or disorder or affected by inflammation. In certain embodiments, the treatment method 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 treatment method 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 under the control of a tissue- or organ-specific promoter and driven by an inducible element, such as a tetracycline-inducible element. In a further embodiment, the method of treatment defined herein comprises administering to a subject in need thereof a neurotropic virus comprising a gene encoding IL-2, the expression of which is driven by a tissue- or organ-specific promoter, such as administration of PHP.B-GFAP-IL2.

[0060] In one embodiment, the subject in need thereof is suffering from a disease or disorder mediated by inflammation. In a further embodiment, the subject in need thereof is suffering from inflammation. In a further embodiment, the subject in need thereof is suffering from an autoimmune disease or disorder. In one embodiment, the disease or disorder is a disease or disorder of the nervous system, such as the central nervous system and / or the peripheral nervous system. In a further embodiment, the disease or disorder is a disease or disorder of the brain. In a further embodiment, the disease or disorder is a neurological disease or disorder other than (i.e., not) a neurodegenerative disease or disorder, such as Alzheimer's disease or Parkinson's disease. In another embodiment, the inflammation is neuroinflammation, such as brain inflammation. In one embodiment, the inflammation is brain inflammation due to brain or head injury, such as traumatic brain injury or stroke. Thus, in some embodiments, the inflammation is brain inflammation due to acute traumatic injury. [Example]

[0061] (Example) Example 1: Regulatory T cells are present in the brain parenchyma of healthy mice To investigate the presence of regulatory T cells in the brain, tissues traditionally considered isolated from the immune system, such as the parenchyma, perivascular space, and intravascular regions of mouse brains, were prepared for confocal microscopy and immunostained for CD4 (a T cell marker) and FoxP3 (a specific marker for regulatory T cells). These tissues were further stained with fluorescently labeled lectins to label vascular structures and with DAPI to identify cell nuclei. Representative images are shown in Figure 1A, and enlarged and 3D reconstructions are shown in Figure 1B. Figure 1C shows the number of regulatory T cells in a perfused mouse brain as determined by flow cytometry.

[0062] As can be seen from the data, regulatory T cells can be readily identified in the brains of healthy mice by both microscopy and flow cytometry. Depending on the age of the mice analyzed, the number of detectable regulatory T cells in the brain ranges from approximately 100 to over 2,000 cells, with the majority of mice containing approximately 100 to 1,000 regulatory T cells in the brain.

[0063] Example 2: Brain-resident regulatory T cells acquire a resident phenotype in situ during long-term brain migration To determine whether regulatory T cells disseminate from the periphery to the brain and whether they can acquire a resident-like phenotype, we performed a parabiotic experiment. Parabiotic pairs were established using CD45.1+ and CD45.2+ mice, and the brains of each mouse were sampled at 2, 4, 8, and 12 weeks (Figure 2A). As can be seen, both CD69+ and CD69- regulatory T cells from donor mice could be identified in the brain and blood (Figure 2B). We measured the proportion of regulatory T cells present in the brain and blood of donor mice (determined using CD45.1 or CD45.2 expression) and determined their phenotype (Figures 2C and 2D).

[0064] As demonstrated by the data and population flow diagram generated from the data (Figure 2E), regulatory T cells can disseminate from the periphery to the brain and be detected as originating from the parabiotic donor mouse. Donor-derived regulatory T cells in the brain exhibit a tissue-resident phenotype, indicating that this can be acquired during brain migration. However, the data demonstrate that the naive regulatory T cell population, disproportionately expanded by IL-2 administration, disseminates to the brain approximately 10-fold less efficiently than activated regulatory T cells (Figure 2E). Therefore, it is necessary to expand regulatory T cells specifically in the brain without significantly expanding the peripheral regulatory T cell population.

[0065] Example 3: Proof-of-principle transgenic mouse model for brain-specific regulatory T cell expansion To test the principle of brain-specific IL-2 delivery to expand regulatory T cell populations specifically in the brain, we developed a transgenic mouse model called Rosa, in which IL-2 expression is switched on by Cre activity under a weak constitutive promoter. fl-Stop-fl We developed IL-2 mice. Expression in this system is approximately four-fold lower per cell than endogenous expression in IL-2-producing cells (Figure 3A). Thus, the system operates by altering the localization of expression rather than overexpression. To test this system in the brain, we used two brain-specific Cre lines, PLP-Cre ER and PLP-Cre ER. T(Figure 3B) and CaMKIIa Cre ER T (Figure 3C) was used to induce restricted expression. Activation of IL-2 production via either transgene expanded the regulatory T cell population in the brain (Figures 3D and 3E). PLP-Cre resulted in a slight increase in peripheral T cells, whereas CaMKIIa Cre did not (Figures 3D and 3E). Therefore, we chose to use the CaMKIIa Cre driver for subsequent experiments. Single-cell RNA-seq was performed on brain CD4 T cells using the 10x genomics Chromium platform. Expanded brain regulatory T cells from the brains of IL-2 aCaMKII Cre mice clustered closely with the (smaller) population of brain regulatory T cells from wild-type mouse brains (Figure 3F). Analysis of single-cell transcriptional data revealed that regulatory T cells in the brains of IL-2 aCaMKII Cre mice expressed known markers, including Ilr2, Gata3, and Ikzf2, indicating the absence of an abnormal proliferative effect on regulatory T cell populations (Figure 3G). Analysis of expressed cytokines demonstrated that the only highly expressed cytokine from these expanded regulatory T cells was the low-affinity epidermal growth factor receptor (EGFR) ligand Areg, implicated in wound healing and tissue repair (Zaiss et al. (2015) doi: https: / / doi.org / 10.1016 / j.immuni.2015.01.020) (Figure 3H). Brain regulatory T cell proliferation was significantly suppressed by IL-2 aCaMKII Cre (αCamKII) mice. IL2 ) did not result in adverse behavioral changes in mice (Figures 3I-3S).

[0066] These data demonstrate that local delivery of IL-2 can specifically expand regulatory T cell populations in the brain without expanding their numbers in the periphery, and that the expanded regulatory T cells maintain a classical regulatory T cell expression profile.

[0067] Example 4: Expanded Brain Regulatory T Cells Protect Against Traumatic Brain Injury To determine the potential of brain-specific regulatory T cell proliferation to attenuate neuroinflammatory damage, we investigated the effects of moderate traumatic brain injury (TBI) delivered by cortical controlled impact. IL2 ) mice and littermate controls had moderate TBI and were examined 15 days after TBI. Wild-type mice showed complete cortical death at the impacted cortical site with no evidence of neuronal recovery, whereas IL-2 aCaMKII Cre mice showed greatly reduced injury at the impact site, with compensatory expansion of the ipsilateral hippocampus, reduced lesion size, and preservation of neural tissue (Figures 4A-4D).

[0068] The data demonstrate that local delivery of IL-2 can create a local anti-inflammatory environment that can prevent neuropathology without increasing the systemic regulatory T cell load.

[0069] Example 5: Astrocyte-specific expression using the GFAP promoter To demonstrate the efficacy of localized IL-2 delivery, we developed a delivery system that can be used in a therapeutic setting. Adeno-associated viruses (AAVs), which cross the blood-brain barrier (BBB), are a promising tool for rapid administration of CNS therapeutics, as they enable the delivery of transgenes encoding large bioactive molecules without the need for invasive surgical procedures. AAV-based vectors are the system of choice in clinical trials due to their long-term transgene expression and excellent safety profile. Because AAVs present a unique opportunity for delivering IL-2 to the CNS in a clinical setting, we used the recently identified AAV variant, PHP.B (Rincon et al., 2018, doi: https: / / doi.org / 10.1038 / s41434-018-0005-z), which has been shown to be efficient at crossing the BBB and confers high levels of transduction throughout the CNS. Because our primary concern here was off-target production of IL-2 in the periphery, we linked the AAV vector to the GFAP promoter, which confers long-term and specific expression only in astrocytes (Figure 5A). The combination of a neurotropic virus and a brain-specific promoter provides a "double lock" on target specificity, limiting or eliminating peripheral expression of the delivered target after systemic delivery. We then generated the AAV-PHP.B virus carrying a transgene for murine IL-2 (NG_06779.1) under the control of the astrocyte-specific GFAP promoter (PHP.B-GFAP-IL2) as follows:

[0070] A classical triple transfection method was used, followed by vector titration using a qPCR-based method (Rincon et al. (2018), doi: https: / / doi.org / 10.1038 / s41434-018-0005-z). The murine IL-2 coding sequence, along with the 5' and 3' UTRs (accession number BC116845), was cloned into a single-stranded AAV2-derived expression cassette containing the full-length GFAP promoter (Brenner et al. (1994), doi: https: / / doi.org / 10.1523 / JNEUROSCI.14-03-01030.1994), woodchuck hepatitis posttranscriptional regulatory element (WPRE), and bovine growth hormone polyadenylation (bGH polyA) sequences. A control vector was prepared by exchanging the IL-2 coding sequence for a sequence encoding enhanced green fluorescent protein (EGFP).

[0071] This therapeutic design allows targeted delivery of self-proteins expressed in the physiological range. Injection of PHP.B-GFAP-IL2 into WT mice successfully promoted brain-specific proliferation of regulatory T cell populations (Figures 6A, 6B, and 6E) without subsequently inducing peripheral proliferation (Figures 6C, 6D, and 6E). Brain-specific proliferation of regulatory T cell populations was also PHP.B-GFAP-IL2 dose-dependent (Figure 6F).

[0072] Taken together, this data provides evidence that the "double-lock" PHP.B-mediated gene delivery of IL-2 to the brain provided herein results in brain-specific expansion of regulatory T cells.

[0073] Unlike classical gene therapy, where the efficiency of cell transduction by viral vectors is crucial, the production of a promising secreted factor means that even a small number of transduced cells can modulate disease. Therefore, a lower dose of 1 x 10 was used to test the effect on experimental autoimmune encephalomyelitis (EAE), the gold standard mouse model of multiple sclerosis. 9We selected the PHP.B-GFAP-IL2 vector genome. Untreated mice developed classic EAE pathology, with severe clinical symptoms (Figure 6G) and severe lymphocytic infiltration in the brain (Figure 6H). In contrast, PHP.B-GFAP-IL2 pre-treated mice were resistant to EAE, with disease severity rapidly plateauing and reversing (Figure 6G) and lymphocytic infiltration being sharply suppressed (Figure 6H). Potential mechanisms include increased expression of AREG and IL-10 (Figure 6G).

[0074] Because the kinetics of EAE are suitable for testing curative effects, we induced EAE in a cohort of mice and then, after the onset of clinical symptoms, administered 1x10 9 Treatment with control vector genome (PHP.B-GFAP-GFP) or "double-locked" PHP.B-GFAP-IL2 (day 10) was performed. Surprisingly, the protective effect of PHP.B-GFAP-IL2 was still observed, with a sharp decline in cumulative clinical scores observed when the clinical time course was separated out to day 15 (Figure 6I).

[0075] The data provide preclinical evidence for "double-lock" gene delivery of IL-2 to the brain as a promising treatment for neuroinflammatory diseases such as multiple sclerosis.

[0076] Example 6: Expansion of regulatory T cells in the brain reduces damage from traumatic brain injury To determine the potential of PHP.B-GFAP-IL2 therapy in reducing progression or reversing injury during traumatic brain injury, 1 x 10 cells were cultured in 10% IL-1+ / 2% PBS containing PHP.B-GFAP-IL2 or control PHP.B without IL-2 (PHP.B-GFAP-GFP). 9 The vector genome was administered to mice prior to traumatic brain injury.

[0077] Microscopic analysis of the brains of control PHP.B-treated mice revealed extensive superficial brain damage at the impact site, whereas treatment with PHP.B-GFAP-IL2 significantly reduced the size of the brain impact site (Figure 7A). Histological analysis identified preservation of the cerebral cortex at the impact site, and regeneration was demonstrated by BrdU incorporation (Figure 7B). There was also reduced neural tissue loss 14 days after injury, as demonstrated by histology (Figures 7B and 7C) and MRI (Figure 7D). Neuroprotective effects were also observed at the behavioral level, with the decline in behavioral performance of mice after TBI being completely reversed in PHP.B-GFAP-IL2-treated mice (Figures 7E and 7F). These effects were likely mediated by alterations to the local environment, as little change in inflammatory influx was observed (Figure 8).

[0078] Thus, these data demonstrate the utility of brain-specific administration of IL-2 and regulatory T cell proliferation in reducing and / or reversing damage during traumatic brain injury.

[0079] Example 7: Expansion of regulatory T cells in the brain reduces stroke severity To extend the above findings to a second indication, we used two independent mouse models of stroke. In both photothrombotic stroke (Figures 9A and 9B) and ischemic stroke (Figures 9C and 9D), PHP.B-GFAP-IL2-treated mice showed significantly higher levels of IL-1 than control (PHP.B-GFAP-GFP; both 1x10 9 A significant reduction in severity was observed compared to when a dose of vector genome was administered.

[0080] Taken together, the results presented herein confirm the therapeutic potential of the "dual lock" PHP.B-GFAP-IL2 system to prevent or treat neurological damage in several independent preclinical models of neuroinflammatory disease without altering peripheral immunity.

[0081] Example 8: Small molecule induction system for brain-specific regulatory T cell proliferation To determine the possibility of temporal control of PHP.B-GFAP-IL2 therapy, wild-type mice were treated with 1 x 10 PHP.B-GFAP-GFP control vector or PHP.B-GFAP-TetR-T2A-rtTA(V7 / V14).TetO-IL2 (PHP.GFAP / TetO-IL2) 9 vector genome (total dose) was administered.

[0082] The PHP.B-GFAP / TetO-IL2 vector contains a TetO sequence upstream of the IL-2-encoding gene, which binds the reverse tetracycline-controlled transactivator (rtTA) protein (expressed under the control of the GFAP promoter) and promotes expression in the presence of tetracyclines such as minocycline / minomycin. Therefore, mice were gavaged daily with minocycline (50 mg / kg) or PBS control (n = 4-5 mice per group), and the proportion of Tregs in the spleen or perfused brain was assessed 14 days after treatment. As seen in Figure 10, administration of minocycline to mice administered the PHP.GFAP / TetO-IL2 vector resulted in substantial expansion of Tregs in the brain, but not in the periphery (spleen).

[0083] Thus, these data indicate that small-molecule-mediated IL-2 expression controlled by a tetracycline-inducible element specifically expressed in astrocytes can be used to specifically increase regulatory T cell populations in the brain without increasing Treg levels in the periphery. The present application provides the following aspects of the invention. (Aspect 1) 1. A method for expanding a population of regulatory T cells in a tissue or organ of a subject in need thereof, comprising administering IL-2 and a targeting moiety specific for said tissue or organ, wherein said tissue or organ is the central nervous system and / or peripheral nervous system. (Aspect 2) The method of embodiment 1, wherein the tissue or organ is the brain. (Aspect 3) 3. The method of embodiment 1 or embodiment 2, wherein administering IL-2 comprises tissue- or organ-specific expression of IL-2 in said tissue or organ of said subject. (Aspect 4) The method of embodiment 3, wherein the tissue- or organ-specific expression of IL-2 is driven by a tissue- or organ-specific promoter. (Aspect 5) 5. The method of any one of aspects 1 to 4, wherein the administration of IL-2 or tissue- or organ-specific expression of IL-2 in said tissue or organ comprises an exogenous IL-2 coding sequence. (Aspect 6) The method of any one of embodiments 1 to 5, wherein said tissue or organ-specific targeting moiety comprises a viral vector. (Aspect 7) The method of embodiment 6, wherein the viral vector is a neurotropic virus or viral vector. (Aspect 8) 8. The method of embodiment 7, wherein the neurotropic virus or viral vector is an adeno-associated virus selected from AAVrh.8, AAVrh10, or AAV9, and variants and derivatives thereof, such as AAVhu68 and PHP.B. (Aspect 9) The method of embodiment 8, wherein the neurotropic virus or viral vector is the adeno-associated virus variant PHP.B. (Aspect 10)

[0023] Aspect 1-9, wherein the tissue or organ-specific targeting moiety or the viral vector crosses a barrier separating the tissue or organ of the subject from other tissues or organs, such as the blood-brain barrier. (Aspect 11) A pharmaceutical composition comprising IL-2 and a targeting moiety specific for a tissue or organ of a subject, wherein said targeting moiety is specific for said central and / or peripheral nervous system. (Aspect 12) 12. The pharmaceutical composition of embodiment 11, wherein said tissue- or organ-specific targeting moiety comprises a viral vector. (Aspect 13) 13. The pharmaceutical composition of embodiment 12, wherein the viral vector is a neurotropic virus or viral vector. (Aspect 14) 14. The pharmaceutical composition of embodiment 13, wherein said neurotropic virus or viral vector is an adeno-associated virus selected from AAVrh.8, AAVrh10 or AAV9, and variants and derivatives thereof, such as AAVhu68 and PHP.B. (Aspect 15) 15. The pharmaceutical composition of embodiment 14, wherein the neurotropic virus or viral vector is the adeno-associated virus variant PHP.B. (Aspect 16) 16. The pharmaceutical composition of any one of aspects 11-15, wherein said tissue or organ-specific targeting moiety or said viral vector crosses a barrier separating said tissue or organ from other tissues or organs of said subject, such as the blood-brain barrier. (Aspect 17) 17. A pharmaceutical composition according to any one of aspects 11 to 16 for use in the treatment of a disease or disorder mediated by inflammation and / or in reducing inflammation. (Aspect 18) 17. A method for treating a disease or disorder mediated by inflammation and / or for reducing inflammation, any of which comprises administering to a subject in need thereof the method according to any one of aspects 1 to 10 or the pharmaceutical composition according to any one of aspects 11 to 16. (Aspect 19) 19. The pharmaceutical composition for use according to embodiment 17 or the method according to embodiment 18, wherein said disease or disorder is a neurological disorder. (Aspect 20) 19. The pharmaceutical composition for use according to embodiment 17 or the method according to embodiment 18, wherein said disease or disorder is multiple sclerosis. (Aspect 21) 19. The pharmaceutical composition for use according to embodiment 17 or the method according to embodiment 18, wherein said inflammation is inflammation of the central nervous system and / or peripheral nervous system, such as neuroinflammation. (Aspect 22) 22. The pharmaceutical composition for use according to embodiment 17 or embodiment 21, or the method according to embodiment 18 or embodiment 21, wherein said inflammation is inflammation of the brain. (Aspect 23) A pharmaceutical composition for use according to aspect 17 or a method according to aspect 18, wherein said brain inflammation is due to an injury to the brain or head, such as a traumatic brain injury or stroke. (Aspect 24) A pharmaceutical composition for use according to aspect 17 or a method according to aspect 18, wherein said brain inflammation is due to an acute injury to the brain or head, such as an acute traumatic injury. (Aspect 25) 19. The pharmaceutical composition for use according to embodiment 17 or the method according to embodiment 18, wherein said disease or disorder and / or inflammation is an autoimmune disease or disorder and / or said inflammation is autoimmune mediated.

Claims

1. A pharmaceutical composition comprising a gene encoding IL-2 and a targeting moiety specific to a tissue or organ of a subject, wherein the targeting moiety is a neurotropic virus or viral vector specific to the central nervous system and / or peripheral nervous system in the subject, and wherein IL-2 is specifically expressed in the tissue or organ of the subject.

2. 2. The pharmaceutical composition of claim 1, wherein the tissue or organ is the brain.

3. 3. The pharmaceutical composition according to claim 1, wherein the tissue- or organ-specific expression of IL-2 is driven by a tissue- or organ-specific promoter.

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 exogenous IL-2 coding sequence.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the neurotropic virus or viral vector is an adeno-associated virus selected from AAVrh.8, AAVrh10 or AAV9 and variants thereof, such as AAVhu68 and PHP.B.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the neurotropic virus or viral vector is the adeno-associated virus variant PHP.B.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the neurotropic virus or viral vector crosses a barrier, such as the blood-brain barrier, that separates the tissue or organ from other tissues or organs of the subject.

8. 8. A pharmaceutical composition according to any one of claims 1 to 7 for use in the treatment of a disease or disorder mediated by inflammation or associated with neuroinflammation due to injury, and / or for reducing inflammation.

9. 9. The pharmaceutical composition for use according to claim 8, wherein the disease or disorder is a neurological disorder.

10. 9. The pharmaceutical composition for use according to claim 8, wherein the disease or disorder is multiple sclerosis.

11. 9. The pharmaceutical composition for use according to claim 8, wherein the inflammation is inflammation of the central and / or peripheral nervous system, such as neuroinflammation.

12. 12. The pharmaceutical composition for use according to claim 8 or claim 11, wherein the inflammation or neuroinflammation is inflammation of the brain.

13. 9. The pharmaceutical composition for use according to claim 8, wherein the brain inflammation or neuroinflammation is due to an injury to the brain or head, such as a traumatic brain injury or stroke.

14. 9. The pharmaceutical composition for use according to claim 8, wherein the brain inflammation or neuroinflammation is due to an acute injury to the brain or head, such as an acute traumatic injury.

15. 9. The pharmaceutical composition for use according to claim 8, wherein the disease or disorder and / or inflammation or neuroinflammation is an autoimmune disease or disorder and / or the inflammation or neuroinflammation is autoimmune.

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