WO2017202814A1 - Methods and pharmaceutical compositions for the treatment of neuropathological disorders characterized by a loss of cortical neurons - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of neuropathological disorders characterized by a loss of cortical neurons. The 10 inventors showed that interleukin 33 promotes axonal outgrowth of embryonic cortical neurons grafted into injured adult motor cortex. Moreover, they explore the effects of transplanted cells on the host local inflammatory environment in order to understand the extent to which inflammation following cortical lesion could influence the survival of grafted neurons and the development of their projections. In particular, the present invention relates 15 to ST2 agonist for use in the treatment of neuropathological disorders characterized by a loss of cortical neurons in a subject in need thereof.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF NEUROPATHOLOGICAL DISORDERS CHARACTERIZED BY A LOSS OF

CORTICAL NEURONS

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of neuropatho logical disorders characterized by a loss of cortical neurons.

BACKGROUND OF THE INVENTION:

Neuropatho logical disorders are a major concern since it exists a wide diversity of pathologies, such as neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease or Huntington's disease) or neuronal injuries following trauma and neuronal cell death following strokes for instance. These pathologies are often accompanied by motor and cognitive dysfunctions with limited treatment options.

In particular, loss of cortical neurons is a common characteristic of numerous neuropatho logical conditions. The inhibitory nature of the adult mammalian central nervous system (CNS) prevents spontaneous axonal regeneration following injury (Davies et al, 1997; 1999). One way to overcome the limited regenerative capacity of the adult CNS is transplantation of embryonic neurons. The inventors have previously reported that embryonic cortical neurons grafted into the adult mouse motor cortex immediately after a cortical lesion allowed reestablishment of the damaged motor pathways. The transplanted neurons develop projections towards all cortical and subcortical targets of the motor cortex, including distant targets such as the spinal cord (Gaillard et al, 2007). While the results of this study were encouraging for CNS repair, a serious limitation to consider such approaches in a clinical setting is the delay of transplantation after injury. The inventors have recently shown that a one-week delay between the cortical lesion and transplantation can significantly enhance graft vascularization, cell proliferation, survival and density of projections developed by grafted neurons, leading to a beneficial impact on functional repair and recovery (Peron et al., 2017). However, mechanisms responsible for this improvement are not well defined. It has been hypothesized that potential benefits of introducing a delay between the lesion and transplantation may be due to the release of trophic factors secreted by cells surrounding the lesion (Nieto-Sampedro et al, 1983), the secretion of pro-angiogenic factors (Skold et al, 2005, Dray et al., 2009), or a decrease in toxin (Gonzalez and Sharp, 1987) and inflammation levels, characterized by activated microglia and astrocytes (Zhang et al., 2010). Microglia, the resident innate immune cells in the brain, is an active contributor to neuron damage in neurodegenerative diseases (Block et al, 2007). In acute injury, microglial response generally reaches its maximum at 5-7 days after injury (Ladeby et al., 2005), before gradually disappearing. After activation, microglial cells proliferate and migrate to the site of injury where they contribute to cell damage by releasing pro-inflammatory cytokines such as interleukin 1 and 6 (IL-1, IL-6), tumor necrosis factor, (TNF) and leukaemia inhibitory factor (LIF) (Chao et al. 1995). Additionally, activated microglia may promote neuronal survival by removing cell debris (Rapalino et al. 1998) and releasing protective neurotrophic factors such as Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) or Glial cell-line Derived Neurotrophic Factor (GDNF) (Madinier et al., 2009, Neumann et al., 2006, Schwartz et al., 2006). Furthermore, microglia cells secrete anti-inflammatory cytokines such as Transforming Growth Factor-β1 (TGF-β1, Kiefer et al, 1995), interleukin 4 and 10 (IL-4, IL- 10).

The astrocyte activation may also have antagonistic effects (Farina et al., 2007). For example, the production of neurotrophic factors by astrocytes may promote neuronal survival (Faulkner et al, 2004, Myer et al, 2006, Sofroniew, 2005, Vinters and Sofroniew, 2010). Conversely, glial scar formation impairs adult CNS regeneration (Itoh et al, 2007, Rolls et al., 2009, Wanner et al, 2008).

Interleukin 33 (IL-33), a newly identified cytokine of the IL-1 superfamily, can function as an alarmin that is released following cell necrosis to alert the immune system to tissue damage or stress. Mouse CNS expresses IL-33 in astrocytes and endothelial cells (Hudson et al., 2008; Yasuoka et al., 2011). Treatment with IL-33 induces proliferation of microglia and enhances production of pro -inflammatory cytokines, such as IL-Ιβ and TNFα, as well as the anti-inflammatory cytokine IL-10 (Yasuoka et al., 2011). It was also demonstrated that IL-33 has a beneficial effect in stroke models such as ischemia (Korhonen et al, 2015) and spinal cord injury (Gadani et al, 2015). These results show that the pro- or anti-inflammatory effects of IL-33 depend on the disease and the model.

The impact of the inflammatory response on neuronal survival seems to depend on a balance between pro- and anti-inflammatory mechanisms induced by different mediators (Bernardino et al., 2005, Vezzani et al, 2008). Considerable effort has been devoted to the study of changes occurring in the post-lesioned environment and their effects on axonal regeneration in adult CNS. However, few studies have been dedicated to the consequences of these changes in axonal growth of transplanted embryonic cortical neurons in the injured adult brain. SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of neuropathological disorders characterized by a loss of cortical neurons. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Surprisingly, the inventors showed that the loss of interleukin 33 decreases axonal outgrowth of embryonic cortical neurons grafted into injured adult motor cortex.

Also, they explore the effects of transplanted cells on the host local inflammatory environment in order to understand the extent to which inflammation following cortical lesion could influence the survival of grafted neurons and the development of their projections.

Methods of the present invention

A first aspect of the present invention relates to a method of treating neuropathological disorders characterized by a loss of cortical neurons in a subject in need thereof, comprising administering a therapeutically effective amount of ST2 agonist.

In some embodiments, the ST2 agonist is administered in combination with intracerebral transplantation of cortical neurons.

As used herein, the term "subject" refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with neurologic disorders.

As used herein, the terms « cortical neurons » refer to neurons of the outer covering of gray matter over the hemispheres of the brain. Cortical neurons refer to neurons of the cerebral cortex.

As used herein, the terms "neuropathological disorders characterized by a loss of cortical neurons" refers to any disorders selected from the group consisting of Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, injuries resulting of trauma, neuronal cell death resulting of stroke, epilepsy.

In particular, the neuropathological disorders characterized by a loss of cortical neurons include any cortical lesion, traumatic brain injury caused by an external mechanical force on the cerebral cortex.

In one embodiment, the neuropathological disorder is selected from the group consisting of Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, injuries resulting of trauma, neuronal cell death resulting of stroke, epilepsy. As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term "ST2" has its general meaning in the art and refers to interleukin 1 receptor- like 1, also known as IL1RL1. ST2 is a member of the Interleukin-1 receptor family and exists in a transmembrane (ST2L) and a soluble form (sST2) due to alternative splicing. ST2 is the receptor of interleukin 33.

Intereleukin-33 exerts its effects by binding to the transmembrane receptor ST2L isoform. sST2 avidly binds to IL-33 competing with ST2L. The interaction of this soluble receptor with IL-33 blocks the IL-33/ST2L system and, as a result, eliminates the effects of IL-33. Therefore, sST2 is considered a decoy receptor (Schmitz et al, 2015). Thus, the ST2 system acts not only as a mediator of IL-33 function in its ST2L transmembrane isoform but also as an inhibitor of IL-33 through its soluble sST2 isoform.

Isoform A ST2 receptor (SEQ ID N: 1) :

As used herein, the term "ST2 agonist" refers to any compound natural or not that is able to bind to ST2 and promotes ST2 activity.

ST2 receptor agonists

In some embodiments, the ST2 agonist is a small organic molecule. The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more in particular up to 2000 Da, and most in particular up to about 1000 Da.

In some embodiments, the ST2 agonist is a ST2 antibody or a portion thereof. In some embodiments, the ST2 agonist is an antibody such as chimeric antibodies, humanized antibodies or full human monoclonal antibodies.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of ST2. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes. Briefly, the recombinant ST2 may be provided by expression with recombinant cell lines. In particular, ST2 may be provided in the form of human cells expressing ST2 at their surface. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

As will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non- human sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.

In another embodiment, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®".

In some embodiments, the ST2 agonist is a polypeptide. In a particular embodiment the ST2 agonist is interleukin 33. In a particular embodiment the ST2 agonist is a functional equivalent of interleukin 33.

As used herein, the terms "interleukin 33" or "IL-33" refer to a cytokine that binds to the IL1RL1/ST2 receptor. Interleukin 33 belongs to the IL-1 cytokine superfamily.

Isoform 1 interleukin 33 (SEQ ID N:5)

As used herein, a "functional equivalent of interleukin 33" is a polypeptide which is capable of binding to ST2, thereby promoting a ST2 activity according to the invention. The term "functional equivalent" includes fragments, mutants, and muteins of interleukin 33. The term "functionally equivalent" thus includes any equivalent of interleukin 33 obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to ST2 and promote an ST2 activity according to the invention. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence.

In some embodiments, the ST2 agonist is a polypeptide having at least 80% of identity with SEQ ID NO:5, or SEQ ID NO:6, or SEQ ID NO:7 or SEQ ID NO:8.

In some embodiments, the functional equivalent of a polypeptide is at least 80% homologous to the corresponding protein.

In some embodiments, the functional equivalent of a polypeptide is at least 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o or 99% homologous to the corresponding protein.

In a preferred embodiment, the functional equivalent of a polypeptide is at least 90% homologous as assessed by any conventional analysis algorithm such as for example, the Pileup sequence analysis software (Program Manual for the Wisconsin Package, 1996).

In some embodiments, the present invention provides a polypeptide which comprises consecutive amino acids having a sequence which corresponds to the sequence of at least a portion of interleukin 33, which portion binds to ST2 and promotes the ST2 activity according to the invention.

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptides or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. In particular, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is in particular generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

In some embodiments, the ST2 agonist of the invention is an immunoadhesin.

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin" which is able to bind to ST2) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity to ST2 (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site for ST2. In one embodiment, the adhesin comprises the polypeptides characterized by SEQ ID NO:2. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

The immunoglobulin sequence typically, but not necessarily, is an immunoglobulin constant domain (Fc region). Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to the patient, and are safe for chronic or repeated use.

The polypeptides of the invention, fragments thereof and fusion proteins (e.g. immunoadhesin) according to the invention can exhibit post-trans lational modifications, including, but not limited to glycosylations, (e.g., N-linked or O-linked glycosylations), myristylations, palmitylations, acetylations and phosphorylations (e.g., serine/threonine or tyrosine). In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

In one embodiment, the ST2 agonist is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods.

In a particular embodiment, the ST2 agonist is interleukin 33.

Cortical neurons transplantation

In some embodiments, the ST2 agonist is administered in combination with intracerebral transplantation of cortical neurons.

In some embodiments, the cells overexpressing ST2 are transplanted.

In a particular embodiment, the cortical neurons derived from stem cells.

The terms "stem cell" as used herein, refer to a cell in an undifferentiated or partially differentiated state that has the property of self-renewal and has the developmental potential to differentiate into multiple cell types, without a specific implied meaning regarding developmental potential (i.e., totipotent, pluripotent, multipotent, etc.). A stem cell is capable of proliferation and giving rise to more such stem cells while maintaining its developmental potential. In theory, self-renewal can occur by either of two major mechanisms. Stem cells can divide asymmetrically, which is known as obligatory asymmetrical differentiation, with one daughter cell retaining the developmental potential of the parent stem cell and the other daughter cell expressing some distinct other specific function, phenotype and/or developmental potential from the parent cell. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. In some embodiments, the term stem cell refers generally to a naturally occurring parent cell whose descendants (progeny cells) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. Cells that begin as stem cells might proceed toward a differentiated phenotype, but then can be induced to "reverse" and re-express the stem cell phenotype, a term often referred to as "dedifferentiation" or "reprogramming" or "retrodifferentiation" by persons of ordinary skill in the art.

In a particular embodiment, the cortical neurons derived from induced pluripotent stem cells.

As used herein, the terms "iPSC" and "induced pluripotent stem cell" are used interchangeably and refers to a pluripotent stem cell artificially derived (e.g., induced or by complete reversal) from a non-pluripotent cell, typically an adult somatic cell, for example, by inducing a forced expression of one or more genes.

In a particular embodiment, the cortical neurons derived from embryonic stem cells. The term "embryonic stem cell" as used herein refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see, for e.g., U.S. Pat. Nos. 5,843,780; 6,200,806; 7,029,913; 7,584,479, which are incorporated herein by reference). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein by reference). Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta, i.e., are not totipotent.

Cell transplantation therapies typically involve the intraparenchymal (e.g., intracerebral) grafting of the replacement cell populations into the lesioned region of the nervous system, or at a site adjacent to the site of injury. Most commonly, the therapeutic cells are delivered to a specific site by stereotaxic injection. Conventional techniques for grafting are described, for example, in Bjorklund et al. (Neural Grafting in the Mammalian CNS, eds. Elsevier, pp 169-178, 1985), Leksell et al. (Acta Neurochir., 52:1-7, 1980) and Leksell et al. (J. Neurosurg., 66:626-629, 1987). Identification and localization of the injection target regions will generally be done using a non-invasive brain imaging technique (e.g., MRI) prior to implantation (see, for example, Leksell et al, J. Neurol. Neurosurg. Psychiatry, 48:14-18, 1985).

Briefly, administration of cells into selected regions of a patient's brain may be made by drilling a hole and piercing the dura to permit the needle of a microsyringe to be inserted. The cell preparation of the invention permits grafting of the cells to any predetermined site in the brain or spinal cord. It also is possible to effect multiple grafting concurrently, at several sites, using the same cell suspension, as well as mixtures of cells.

Following in vitro cell culture and isolation as described herein, the cells are prepared for implantation. The cells are suspended in a physiologically compatible carrier, such as cell culture medium (e.g., Eagle's minimal essential media), phosphate buffered saline, or artificial cerebrospinal fluid (aCSF). Cell density is generally about 50.000-200.000 cells/μΐ (Gaspard et al, 2008). The volume of cell suspension to be implanted will vary depending on the site of implantation, treatment goal, and cell density in the solution.

Several injections may be used in each brain area, particularly if the lesioned brain region is large. In contrast, relatively fewer injections are needed if the cells are transplanted into a smaller area.

In some embodiments, the cells are encapsulated within permeable membranes prior to implantation. Encapsulation provides a barrier to the host's immune system and inhibits graft rejection and inflammation. Several methods of cell encapsulation may be employed. In some instances, cells will be individually encapsulated. In other instances, many cells will be encapsulated within the same membrane. Several methods of cell encapsulation are well known in the art, such as described in European Patent Publication No. 301,777, or U.S. Pat. Nos. 4,353,888, 4,744,933, 4,749,620, 4,814,274, 5,084,350, and 5,089,272.

In some embodiments, the ST2 agonist and the intracerebral transplantation of cortical neurons occur simultaneously, at essentially the same time, or sequentially. In a particular embodiment, the ST2 agonist is administrated before the intracerebral transplantation of cortical neurons.

In one embodiment, the intracerebral transplantation of cortical neurons occurs on the lesion site.

In still another embodiment, there is a delay between lesion and the intracerebral transplantation. In a particular embodiment, the delay between lesion and intracerebral transplantation of cortical neurons is equal to 0 (no delay), 1-15 (short time delay) or 15-30 (long time delay) days. In a particular embodiment, the delay between lesion and intracerebral transplantation of cortical neurons is between 0 and 30 days. In a particular embodiment, the delay between lesion and intracerebral transplantation of cortical neurons is equal to 7 days.

Pharmaceutical compositions

In some embodiments, the ST2 agonist or the combination of ST2 agonist with cortical neurons of the invention is administered to the subject with a therapeutically effective amount.

By a "therapeutically effective amount" is meant a sufficient amount of ST2 agonist or a sufficient amount of ST2 agonist and cortical neurons to treat neuropatho logical disorders characterized by a loss of cortical neurons at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the ST2 agonist may be varied over a wide range from 0.01 to 1,000 mg per adult per day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The compositions according to the invention are formulated for parenteral, transdermal, oral, rectal, subcutaneous, sublingual, topical or intranasal administration.

Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

In a particular embodiment, the pharmaceutical compositions are formulated for parenteral administration. The pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

In a particular embodiment, the ST2 agonist or the combination of ST2 agonist and cortical neurons are administrated by intracerebral route.

The ST2 agonist or the combination of ST2 agonist and cortical neurons of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In some embodiments, the ST2 agonist or the combination of ST2 agonist and cortical neurons of the invention may be administered in combination with conventional treatment usually used for treating neuropathological disorders characterized by a loss of cortical neurons.

Cells containing an expression vector expressing IL-33 gene

Another aspect of the invention relates to a transduced or transfected cells containing an expression vector which expresses IL-33 gene.

As used herein, the term "transduction" refers to the delivery of a gene using a retroviral vector particle by means of infection, in particular, introduction of a gene carried by the retroviral vector into a cell via lentivirus or vector infection and pro virus integration.

The term "transfection" as used herein refers to the use of methods, such as chemical methods, to introduce exogenous nucleic acids, such as the synthetic, modified R As described herein, into a cell, preferably a eukaryotic cell. As used herein, the term transfection does not encompass viral-based methods of introducing exogenous nucleic acids into a cell. Methods of transfection include physical treatments (electroporation, nanoparticles, magnetofection), and chemical-based transfection methods. Chemical-based transfection methods include, but are not limited to, cyclodextrin, polymers, liposomes, and nanoparticles. In some embodiments, cationic lipids or mixtures thereof can be used to transfect the synthetic, modified R As described herein, into a cell, such as DOPA, Lipofectamine and UptiFectin. In some embodiments, cationic polymers such as DEAE-dextran or polyethylenimine, can be used to transfect a synthetic, modified RNAs described herein.

As used herein, the term "expression vector", encompass vectors such as plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. Such system typically includes a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of RNA expression, a sequence that encodes a suitable ribosome binding site, RNA splice junctions, sequences that terminate transcription and translation and so forth. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell.

Plasmids are the most commonly used form of vector but other forms of vectors which serves an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y.; and Rodriquez, et al. (eds.) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988), which are incorporated herein by reference.

The term "operably linked" is used herein for indicating that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURE:

Figure 1: Absence of IL-33 alters axonal outgrowth of grafted neurons.

This figure of coronal sections illustrates the projections developed by the grafted neurons in the striatum, 14 days after transplantation with a 7 days delay between motor cortical lesion and transplantation in the motor cortex of wild type (A, C) or IL-33 KO (B, D) mice. Scale bars: (A, B) 120μm (C, D) 90μm.

EXAMPLE:

Material & Methods

Animals

All animal experimental procedures and housing were carried out in accordance with the guidelines of the French Agriculture and Forestry Ministry (decree 87849) and the European Communities Council Directive (2010/63/EU). All experiments were conducted in compliance with current Good Clinical Practice standards and in accordance with relevant guidelines and regulations and the principles set forth under the Declaration of Helsinki (1989). All efforts were made to reduce the number of animals used and their suffering. A total of 84 C57BL/6 and 20 IL-33 KO mice were used in this study, of which 12 mice were used as controls (without a lesion), 36 mice were lesioned and 56 mice were lesioned and transplanted.

Lesion and transplantation procedures

Adult (4-6 months old) C57BL/6 mice (n=72, Janvier, France) and IL-33 KO mice ( Pichery et al., 2012) were lesioned. Briefly, animals were anaesthetized with avertin (intraperitoneal, ip., 250 mg per kg of body weight) and the motor cortex was aspirated from 0.5- 2.5 mm rostral to the Bregma and from 0.5-2.5 mm lateral to the midline, with the corpus callosum left intact. Among these mice, 36 were transplanted as described previously (Ebrahimi-Gaillard et al., 1995; Gaillard et al., 1998). Motor cortical tissue was obtained from embryonic day 14 transgenic mice embryos overexpressing the enhanced green fluorescent protein (EGFP) under the control of a chicken beta-actin promotor (C57BL/6-TgN(beta-act- EGFP) Osb strain (Okabe et al, 1997). Motor cortical tissue was deposited into the host lesion cavity either immediately or 7 days after the lesion. Care was taken to maintain the original dorso-ventral and antero-posterior orientations of the cortical fragments during the transplantation procedure.

Tissue processing

Mice were injected with a lethal dose of avertin and perfused transcardiacally with 150 ml of saline (0.9%), followed by 300 ml of ice-cold paraformaldehyde (PFA, 4%) in 0.1 M phosphate buffer (PB, pH 7.4). Brains were removed, post-fixed in 4% PFA overnight at 4°C, cryoprotected in 30%> (w/v) sucrose, 0.1 M sodium phosphate solution (pH 7.4). For immunohistochemistry experiments, brains were cut in 6 series on a freezing microtome (Microm HM450, Thermo Scientific) in 40μm-thik coronal sections and stored in a cryoprotective solution (20%> glucose, 40%> ethylene glycol, 0.025%> sodium azide, 0.05M phosphate buffer, pH 7.4).

Immunohistochemistry (IHC)

Free-floating sections were incubated in a blocking solution (3% bovine serum, 0.3%> triton X-100 in PBS 0.1M, pH 7.4) for 90 minutes at room temperature (RT). The following antibodies were used in this study to label activated microglia, hematopoietic cells, astrocytes, oligodendrocytes and neurons, respectively: rabbit anti-Ibal (1 :500, Wako), rat anti-CD45 (1 :500, Abeam), chicken anti-GFAP (1 : 1000, Abeam), rabbit anti-olig2 (1 :500, Millipore) and mouse anti-NeuN (1 :500, Millipore). Chicken anti-GFP (1 :1000, Abeam) or Rabbit anti- GFP (1 : 1000, Invitrogen) were used to label transplanted cells. For IL-33 staining, goat anti- IL-33 (1 :500, R&D systems) was used, and nuclei were labelled with DAPI (1 :2000, Sigma). Appropriate secondary antibodie were diluted in blocking solution and applied for lh at RT. The sections were covered with DePeX (VWR) mounting medium.

In situ hybridization (ISH)

ISH was performed in order to characterize the spatiotemporal expression of pro- inflammatory cytokines IL-1α, IL-Ιβ, IL-6, TNFα, and LIF and anti-inflammatory cytokines: TGFβ1, IL-4 and IL-10, several days after cortical lesion. Specific digoxygenin- labeled cRNA probes were prepared from cDNA fragments (450bp to 800bp) of these murine cytokines. cDNAs were amplified by PCR using specific primers from cDNA banks obtained from different sources (Brain, liver, spleen, skin, adipose tissue and LPS-treated Bone marrow-derived macrophages). cDNA fragments were then cloned in pGEM®-T Easy vectors (Promega, Charbonnieres-les-Bains, France) and verified by sequencing. Complementary (antisense) and non-complementary (sense) RNA probes were produced using T7 or SP6 RNA polymerase (Riboprobes® System-T7, Promega Corporation, Madison, USA). Before being exposed to the probes, sections were digested by proteinase K (5μg/ml) for 10 min at 37°C followed by an acetylation step in triethanolamine buffer (100mM triethanolamine, 0.25% acetic anhydride) to reduce non-specific binding. Hybridization was carried out overnight at 65°C in a humidified chamber using probes at a final concentration of 500 ng/ml diluted in hybridization buffer containing 50% formamide, IX Denhardt's solution, 10% dextran sulfate, lmg/ml yeast tRNA in salt solution (200mM NaCl, lOmM Tris-HCl pH 7.5, lOmM phosphate buffer pH 7.4, 5mM EDTA pH 8). The following day, sections were washed in IX sodium saline citrate (SSC), 50%> formamide, 0.1 % Tween 20 at 65 °C, and in MABT buffer (0.15 M NaCl, 0.1 M Maleic acid, 0.2 M NaOH, 0.1% Tween 20, pH 7.5) at RT. After blocking in 10% B10 reagent (Roche Diagnostics, Mannheim, Germany) and 10% sheep serum, sections were incubated overnight at RT with an alkaline phosphatase-labeled anti-digoxigenin antibody (Roche Diagnostics, Mannheim, Germany) diluted 1 :2000 in blocking buffer. Sections were finally washed with NTMT buffer (0.1 M NaCl, 0.1 M Tris- HCl pH 9.5, 0.05 M MgC12, 0.1 M Tween 20, pH 9.5) before being incubated in detection buffer containing 0.045% nitroblue tetrazolium, 0.35% 5-bromo-4-chloro-3-idolyl phosphate (Roche Diagnostics, Mannheim, Germany) and 0.1% levamisole (Sigma) in NTMT buffer. Slides were then dried and mounted with Depex (BDH Laboratories, Poole, England). All results with antisense probes were compared with sense probes and were confirmed by testing six animals for each group.

Anti-Ibal antibody was used to define whether macrophage/microglia could be the source of IL-Ιβ and TGF-βΙ . Briefly, after ISH, sections were incubated in blocking solution (3% bovine serum, 0.3% triton X-100 in PBS 0.1M, pH 7.4) at RT for 90 minutes. Anti-Ibal primary antibody (1 :500, Wako) diluted in blocking solution was applied overnight at 4°C. Sections were then incubated with biotinylated goat anti-rabbit antibody (1 :200; Vector Burlingame, CA) at RT for lh30 minutes. After washing, sections were treated with 0.3% hydrogen peroxide (Sigma, Seelze, Germany) to quench endogenous peroxidases and were reacted with avidin-biotin peroxidase complex (Vectastain® ABC Kit, Vector, Burlingame, CA) at RT for lh. The sections were subsequently incubated in 0.1 M PB containing 0.33 mg/ml 3-3-diaminobenzidine tetrahydrochloride (Sigma, St Louis, USA) and 0.0006%) hydrogen peroxide. Mounted sections were dried and covered with Depex (BDH, Poole, England).

Data acquisition and quantification

For each mouse, mosaic images of injury area were acquired with a Zeiss Axio Imager.M2 Apotome microscope at x20 magnification, at the rostral middle and caudal part of the lesion or the graft. On mosaic acquisition, six images corresponding to the areas of interest were used for all quantifications using ZEN software (Zeiss). For control mice, equivalent sections were selected at the same antero-posterior coordinates as the lesioned sections. Areas of interest were further analyzed and photographed with a confocal laser- scanning microscope FV1000 (Olympus, France).

Statistical analysis

Statistical analyses were performed using a two-tailed student's t test or two-way analysis of variance (ANOVA) followed by a Bonferroni correction. Data are expressed as mean SEM. Differences were considered statistically significant when p<0.05, p<0.001, p<0.0001 (*, **, respectively).

Results

Cortical lesion increases brain resident immune and peripheral infiltrating cells

To examine the effects of cortical lesion on the number of resident immune cells and peripheral infiltrating cells in the injured cortex, IHC was used to identify GFAP+ astrocytes, Iba-1+ microglial cells/macrophages, 01ig2+ oligodendrocytes and CD45+ hematopoietic cells. Mice were divided into 3 groups: control group (without lesion), Day 0 lesion group and Day 7 lesion group, which also correspond to the time of grafting after lesion. As expected, the basal number of GFAP+ cells in the cortex of control group was low (13±2) and immunoreactivity was also low for Ibal (279±29); 01ig2+ (240±17) and CD45 (17± 2). At the day of lesion (day 0), the number of cells slightly increased around the cortical lesioned area (GFAP: 31±6; Ibal : 415±34; 01ig2: 277±28; CD45: 32± 3). At day 7, the number of astrocytes (721±43), microglia (1907±82), oligodendrocytes (925±36) and hematopoietic CD45+ cells (683±51) was significantly increased in comparison to control and day 0 groups (***, p<0.0001). In addition, microglia showed morphological changes, became activated and presented an amoeboid morphology. Thus, a delay of one week after cortical lesion results in the recruitment and activation of inflammatory brain resident mediators and peripheral infiltrating cells.

Cortical transplantation modified brain resident immune and peripheral infiltrating cells

The effects of cortical transplantation, with or without delay after the lesion, was then examined on the number of resident and peripheral infiltrating immune cells, 7 days after transplantation in the injured cortex as well as in the graft. To achieve this, the same previous markers were used in two groups of transplanted animals.

The number of microglial and hematopoietic cells were not significantly different between the two groups of transplanted animals whether in the host cortex adjacent to the transplant (Ibal+ cells, No delay: 1329±57; delay: 1349±47) (CD45+ cells, No delay: 666± 38; delay: 596± 30) or within the transplant (Ibal+ cells, No delay: 196±15; delay: 191±11), (CD45+ cells, No delay: 191± 15; delay: 149±11). Furthermore, a significant increase in the number of astrocytes was observed in the host cortex adjacent to the transplant in the group of animals transplanted with a one-week delay, compared to the group without delay (GFAP cells, No delay: 632±75; delay: 1047±61). A similar significant increase in astrocytes was also detected in the transplant (No delay: 68±16; delay: 610±109). Moreover, while numerous oligodendrocytes were detected in the host cortex adjacent to the transplant, no significant difference was found between the two groups (01ig2, No delay: 740±50; delay: 729±58). However, a significant increase was observed in the transplant for the group of mice transplanted with a one-week delay, in comparison to that without delay (01ig2, No delay: 209±20; delay: 1163±84). Our results showed that a one-week delay between lesion and transplantation enhanced the number of astrocytes in the host adjacent cortex as well as in the graft, whereas oligodendrocytes increased only within the graft.

Expression of IL-33 following cortical lesion

IL-33 was found to be expressed in control adult cortex without any lesion (126±14) and cortical lesion induced a significant increase in the number of IL-33+ cells immediately after lesion (Day 0 group) (381±50; p<0.0001) confirming the alarmin role of IL-33 in the CNS. Moreover, the expression of IL-33 was further significantly increased after 7 days of lesion (543±36), in comparison to lesion at day 0 (*, p<0.05) and control groups (***, p<0.0001). The number of IL-33+ cells returned to basal levels after 45 days of cortical lesion (130±6).

In order to investigate the cellular sources of IL-33 in the cortex, its immunolabelling was combined with markers for astrocytes (GFAP), microglia/macrophages (Iba-1), oligodendrocytes (01ig2), neurons (NeuN) and hematopoietic cells (CD45). Results showed that at day 7 post-lesion, only few IL33+ cells co-expressed GFAP (0.88±0.06%), Ibal (1.76±0.2) and NeuN (0.40±0.08%). However, about 18% of 01ig2+ (18.13±2.42%) and 4% CD45+ cells (4±0.43%) co-expressed IL-33.

IL-33 expression increases in the transplant, but not in the host, in the group with delay following cortical transplantation

The effects of transplantation on the number IL33+ cells in the host cortex as well as within the graft was then investigated in two groups of transplanted animals, seven days post- grafting. In both transplanted groups, the mean number of IL-33+ cells decreased in the host cortex adjacent to the transplant and was comparable to the level in the control group (126±14). There was no significant difference in the expression of IL-33+ cells in the host cortex between the two groups of transplanted animals (No delay: 249±14; delay: 200±16). IL-33+ cells were found in the transplant in both groups and their number increased significantly following a delay between lesion and transplantation (No delay: 65±6; delay: 101±3).

Moreover, the majority of IL-33+ cells within the graft did not express GFP, indicating probably hat they are of host origin. To investigate graft-versus-host origin of IL- 33 cells observed within the graft, E14 GFP motor cortical tissues were transplanted into the lesioned motor cortex of IL-33 KO mice, with or without a 7 days delay. Results showed the presence of IL-33+ cells within the grafts, with no significant difference between the two transplanted groups (No delay: 14±3; delay: 18±2). Furthermore, the number of IL-33+ cells within the transplant of wild type were compared to those of IL-33 KO mice. The number of IL-33+ cells was significantly lower in IL-33 KO compared to wild type mice, suggesting that a part of IL-33+ cells observed within the grafts originated from host IL-33+ cell migration into the graft.

In order to reveal the cellular sources of IL-33 after cortical transplantation, the phenotype of IL-33+ cells was determined. There was no difference in the percentage of IL- 33+ cells co-expressing the markers of astrocyte (1.2± 0.37%), microglia (2.66±0.49%), neurons (0.33±0.06%) or hematopoietic cells (4.5±0.15%) between the lesioned and transplanted groups, showing again that none of these cells was the main source of the IL-33 detected following transplantation. Strikingly, the percentage of oligodendrocytes co- expressing IL-33 increased in both groups of transplanted animals (No delay: 64±3 %; delay: 72±3%).

Absence of IL-33 alters axonal outgrowth of grafted neurons

Given the increased expression of IL-33 after cortical injury, the effect of IL-33 removal on axonal outgrowth of transplanted cortical neurons was analysed. In order to address the role of IL-33 in axonal outgrowth of transplanted neurons, we grafted embryonic cortical tissue obtained from E14 GFP mice into lesioned cortex of IL-33 KO mice, immediately or 7 days following the lesion (Figure 1). Analysis of the projections of grafted neurons 14 and 30 days after transplantation showed a substantial decrease in the number of projections in the group with delay, in comparison to no delay group. Moreover, we have not observed any projections beyond the striatum indicating that these projections did not extend far from the grafted area and did not reach distant targets.

Differential expression kinetics of IL-Ιβ and TGF-βΙ following cortical lesion To evaluate the level of neuroinflammation following cortical lesion, we investigated the spatiotemporal mRNA expression profile of pro- and anti-inflammatory cytokines. IL-Ιβ and TGF-βΙ exhibited a differential expression at the lesion site, whereas they were neither present in the contralateral side nor outside the lesion site.

Expression of IL-Ιβ and TGF-βΙ, using ISH, was not detected in the control cortex without lesion or in the cortex at the day of lesion. However, a strong expression of IL-Ιβ was observed 4 days after lesion, within the cortex around the lesion cavity. IL-Ιβ expression was undetectable 7 days after the lesion. In addition, TGF-βΙ mRNAs were found to be concentrated in the vicinity of the cortical lesion and to a lesser extent in the corpus callosum adjacent to the lesion site, 4 days after injury. Seven days after the lesion, TGF-βΙ expression decreased, in comparison to day 4, but was still present in the cortex adjacent to the lesion cavity. Furthermore, microglial/macrophagic phenotype of the cells expressing IL-Ιβ and TGF-βΙ was analyzed 4 days after the lesion, using anti-Ibal IHC in combination with ISH. We found that only TGF-β 1+ cells expressed Ibal marker. IL-la, IL-4, IL-6, IL-10, TNFα and LIF transcripts were not detected at any time point tested.

Discussion

We have recently shown that a 7 days delay between the lesion of motor cortex in the adult mouse and homotopic cortical transplantation of embryonic cells can significantly enhance graft vascularization of grafted cells, proliferation and survival as well as density projections developed by grafted neurons. Moreover, we have also shown that this delay has beneficial impacts on functional repair and recovery (Peron et al, 2017). The mechanism of action that leads to these positive outcomes has not been yet not defined. We hypothesized that potential benefits of introducing a delay between the lesion and the transplantation may be due, at least in part, to the modulation of neuroinflammation. Thus, the present study was designed to determine how a delay between lesion and transplantation could modulate posttraumatic inflammation and whether these modulations have beneficial or deleterious effects.

It has been postulated that potential benefits of introducing a delay between the lesion and the transplantation may result from the release of trophic factors secreted by cells surrounding the lesion (Nieto-Sampedro et al, 1983), the secretion of pro-angiogenic factors (Skold et al, 2005, Dray et al, 2009), the decrease of toxin levels (Gonzalez and Sharp, 1987) or the modulation of inflammation level.

Here we show that the number of astrocytes, microglia, oligodendrocytes and CD45+ cells was significantly increased seven days after the lesion, in comparison to the control (without lesion) and day of lesion (DO) groups. In fact, microglial response to cortical injury reaches its maximum level at 7 days after cortical lesion and then gradually disappears, in agreement with a previous study by Ladeby et al. (2005). While activated microglial cells express pro- and anti-inflammatory cytokines, we have found that the expression of proinflammatory cytokine IL-Ιβ starts 1 day after the lesion, increased at day 4 and became undetectable 7 days after the lesion. In addition, we have found a robust expression of antiinflammatory cytokine TGF-β1, 4 days after lesion, which decreased significantly seven days after the lesion, but was still present at detectable levels. Interestingly, 19% of TGF-β1+ cells co-expressed Ibal, which supports the hypothesis that microglial activation is also neuroprotective (Lai et al, 2006).

We have recently reported that a delay of one week between cortical lesion and transplantation leads to a transient but significant increase in graft vascularization. Increasing evidences have demonstrated that TGF-βΙ is pro-angiogenic in vivo and induces angiogenesis (Madri et al, 1988; Roberts et al, 1986; Yang & Moses 1990; Evrard et al, 2012). Our results suggest that an increase in graft vascularization observed in delay group could be in part the consequence of an increase of the expression of TGF-βΙ promoting graft vascularisation.

Surprisingly, this study demonstrated, for the first time, that IL-33 is implicated in the development of axons of grafted neurons as its absence was shown to alter the axonal outgrowth of transplanted neurons.

There are several possible explanations for this. First, IL-33 may have a role in the expression of pro-inflammatory cytokines. Indeed, it has been reported that IL-33 treatment, after spinal cord contusion, reduced the expression of pro-inflammatory TNF-α and promoted the activation of anti-inflammatory arginase-1 positive M2 microglia/macrophages (Pomeshchik et al., 2015). We observed that host IL-33 deletion impaired axonal outgrowth of transplanted neurons, which suggests that absence of IL-33 increases the levels of proinflammatory cytokines, thus creating an environment hostile for axonal outgrowth of grafted neurons. Second, previous studies have suggested that IL-33 signalling induces angiogenesis (Choi, et al, 2009; Maywald et al, 2014). We hypothesize that deletion of IL-33 may decrease the vascularization of the graft therefore decreasing the survival of the grafted neurons. Finally, two recent reports showed that IL-33 treatment is protective in mouse brain after cerebral ischemia, through induction of IL-4 secretion (Korhonen et al, 2015), or in mice subjected to middle cerebral artery occlusion (Lu et al, 2015). A major problem in cell transplantation is low cell survival rate following implantation, (Emgard et al., 2003; Hicks et al., 2009). As IL-33 may have a protective effects against cell loss of grafted cells, its absence may decrease grafted cells survival.

Collectively, these results suggest a beneficial effect of IL-33 in axonal outgrowth of grafted neurons. Cell transplantation in combination with IL-33 treatment may offer a novel therapeutic strategy for cortical injury.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating neuropathological disorders characterized by a loss of cortical neurons in a subject in need thereof, comprising administering a therapeutically effective amount of ST2 agonist.

2. The method of claim 1 wherein the ST2 agonist is a small organic molecule, an antibody, a polypeptide or an aptamer.

3. The method of claim 1 wherein the ST2 agonist is an antibody such as chimeric antibodies, humanized antibodies or full human monoclonal antibodies.

4. The method according to claim 1 wherein the ST2 agonist is interleukin 33 or a functional equivalent of interleukin 33.

5. The method of claim 1 wherein the ST2 agonist is a polypeptide having at least 80% of identity with SEQ ID NO:5, or SEQ ID NO:6, or SEQ ID NO:7 or SEQ ID NO:8.

6. The method of claim 1 wherein the ST2 agonist is an immunoadhesin.

7. The method according to one of the preceding claims, wherein the subject is also administered with a conventional treatment of neuropathological disorders characterized by a loss of cortical neurons.

8. The method according to one of the preceding claims, wherein the ST2 agonist is administered in combination with intracerebral transplantation of cortical neurons.

9. The method according to claim 8, wherein the cortical neurons derived from stem cells, induced pluripotent stem cells, or embryonic stem cells.

10. The method according to one of the preceding claims, wherein the ST2 agonist is administrated before the intracerebral transplantation of cortical neurons.

11. The method according to one of the preceding claims, wherein the intracerebral transplantation of cortical neurons occurs on the lesion site.

12. The method according to one of the preceding claims, wherein there is a delay between lesion and the intracerebral transplantation, said delay is comprised between 0 and 30 days.

13. The method according to claim 12, wherein the delay between lesion and intracerebral transplantation of cortical neurons is equal to 7 days.

14. The method according to one of the preceding claims, wherein the neuropatho logical disorder is selected from the group consisting of Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, injuries resulting of trauma, neuronal cell death resulting of stroke, epilepsy.