WO2017001568A1 - Treatment and prevention of amyotrophic lateral sclerosis - Google Patents

Abstract

The invention relates to the use of interleukin 4 for preventing and treating amyotrophic lateral sclerosis. In particular, the invention relates to the use of an interleukin 4 polypeptide, a nucleic acid encoding said polypeptide or a cell expressing said polypeptide, for preventing and treating amyotrophic lateral sclerosis.

Description

TREATMENT AND PREVENTION OF AMYOTROPHIC LATERAL

SCLEROSIS

FIELD OF THE INVENTION

The invention relates to the field of therapeutics and prevention, more specifically to the treatment and prevention of amyotrophic lateral sclerosis.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disorder, first described in 1869 by Jean Martin Charcot. ALS is characterized by progressive muscular paralysis which reflects the degeneration of motor neurons in the primary motor cortex, brainstem and spinal cord; it has an enormous impact on the quality of life of patients and their careers. ALS is familial in approximately 10% of patients. Although it is usually inherited in an autosomal dominant way, recessive and even X- linked forms also exist. Mutations of the superoxidedismutase 1 (SOD1) gene (chromosome 21) account for about 20 % of familial ALS. These are found in about 20% of families and thus account for 2% of all cases of ALS. SOD1 is an enzyme with 153 amino acid residues which is ubiquitously expressed and acts as a homodimer. It catalyzes the conversion of superoxide free radicals to hydrogen peroxide, which can then be further detoxified to water and oxygen by glutathione peroxidase or catalase. More than 150 different mutations have been reported to be pathogenic.

Currently, there is no effective treatment to prevent or to slow the progression of the disease. Recently, the drug riluzole (Rilutek®) from Sanofi-Aventis has been aproved as a treatment for ALS. However, the efficiency of this treatment is low.

In humans, the earliest symptoms of ALS may include fasciculations, cramps, tight and stiff muscles (spasticity), muscle weakness affecting an arm or a leg, slurred and nasal speech, or difficulty chewing or swallowing. These general complaints then develop into more obvious weakness or atrophy that may cause a physician to suspect ALS. To be diagnosed with ALS, people must have signs and symptoms of both upper and lower motor neuron damage that cannot be attributed to other causes. Mutations in the human SOD1 gene, once transferred to transgenic animals (rats or mice), can reproduce the disease in these animals which are currently the most used model in preclinical ALS studies.

The promotion of the microglial M2 phenotype by chemical agents is another presumably valuable approach in ALS therapy. In addition to their cholesterol-reducing properties, it has been revealed that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e. statins) have an anti-inflammatory activity capable of ameliorating the pathology of Alzheimer's disease and those of other neurological disorders in experimental models. It has been shown that the ant i-neuro inflammatory action of atorvastatin, which is a drug belonging to the statin family, is related to its ability to trigger production of IL-4 without altering the concentration of cholesterol in the brain. Atorvastatin also protected cultured neurons from glutamate-excitotoxicity via a cholesterol- independent mechanism and showed neuroprotective effects for enervated muscles and degenerating motor neurons in wobbler mice. However, clinical trials have shown atorvastatin treatments to be ineffective (Zheng et al, Amyotroph Lateral Scler Frontotemporal Degener, 2013, 14: 241-245) or even to have detrimental effects (Zinman et al, Amyotroph Lateral Scler 2008, 9: 223-228) for ALS patients. Another anti- inflammatory compound that skews microglial activation toward an M2 is NP001. Despite having showed promising results in preliminary ALS clinical trials (Neuraltus Pharmaceuticals, Dec. 9 2013 release; Miller et al, 2011), this drug failed to meet endpoints in a phase II clinical trial (http://www.als.net/ALS-Research/NP001/ALS- Topics/).

Therefore, there is a need of finding a more effective treatment to prevent the disease and to stop or at least slow down the progression of the disease once diagnosed.

BRIEF DESCRIPTION OF THE INVENTION

The inventors of the present invention have discovered that, surprisingly, administration of mouse recombinant IL4 (rIL4) to mSODl^G93A mice, which is the most widely used experimental model for ALS, is able to prolong survival (Fig. 1), delay the manifestation of the most apparent symptoms of the disease (Fig. 2) and improve the clinical course of the disease attenuating body weight loss (Fig.3) and improving the motor behavior (Fig. 7). Thus, the invention relates to an interleukin 4 polypeptide, or a nucleic acid encoding said interleukin 4 polypeptide, or a cell expressing said polypeptide, for use in the prevention and/or treatment of amyotrophic lateral sclerosis. BRIEF DESCRIPTION OF THE FIGURES

Fig.l (A) Kaplan-Meier plot of survival (rIL4 treated vs. control animals) showing a significant increase in survival after chronic administration of rIL4. (n=8) (B) Gender- specific therapeutic effects of rIL4 treatment in S0D1^G93A mice. Survival graphs comparing untreated animals vs. a sex-segregated rIL4-treated group. Although chronic administration of rIL4 is effective in both cases, the response in the female group is significantly better. (C) Data for individual animals are plotted as black spots with mean ± SEM in lines and bars (C) * * * = p < 0.0001 vs. control (ANOVA test).

Fig.2 - A, B Comparison between control animals and rIL4-treated group (the discriminating point for the disease being obvious is arbitrarily established at the time when the clinical score reaches 3. This time -point is considered the Disease Onset for the sake of the experimental comparison between groups). C, D Disease onset graphs plotted after the segregation of groups by sex. *** = p < 0.0001 vs. control (B) and ** = p < 0.01 vs. control (D) (ANOVA test).

Fig.3- Attenuation of body weight loss after chronic administration of rIL4 in mSODl^G93A mice. (A) The percentage of weight loss is plotted for the entire group of animals and shows a significant decrease in weight loss as a result of the rIL4 treatment. (B) Gender-specific differences in the percentage of weight loss after rIL4 treatment. * = p < 0.05 vs. control (A, B) (ANOVA test).

Fig.4- (A) Kaplan-Meier plot of survival after AT treatment (treated vs. control animals) showing no significant differences between groups (n=5). (B) Disease onset graph comparing untreated animals vs the AT -treated group (Disease onset is considered when the clinical score reaches 3). (C) Treatment with AT does not significantly affect body weight loss in mSODl^G93A mice reaching the end stage of the disease.

Fig.5- Effects of rIL4 treatment on neuroinf ammatory markers in spinal cord ventral horn from mSODl^G93A end terminal mice. Spinal cord sections were immunostained for GFAP (A-D), Iba-1 (F-I), Mac2 (K-N), AJ10 (P-S) and IgG (U-X). Samples were taken either from control (A, C, F, H, K, M, P, R, U, W) or rIL4-treated animals (B, D, G, I, L, N, Q, S, V, X). Immunostaining was quantified for each condition as indicated in the graphs for each panel (E, J, O, T, Y). Note the significant reduction in microglial activation detected by Iba-1 and Mac2 immunostaining in rIL4-treated animals accompanied by a decrease in the accumulation of neurotoxic misfolded SOD1 detected by AJ10 immunostaining. The deposition of IgG in ventral horn was also significantly reduced in rIL4-treated animals. Dashed lines delimitate the margins of the ventral horn. Bar in B = 100 urn (valid for A, B, F, G, K, L, P, Q, U and V). Bar in D = 150 urn (valid for C, D, H, I, M, N, R, S, W and X). J, O * = p<0.05; T ** = p<0.01 and W **** = pO.0001.

Fig.6- Effects of the AT treatment on the neuroinflammatory response in spinal cord ventral horn from mSODl^G93A end terminal mice. Spinal cord sections were immunostained for GFAP (A-D), Iba-1 (F-I), Mac2 (K-N), AJ10 (P-S) and IgG (U-X). Samples were taken either from control (A, C, F, H, K, M, P, R, U, W) or AT -treated animals (B, D, G, I, L, N, Q, S, V, X). Immunostaining was quantified for each condition as indicated in the graphs for each panel (E, J, O, T, Y). Note the absence of significant changes in all of the markers studied except the case of Mac2. Dashed lines delimitate the margins of the ventral horn. Bar in B = 100 um (valid for A, B, F, G, K, L, P, Q, U and V). Bar in D = 150 um (valid for C, D, H, I, M, N, R, S, W and X). O * = p<0.05 (ANOVAtest AT vs. control).

Fig.7- Data from CatWalk XT test recordings showing changes in the gait pattern of mSODl^G93A mice after treatment with rIL4. The interpodal distance was measured at P90 (A) and P120 (B) (untreated vs. rIL4 group). As the control group was unable to complete the test beyond P90, the PI 20 graph uses the P90 control group as a control vs. the P120 rIL4 group. C) Representative recording of abnormal gait pattern seen at P90 in untreated animal. D) The considerable alterations seen in C) are prevented by treatment with rIL4.

Fig.8- Data from CatWalk XT test recordings showing changes in the gait pattern of mSODl^G93A mice after treatment with AT. The interpodal distance was measured at P120 (untreated vs. AT group). No significant differences were observed between groups. Fig. 9- Sex-segregated survival plot (rIL4 treated vs control animals). rIL4 was administrated subcutaneously from day 50 (50 ng, 3 injections per week). Data for individual animals are plotted as black spots with mean ± SEM in lines and bars.

Fig. 10- Non sex-segregated survival plot (rIL4 treated vs control animals). rIL4 was administrated subcutaneously from day 50 (50 ng, 3 injections per week). Data for individual animals are plotted as black spots with mean ± SEM in lines and bars. *=p < 0.05 vs. control (ANOVA test).

Fig. 11- Survival plot (rIL4 treated vs control animals) in females group. rIL4 was administrated subcutaneously from day 50 (500 ng, 3 injections per week). Data for individual animals are plotted as black spots with mean ± SEM in lines and bars. ***= p < 0.0001 vs. control (ANOVA test). Compared to control group (non-treated) this high dose significantly increases survival of females (+17 days vs +6 days for group of 50ng).

Fig. 12- Comparison between control animals and rIL4-treated group (50 ng subcutaneous from day 50). Data for individual animals are plotted as black spots with mean ± SEM in lines and bars. The discriminating point for the disease being obvious is arbitrarily established at the time when the clinical score reaches 3 (paralysis of a single limb). Palsy graphs plotted after the segregation of groups by sex.

Fig. 13- Comparison between control animals and rIL4-treated group (50 ng subcutaneous from day 50). Data for individual animals are plotted as black spots with mean ± SEM in lines and bars. The discriminating point for the disease being obvious is arbitrarily established at the time when the clinical score reaches 3 (paralysis of a single limb). Palsy graphs without sex segregation. *** = p < 0.0001 vs. control (ANOVA test). The treatment with rIL4 delays the apparition of illness' symptoms.

Fig. 14- Data from CatWalk XT test recordings showing changes in the gait pattern of S0D1^G93A mice after subcutaneous treatment with rIL4 in females. Mean values ± SEM in bars. This is an analysis of left and right limbs. The interpodal distance was monitored from the start of treatment until day 125 (untreated vs. rIL4 group).

Fig. 15- Data from CatWalk XT test recordings showing changes in the gait pattern of S0D1^G93A mice after subcutaneous treatment with rIL4 in males. Mean values ± SEM in bars. This is an analysis of left and right limbs. The interpodal distance was monitored from the start of treatment until day 125 (untreated vs. rIL4 group). Observe that no statistical analysis was performed at day 125 due to the small number of remaining animals. The alterations seen in the non-treated group are considerably prevented after subcutaneous administration of rIL4.

Fig. 16- Attenuation of body weight loss after subcutaneous chronic administration of rIL4 in mSODl^G93A mice (grouped by gender). The percentage of weight loss is plotted for the entire group of animals and shows a decrease in weight loss as a result of the rIL4 treatment.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an interleukin 4 polypeptide, or a nucleic acid encoding said interleukin 4 polypeptide, or a cell expressing said polypeptide, for use in the prevention and/or treatment of amyotrophic lateral sclerosis.

Alternatively, the invention relates to the use of an interleukin 4 polypeptide, or a nucleic acid encoding said interleukin 4 polypeptide, or a cell expressing said polypeptide, for the manufacture of a medicament for treating and/or preventing amyotrophic lateral sclerosis.

Alternatively, the invention relates to a method of prevention and/or treatment of amyotrophic lateral sclerosis in a subject, comprising administering to said subject a therapeutically effective amount of an interleukin 4 polypeptide, or a nucleic acid encoding said interleukin 4 polypeptide, or a cell expressing said polypeptide.

The term "interleukin 4 polypeptide", or "IL-4 polypeptide", as used herein, refers to a polypeptide originally described as a factor produced by EL-4 thymoma cells following activation by TPA, which induced proliferation of murine B-cells -activated by submitogenic doses of anti-Ig (Howard et al, Journal of Experimental Medicine 1982, 155(3): 914-923). IL-4 has also been termed B-cell Growth Factor (BOGF), B- cell Stimulatory Factor (BSF-1), T-cell Growth Factor II (TCGF-II) and Mast cell Growth Factor-II (MCFG-II). IL-4 is synthesized in the cell as a precursor protein including a signal peptide which is subsequently cleaved generating the mature protein which is secreted. The term "IL-4 polypeptide", as used herein, includes both the precursor protein, i.e., the protein wherein the signal peptide is present, and the mature form of the protein which does not include the signal peptide. In a preferred embodiment, the IL-4 polypeptide does not include a signal peptide. The term "signal peptide" as used herein, refers to a peptide which serves to direct a protein containing such a peptide from the endoplasmic reticulum of a cell to the Golgi apparatus and ultimately to a lipid bilayer (e.g. for secretion) and is usually removed during this process, therefore signal peptides are not present in mature proteins. In a more preferred embodiment, the IL-4 polypeptide does not include the signal peptide of SEQ ID NO: 1 or the signal peptide of SEQ ID NO: 2.

The term "IL-4 polypeptide" includes IL-4 polypeptides from any origin, for example human, bovine, murine, equine, canine, etc., as well as functionally equivalent variants of said polypeptides. In a particular embodiment, the IL-4 polypeptide is a human polypeptide. In a more particular embodiment, the IL-4 polypeptide is selected from:

(a) Any of the two iso forms of the human protein with the UniProt accession number P05112 (release of 11 March 2015), which correspond to the precursor protein.

(b) Any of the mature forms, i.e. lacking the signal peptide, of the isoforms of the human protein with the UniProt accession number P05112 (release of 1 1 March 2015).

(c) A functionally equivalent variant of any of the polypeptides mentioned in (a) and (b).

In a particular embodiment, when the IL-4 polypeptide is a human polypeptide, or a functionally equivalent variant thereof, said IL-4 polypeptide is to be used in the treatment and/or prevention of a human subject.

In another particular embodiment, the IL-4 polypeptide is a murine IL-4 polypeptide or a functionally equivalent variant thereof. In a more particular embodiment, the IL-4 polypeptide is selected from:

(a) The mouse protein with the UniProt accession number P07750 (release of 31 March 2015), which corresponds to the precursor protein.

(b) The mature form, i.e., lacking the signal peptide, of the protein with the UniProt accession number P07750 (release of 31 March 2015).

(c) A functionally equivalent variant of any of the polypeptides mentioned in (a) and (b). The IL-4 polypeptide can be obtained from nature, i.e., isolated from a subject producing said polypeptide, or it can be produced by synthetic or recombinant techniques. In a particular embodiment, the IL-4 polypeptide is a recombinant polypeptide, the term "recombinant polypeptide", as used herein, means that the IL-4 polypeptide is obtained by the expression of a nucleic acid encoding the amino acid sequence of said IL-4 polypeptide in a heterologous organism, like for example bacteria, yeast, insect cells or mammal cells.

In a more particular embodiment, the recombinant polypeptide is a human recombinant polypeptide. In an even more particular embodiment, the human recombinant polypeptide comprises the sequence SEQ ID NO: 3.

In another particular embodiment, the recombinant polypeptide is a murine recombinant polypeptide. In an even more particular embodiment, the murine recombinant polypeptide comprises the sequence SEQ ID NO: 4.

The term "functionally equivalent variant", as used herein, is understood as any polypeptide the sequence of which can be obtained by means of an insertion, substitution or elimination of one or more amino acids of the sequence of an IL-4 polypeptide of reference, for example a human or murine IL-4 polypeptide, and which at least partly conserves the capacity to perform one of the biological activities of IL-4. The biological activities of IL-4 can be measured by any technique known by the skilled person, for example one of the following assays:

- Promote proliferation and differentiation of activated B-cells (Callard et al, Assay for human B cell growth and differentiation factors. Clemens MJ et al (editors). Lymphokines and Interferons. A practical Approach, pp. 345-64, IRL Press, Oxford (1987)).

- Induction of CD23 in a number of B-cell lines with CD23 detected either by flow-through cytometry or by a fluorescence immunoassay (Defrance et al, J.

Exp. Med. 1987, 165:1459-1467).

In another particular embodiment, the functionally equivalent variant is a functionally equivalent variant of a human IL-4 polypeptide. In a more particular embodiment, the functionally equivalent variant is a functionally equivalent variant of any of the two iso forms of the human protein with the UniProt accession number P05112 (release of 11 March 2015) or any of the mature forms, i.e. lacking the signal peptide, of the isoforms of the human protein with the UniProt accession number P05112. In another particular embodiment, the functionally equivalent variant is a functionally equivalent variant of the human IL-4 polypeptide of sequence SEQ ID NO: 3.

In a particular embodiment, the functionally equivalent variant is a variant of the murine IL-4 polypeptide. In a more particular embodiment, the functionally equivalent variant is a variant of the mouse protein with the UniProt accession number P07750 (release of 31 March 2015), which corresponds to the precursor protein or the mature form, i.e., lacking the signal peptide, of the protein with the UniProt accession number P07750 (release of 31 March 2015). In another particular embodiment, the functionally equivalent variant is a variant of the murine IL-4 polypeptide of sequence SEQ ID NO: 4.

Functionally equivalent variants of a IL-4 polypeptide according to the invention include sequences having at least approximately 50%, at least 60%, at least 70%>, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequences of the IL-4 polypeptide of reference, for example a human IL-4 polypeptide, or a murine IL-4 polypeptide. It is also contemplated that functionally equivalent variants of a of a IL-4 polypeptide comprise additions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the amino acid sequence of the IL-4 polypeptide of reference, for example a human IL-4 polypeptide, or a murine IL-4 polypeptide. Likewise, it is also contemplated that variants comprise deletions consisting of at least 1 amino acid, or at least 2 amino acids, or at least 3 amino acids, or at least 4 amino acids, or at least 5 amino acids, or at least 6 amino acids, or at least 7 amino acids, or at least 8 amino acids, or at least 9 amino acids, or at least 10 amino acids or more amino acids at the N-terminus, or at the C-terminus, or both at the N- and C-terminus of the IL-4 polypeptide of reference, for example a human IL-4 polypeptide, or a murine IL-4 polypeptide. The degree of identity between the variants and the IL-4 polypeptide of reference is determined by using algorithms and computer methods which are widely known by the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J Mol Biol, 215: 403- 410 (1990)].

Functionally equivalent variants of a IL-4 polypeptide according to the invention will preferably maintain at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200% or more of the biological activity of the IL-4 polypeptide of reference, for example a human IL-4 polypeptide, or a murine IL-4 polypeptide. Assays for determining the biological activity of a IL-4 polypeptide have been previously mentioned. Such assays can be used by the skilled person to determine the biological activity of a variant of an IL-4 polypeptide, compare its activity with the activity of the IL-4 polypeptide from which it derives and determine if such variant substantially maintains the biological activity of the IL-4 polypeptide and, therefore, is a functionally equivalent variant of an IL-4 polypeptide. Since IL-4 activity is species-specific, the skilled person will be aware that the assays to determine the activity of a variant of an IL-4 polypeptide shall be performed in cells of the same species as the IL-4 polypeptide from which the variant derives. Thus, the activity of a variant of a human IL-4 polypeptide shall be assessed in human cells and the activity of a variant of a murine IL-4 polypeptide shall be assessed in mouse cells.

In another embodiment, the IL-4 polypeptide is a variant showing decreased toxicity, such as the variants described in W09747744 or a variant showing improved affinity for its receptor, such as those described in EP0939817.

In another embodiment, the IL-4 polypeptide is a fusion protein comprising IL-4 and an IgG Fc region which is characterized by showing improved half-life in serum. Illustrative examples of these fusion proteins are those described by Nickerson et al.

(Transplant Immunology, 1996, 4: 81-85) or by Walz et al. (Hormone and Metabolic

Research, 2002, 34: 561-569). In another embodiment, the IL-4 polypeptide is a circularly permutated IL-4 showing increased receptor binding as described by Kreitman et al. (PNAS, 1994, 91 : 6889-6893).

In another embodiment, the IL-4 polypeptide is an IL-4 variant that selectivity binds to the receptor IL-2Ry in T cells, B cells and monocytes but showed no activity on endothelial cells as described by Shanafelt et al .(PNAS, 1998, 95: 9454-58).

In another embodiment, the IL-4 polypeptide is an IL-4 mimetic. The term "IL-4 mimetic", as used herein, means that the peptide mimics the binding site for the specific receptor of IL4, for example the IL-4Ra receptor for human IL4. Illustrative examples of IL-4 mimetics are those described in WO200212337.

In a particular embodiment, the IL-4 polypeptide is not fused to an anti-apoptotic BCL-2 family polypeptide as defined in WO 2015/042706 Al. In a more particular embodiment, the IL-4 polypeptide is not fused to BC1-X_L, Bcl-w or Bcl-2 as defined in WO 2015/042706 Al.

The invention also contemplates a nucleic acid encoding an IL-4 polypeptide for use in the prevention and/or treatment of ALS.

The term "nucleic acid encoding an interleukin 4 polypeptide", as used herein, refers to a nucleic acid, either a deoxyribonucleic acid or a ribonucleic acid, comprising a coding sequence for an IL-4 polypeptide as defined above. Said nucleic acid can include, but is not limited to: the coding sequence of the IL-4 polypeptide, including the pre-IL4 (containing the signal sequence) or the mature IL-4; the coding sequence of the IL-4 polypeptide and additional coding sequence; the coding sequence of the IL-4 polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or other non-coding sequences in 5' or 3 ' of the coding sequence of the IL-4 polypeptide, for example, one or more regulatory sequences like a regulated or regulatable promoter, enhancer or other transcriptional regulatory sequences, repressor binding sequences translation regulatory sequences or any other nucleic acid regulatory sequence.

Therefore, the term "nucleic acid encoding said IL-4 polypeptide" includes a nucleic acid comprising only the coding sequence of an IL-4 polypeptide as well as a nucleic comprising additional coding and/or non-coding sequences. The nucleic acid encoding an IL-4 polypeptide can be incorporated into a vector. The term "vector", as used herein, refers to a construct capable of delivering, and preferably additionally expressing, one or more polynucleotides of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. This term also relates to targeting constructs which allow for random or site-directed integration of the targeting construct into genomic DNA. Such targeting constructs, preferably, comprise DNA of sufficient length for either homologous recombination or heterologous integration.

The invention also contemplates a cell expressing an IL-4 polypeptide for use in the prevention and/or treatment of ALS.

The term "cell expressing an IL-4 polypeptide", as used herein, refers to a cell that is able to express and secrete the IL-4 polypeptide as defined above or a cell than contains a nucleic acid encoding an IL-4 polypeptide as defined above. In a particular embodiment, the cell expressing an IL-4 polypeptide is a human cell. The cell expressing an IL-4 polypeptide can be a cell with endogenous expression of the IL-4 polypeptide, for example, a CD4+ T cell, either a T helper 1 (Thl) or a T helper 2 (Th2), a CD4+CD25+ T cell or T regulatory cell, a mast cell, a polymorphonuclear leukocyte (PMN) or a recombinant cell, which may or may not endogenously express an IL-4 polypeptide but which has been genetically modified in order include an exogenous nucleic acid encoding an IL-4 polypeptide and in which the exogenous nucleic acid leads to the expression of the IL-4 polypeptide. In a particular embodiment, the cell expressing an IL-4 polypeptide is a recombinant cell. Said recombinant cell can be obtained by any technique known by the skilled person to introduce a foreign nucleic acid into a cell and make said cell express the polypeptide encoded by said nucleic acid. For example, a nucleic acid encoding an IL-4 polypeptide, or a vector comprising such nucleic acid, can be introduced into a cell, preferably a human cell, by well-known techniques such as infection, transduction, transfection, electroporation and transformation. Illustrative non- limitative examples of cells that can be used to obtain recombinant cells expressing an IL-4 polypeptide for the uses of the invention include T cells, dendritic cells, tumor cells, Human Embryonic Kidney 293 (HEK293) cells, Chinese Hamster Ovarian (CHO) cells, etc.

In a particular embodiment, the cell expressing an IL-4 polypeptide is an isolated cell. The term "isolated" means that the cell is not forming part of the human or animal body.

In a particular embodiment, the cell expressing an IL-4 polypeptide does not have endogenous expression of IL-4. In a more particular embodiment, the cell expressing an IL-4 polypeptide is not a T cell which endogenously expresses IL-4. In an even more particular embodiment, the cell expressing an IL-4 polypeptide is not a CD4+ T cell (T helper cell) or a CD4+CD25+ T cell (T regulatory cell).

The term "amyotrophic lateral sclerosis" or "ALS", as used herein, refers to the neurodegenerative disease characterized by progressive muscular paralysis due to the degeneration in the primary motor cortex, brainstem and spinal cord. ALS includes both the sporadic and familial forms, as well as forms that predominantly affect either the lower motorneurons (e.g., progressive muscular atrophy) and forms that predominantly affect the lower brainstem cranial motor nuclei (e.g., progressive bulbar palsy and bulbar amyotrophic lateral sclerosis). ALS is usually inherited in an autosomal dominant way, although there also exist recessive and X-linked forms.

In one embodiment, the ALS is sporadic ALS. In another embodiment, the ALS is a familial ALS. The term "familial ALS" or "FALS", as used herein, refers to a genetic associated form of ALS which exhibits a family history. An ALS is considered not familial or sporadic unless there are at least two people in the family who have or have had ALS and these two people are blood relatives. In about 20% of all families with ALS, the disease is caused by a mutation in the superoxide dismutase, or SOD1, gene. Mutations on C90RF72 (Chromosome 9 open reading frame 72) have also been described in about 40% of FALS. Other genes that may cause familial ALS include the FUS (fused in sarcoma) and the TDP-43 (tar-DNA binding protein-43) genes. Each of these genes accounts for 3-5% of patients with familial ALS. Mutations in a variety of other genes have been identified in very small numbers of people with familial ALS. These include genes such as FIG4, ANG, VAPB, ATXN2, UBQLN2, OPTN, VCP, DAO and senetaxin. In a particular embodiment, the familial ALS is caused by a mutation in the superoxide dismutase 1 (SOD1) gene.

The term "superoxide dismutase 1" or "SODl", as used herein, refers to a gene encoding the enzyme superoxide dismutase 1 (SOD1) which binds copper and zinc ions and is one of the superoxide dismutases responsible for destroying free superoxide radicals converting them to molecular oxygen and hydrogen peroxide.

The earliest signs and symptoms of ALS include muscle twitching, cramping, stiffness, or weakness. Affected individuals may develop slurred speech and, later, difficulty chewing or swallowing (dysphagia). Many people with ALS experience malnutrition because of reduced food intake due to dysphagia and an increase in their body's energy demands (metabolism) due to prolonged illness. Muscles become weaker as the disease progresses, and arms and legs begin to look thinner as muscle tissue wastes away (atrophies). Individuals with ALS lose their strength and the ability to walk. Affected individuals eventually become wheelchair-dependent. Over time, muscle weakness causes affected individuals to lose the use of their hands and arms. Breathing becomes difficult because the muscles of the respiratory system weaken. Most people with ALS die from respiratory failure within 2 to 10 years after the signs and symptoms of ALS first appear; however, disease progression varies widely among affected individuals.

In another embodiment, the ALS is an ALS-FTD, i.e. an ALS which is associated with frontotemporal dementia (FTD). Approximately 20% of individuals with ALS also develop a condition called frontotemporal dementia (FTD), which is a progressive brain disorder that affects personality, behavior, and language. Changes in personality and behavior may make it difficult for affected individuals to interact with others in a socially appropriate manner. People with FTD increasingly require help with personal care and other activities of daily living. Communication skills worsen as the disease progresses. It is unclear how the development of ALS and FTD are related. Individuals who develop both conditions are diagnosed as having ALS-FTD.

The term "prevention", as used herein, refers to the capacity of the interleukin 4 polypeptide, nucleic acid encoding said interleukin 4 polypeptide, or cell expressing said polypeptide, to prevent, minimize or hinder the onset or development of ALS, before its diagnosis. According to the invention, the interleukin 4 polypeptide, nucleic acid encoding said interleukin 4 polypeptide, or cell expressing said polypeptide, will slow the development of ALS symptoms, delay the onset of ALS, or prevent the individual from developing ALS.

The diagnose of the disease in humans is done based on the "El Escorial World Federation of Neurology Criteria For the Diagnosis of ALS". According to these criteria, and without excluding the possibility of these to be changed in the future upon a better understanding of the mechanistic or genetic basis of the disease or the improvement of the available diagnostic or prognostic methods, the current diagnosis of ALS requires the presence of:

1) Signs of lower motor neuron (LMN) degeneration by clinical, electrophysiological or neuropathologic examination,

2) Signs of upper motor neuron (UMN) degeneration by clinical examination, and

3) Progressive spread of signs within a region or to other regions, together with the absence of

4) Electrophysiological evidence of other disease processes that might explain the signs of LMN and/or UMN degenerations; and

5) Neuroimaging evidence of other disease processes that might explain the observed clinical and electrophysiological signs.

The clinical features required for the current diagnosis of ALS are:

1) Signs of LMN degeneration (weakness, wasting and fasciculation) in one or more of the four regions (bulbar, cervical, thoracic, lumbosacral). LMN findings in a region are without regard to right or left, but are indicative of the level of neuraxis involved. Therefore, spread of weakness, wasting and fasciculation' s to another region is more important than spread from right to left or vice-versa.

2) Signs of UMN degeneration (increased or donic tendon reflexes, spasticity, pseudo bulbar features, Hoffmann reflex and extensor plantar response) in one or more of the four regions. These UMN signs are clinically appreciated best in the bulbar, cervical and lumbosacral regions. UMN findings in a region are also without regard to right or left. Once the physical and neurological examinations provide information on the presence or absence of LMN and UMN signs in the four regions (bulbar, cervical, thoracic, lumbosacral) they must be ordered topographically in the manner to determine the certainty of the diagnosis of ALS.

The topographical location of certain UMN and LMN signs in four regions of the CNS together with progression of these signs determines the certainty of the diagnoses of ALS. Progression is a cardinal feature of the clinical diagnosis of ALS. Progression of signs within a region and progression of signs to involve other regions are crucial to the diagnosis. In one embodiment, the interleukin 4 polypeptide, nucleic acid or cell for use according to the present invention is administered before the appearance of at least one, at least two, at least three or more of the above signs and clinical manifestations.

In a particular embodiment, the interleukin 4 polypeptide, nucleic acid or cell for use according to the present invention is administered after the appearance of at least one, at least two, at least three or more of the above signs and clinical manifestations.

In a more particular embodiment, the interleukin 4 polypeptide, nucleic acid or cell for use according to the present invention is administered at advanced stage of the disease. The term "advanced stage of the disease", as used herein, refers to stage characterized by loss of motor function and hind-limb muscle innervation in the patient.

The term "treatment", as used herein, refers to any process, action, application, therapy, or the like, wherein a subject (or patient), including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject, or ameliorating at least one symptom of the disease or disorder under treatment.

In a particular embodiment, the IL-4 polypeptide, nucleic acid encoding said polypeptide or cell expressing said polypeptide for use in the treatment of ALS is administered after the diagnosis of the disease.

The term "patient" or "subject", as used herein, refers to any animal, preferably a mammal and includes, but is not limited to, domestic and farm animals, primates, and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents like rats and mice. In a preferred embodiment, the subject is a human being of any age or race. In a particular embodiment, the subject suffers from ALS. In another particular embodiment, the subject has not been diagnosed as suffering from ALS but is considered to be at increased risk of developing ALS, for example, the subject belongs to a family with a history of ALS and/or the subject has been determined to have a mutation in at least one of the following genes, preferably one mutation that has been reported to cause ALS, such as SOD1, ALS2, ANG, ATXN2, C9orp2, DCTN1, FIG4, FUS, NEFH, OPTN, PRPH, SETX, SIGMAR1, SMN1, SPG11, TARDBP, UBQLN2, VAPB or VCP. In a preferred embodiment, the patient is a female, preferably a human female. In another embodiment, the patient is a male, preferably a human male.

In a particular embodiment, the subject has a mutation in the SOD1, preferably one of the mutations in the SOD1 that have been reported to cause ALS, more preferably a mutation selected from the group A4V, H46R and G93A.

The method of administration of the IL-4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide, will depend on factors such as duration of the therapy and whether the IL-4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide, will be administered for preventing or treating purposes. Thus, the IL-4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide can be administered chronically, sub-chronically or acutely. In a preferred embodiment, the IL-4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide is administered chronically.

The term "chronically", as used herein, refers to a method of administration wherein the IL-4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide, is administered continuously to the patient for extended periods of time in order to maintain the therapeutic effect during this period. Chronic administration forms include the daily administration of one or multiples doses of the compound, twice daily, three times daily or more frequently. Chronic administration includes also repeated administrations during multiple weeks. Rest periods can be used as part of chronic dispensation. Alternatively, chronic administration can involve the administration as a bolus or by continuous transfusion or subcutaneous ly which can be performed daily, every two days, every 3 to 15 days, every 10 days or more. Typically, chronic administration is continued for at least one week, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least four months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least two years or more.

In a particular embodiment, the IL-4 polypeptide, nucleic acid encoding said IL- 4 polypeptide, or cell expressing said polypeptide is administered thrice weekly for 3 weeks, followed by a 2 week rest period.

The IL-4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide may be administered by any suitable administration route, such as, but not limited to, parenteral, oral, topical, nasal, rectal route. In a particular embodiment, the interleukin 4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide, is administered intraperitoneally, intravenously, subcutaneously, intradermically or intramuscularly. In a preferred embodiment, the interleukin 4 polypeptide, nucleic acid encoding said IL-4 polypeptide, or cell expressing said polypeptide, is administered subcutaneously.

The term "therapeutically effective amount", as used herein, refers to the sufficient amount of the compound to provide the desired effect and will generally be determined by, among other causes, the characteristics of the compound itself and the therapeutic effect to be achieved. It will also depend on the subject to be treated, the severity of the disease suffered by said subject, the chosen dosage form, administration route, etc. For this reason, the doses mentioned in this invention must be considered only as guides for the person skilled in the art, who must adjust the doses depending on the aforementioned variables. In a particular embodiment, the therapeutically effective amount produces the amelioration of one or more symptoms of ALS. In a particular embodiment, the therapeutically effective amount of the IL-4 polypeptide is from about 0,5 μg/kg/day to about 10 μg/kg/day, preferably from about 1 μg/kg/day to about 5 μg/kg/day.

In a particular embodiment, desirable serum levels of IL-4 in the subject are from about 0,002 ng/mL to about 10 ng/mL, preferably from about 0,01 ng/mL to about 7,5 ng/mL, more preferably from about 0,5 ng/mL to about 5 ng/mL, even more preferably from about 1 ng/mL to about 2,5 ng/mL. Therefore, in a particular embodiment, the interleukin 4 polypeptide, nucleic acid or cell is administered so that serum levels of interleukin 4 in the subject are from about 0,002 ng/mL to about 10 ng/mL, preferably from about 0,01 ng/mL to about 7,5 ng/mL, more preferably from about 0,5 ng/mL to about 5 ng/mL, even more preferably from about 1 ng/mL to about 2,5 ng/mL.

Serum levels of IL-4 can be determined by any suitable technique known by the skilled person, for example by ELISA.

In a particular embodiment, the IL-4 polypeptide is administered by daily subcutaneous injections at a maximum dose of 5 μg/Kg, so that serum IL-4 levels of at least 0,002 ng/mL in the subject are obtained for at least 8 hours after a single injection.

Unless otherwise indicated, the doses mentioned herein are human doses. The doses mentioned herein can be adapted for any mammal according to, but not limited to, the guidelines of the FDA for conversion of doses based on body surface area (Guidance for Industry, Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005, see Table 1).

In a particular embodiment, the IL-4 polypeptide, nucleic acid or cell is administered subcutaneously to a female subject.

In a particular embodiment, the IL-4 polypeptide, nucleic acid or cell is administered subcutaneously to a subject after the clinical manifestation of the disease, in particular at an advanced stage of the disease.

In a particular embodiment, the IL-4 polypeptide, nucleic acid or cell is administered to a female subject after the clinical manifestation of the disease, in particular in an advanced stage of the disease.

In a particular embodiment, the IL-4 polypeptide, nucleic acid or cell is administered subcutaneously to a female subject after the clinical manifestation of the disease, in particular in an advanced stage of the disease.

EXAMPLES

Materials and Methods

Animal and tissue preparation

The transgenic animals used in this study were: B6SJL-Tg (SOD1-G93A) IGur/J (S0D1^G93A) mice obtained from Jackson Laboratory (Bar Harbor, ME.). Once symptoms had developed, disease progression was quite rapid and caused the death of most of the animals within 128.9 ± 9.1 days.

The animals were deeply anaesthetized with pentobarbital and then transcardially perfused with physiological saline solution followed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB) at pH 7.4 for light microscopy immunocytochemistry. After 24 hours in PFA, the samples were transferred to 30% sucrose in 0.1M PB and 0.02% sodium azide for cryoprotection and were then frozen for cryostat sectioning. Semithin sections (l-μιη thick) stained with methylene blue were also examined.

Adult male C57BL/6J mice were purchased from Charles Rivers Laboratory

(Wilmington, MA) at 2 months of age. Appropriate rules and procedures were followed (Generalitat de Catalunya DOGC 2073, 1995) and approved by the ethical committee for animal testing of the Universitat de Lleida. Immunohistochemical procedures

The spinal cord was cut into 16 μιη transverse sections. This was done on a cryostat and the sections were then mounted on gelatinized slides and stored at -80°C before use. The sections were pretreated with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 1 hour, blocked with 10% normal goat serum (NGS) for 1 hour, and then incubated overnight at 4°C with an appropriate primary antibody.

The following primary antibodies were used: anti-glial fibrillary acidic protein (anti-GFAP) chicken polyclonal antibody (1 : 1000, Abeam, Cambridge, United Kingdom); anti-Iba-1 polyclonal rabbit antibody (1 :500, Wako Chemical, Neuss, Germany); anti-Mac2 rat monoclonal antibody (1 :800, antibodies-online GMBH, Aachen, Germany) and anti-misfolded SODl (AJ10 antibody, Sabado et al, Journal of Neuropathology and experimental neurology 2013, 72: 646-661; 1 : 1000). After previous washing with PBS, the samples were incubated for 1 hour with an appropriate fluorescent secondary antibody and labeled with one of the following fluorochromes: Alexa fluor 488, Alexa fluor 546, (Molecular Probes, Eugene, OR), Cy3, Cy5, or DyLight 488 (Jackson Immuno Research Laboratories, West Grove, PA). Nuclear counterstaining was performed using 4', 6-diamidino-2-phenylindole dihydro chloride (DAPI; 50 ng/ml, Molecular Probes). Images were obtained using an Olympus BX51 (Hamburg, Germany) epifluorescence microscope, equipped with a cooled CCD camera (DP30BW). A FluoView 500 Olympus confocal laser-scanning microscope was also used. The digital images were analyzed with Visilog 6.3 software (Noesis, Orsay, France).

Phenotypical score and behavior analysis

In order to assess the immunomodulatory effect, a number of additional tests were performed. One of these, called the "Clinical Score", consists of conducting a visual observation of the phenotypical condition of the animal. When the animal has no visible affectation, it receives a Clinical Score (CS) of 4. When first hind limb paralysis appears, the CS is 3. A CS of 2 is assigned when the second limb starts to show signs of paralysis and, finally, when the animal shows signs of symmetrical paralysis, a CS of 0 is awarded. A CS of 3 is arbitrarily established as the onset of disease in order to make comparison between different groups and treatments across independent experiments. Behavioral analysis was performed using Cat Walk XT® Software (which is similar to the Paw Print Test). CatWalk XT is a system for the quantitative assessment of footfalls and gait in rats and mice. As the software showed certain limitations for studying a neurodegenerative disease like ALS, although the interpodal distance was measured from 8 weeks onwards, only pre-terminal stage data was analyzed (the pre-terminal stage is the last point at which the animal is able to complete the test). CS was obtained once a month from the age of 4 weeks and then once a week from the age of 16 weeks, while the CatWalk XT was performed once a month throughout the life of the animal.

In vivo administration of recombinant IL4 (rIL4) to mSODl^G93A mice: start of treatment at day 30, IP administration.

Female and Male mSODl^G93A mice were given intraperitoneal injections (ip) three times a week, from 30 days to the end terminal stage, with 50 ng murine rIL4 (Gibco, Gran Island, NY) in 200μ1 phosphate-buffered saline (PBS), containing 1% serum obtained from non-transgenic mice that were over 4 weeks old.

In vivo administration of recombinant IL4 (rIL4) to mSODl^G93A mice: start of treatment at day 50, IP and subcutaneous administration) Female and Male mSODl^G93A mice were given intraperitoneal (IP) and subcutaneous injections (SC) three times a week, from 50 days up to the end terminal stage, with 50 ng or 500 ng of rIL4. Murine rIL4 (ProSpecBio, ProSpec-Tany TechnoGene Ltd. Israel) was conditioned in 200μ1 phosphate-buffered saline (PBS), containing 1% serum obtained from non-transgenic mice that were over 4 weeks old.

In vivo administration of atorvastatin to mSODl^G93A mice

Female and Male mSODl^G93A mice were given intraperitoneal injections (ip) on a daily basis, from 30 days old to the end terminal stage, with 20mg/day/kg of Atorvastatin Calcium Salt (Calbiochem, Darmstadt, Germany) in 200 μΐ phosphate- buffered saline (PBS).

Statistical analysis

All data are expressed as mean ± SEM. The statistical analysis was assessed using either the Student's t-test or one-way analysis of variance (ANOVA), followed by a post hoc Bonferroni's test. The level of significance was chosen as p < 0.05.

Results

Effects of rIL4 and atorvastatin administration on ALS clinical outcome in S0D1^G93A mice

Lifespan was measured when the animals were at the end terminal stage. This was done using the righting reflex test. The righting reflex, which is also known as the labyrinthine righting reflex, is a reflex that corrects the orientation of the body when it is taken out of its normal upright position. When the animal was unable to right itself within 10 seconds, it was considered that the end terminal point had been reached. After rIL4 treatment, all the experimental mice showed significant increases in lifespan: to an average of seventeen days (17 days), which represented an increase of about 14% (Fig 1A). When the Kaplan-Meier analysis was stratified for gender, females displayed a better response to treatment than males, with their lifespans increasing to 158 days while those of males only reached 136 days (Fig IB, C). The effects of IL-4 treatment on the onset of disease were also evaluated via a CS test (Fig 2). A CS of 3 was associated with the onset of disease. As in the case of lifespan, the onset of disease was significantly delayed in rIL4-treated animals with respect to the control group, with average delay of 23 days (an increase of ~ 22, 22%). Given the previous gender difference that we had observed, we decided to analyze this group individually. This revealed that female treated animals displayed a delay in the onset of disease of about a week (7,7 days) with respect to treated males.

The change in the rate of weight loss was also determined after administering the rIL4 treatment. To normalize the data, average weight was calculated as a percentage of the end terminal weight value with respect to the mean animal weight throughout its life. Treatment with rIL4 also resulted in a loss of weight, with a gender-specific difference consistent with the other clinical assessments that were performed (Fig.3).

For the atorvastatin treatment, the same parameters were measured as described above. In this case, however, no data indicative of a therapeutic effect were observed (Fig. 4).

rIL4 but not atorvastatin inhibits neuro inflammatory response in spinal cord from SOD 1^G93A mice

Immunohistochemical analysis of spinal cord was performed to assess whether the severity of ALS pathology was modified after rIL4 treatment (Fig. 5). In addition of glial markers, neuronal degeneration was monitored by detecting neurotoxic species of misfolded SOD1 with an anti-SODl conformational-specific antibody (AJ10) (Sabado et al, 2013). GFAP immunostaining did not show any changes as a consequence of rIL4 treatment (Fig.5 A-E).

However, the amount of microgliosis revealed by either Iba-1 or Mac2 immunostaining was significantly reduced after rIL4 administration (Fig. 5 F-O). Iba-1 is a general marker for microglial cells but Mac2 is only present in activated and presumably neurotoxic microglia.

An accumulation of misfolded SOD1 is indicative of the severity of the disease process (Saxena et al, Neuron 2013, 80: 80-96). As previously described (Sabado et al, 2013, supra), neurons containing misfolded SOD1 that display strongly positive AJ10 immunostaining were often associated with activated microglia with neuronophagic activity. Treatment with rIL4 showed a reduction in the number of mutant/misfolded neurotoxic SOD1 conformers revealed by the AJ10 antibody (Fig. 5 P-T). Substantial focal IgG deposits were described in spinal cord from SOD1 ALS mice, indicating either the breakdown of the blood brain barrier (Mancuso et al., 2014) and/or an immunologic reaction. IgG deposits were studied in lumbar spinal cord from rIL4-treated mSODl mice and untreated end terminal mice. Once again, rIL4 administration resulted in a significant reduction in these deposits (Fig. 5 U-Y). Overall, we can conclude that rIL4 administration is able to prompt a significant antiinflammatory action involving microglial cells and tissue immunoglobulins that improves MN pathology as determined by a reduction in the accumulation of misfolded SOD1.

On the other hand, the atorvastatin treatment produced no changes in either glial populations or in the quantity of neurotoxic SOD1 conformers. This was in line with previously described clinical observations (Fig. 6).

Motor Behavioral Analysis

Gait analysis was performed by means of Cat Walk XT® system. This test is similar to traditional paw print analysis: it involves the use of a corridor that directs and limits the freedom of movement of mice to a straight line that runs across the walkway. Data indicative of the paw print pattern that were acquired using an electronic device were analyzed using appropriate software. In our case, the interpodal distance (defined as the distance between the front and hind limbs on the same side of the body) was measured for each group (Fig. 7). As previously described, untreated animals developed clinical symptoms earlier than the rIL4 treated animals and were unable to complete the test beyond P90. As a result, P90 and terminal stage animals belonging to the treated group could only be compared with P90 control animals.

Mice have a natural tendency to move their hind limbs towards their front ones; in consequence, any neuromuscular disorder would tend to increase the distance between the paws. As shown in the graph at P90, the rIL4 group was characterized by shorter interpodal distances compared to the control group. This was observed for both paws and on both sides of the animals, indicating that the treated animals displayed an improved phenotype. This is consistent with previously reported data about survival, the onset of disease and spinal cord pathology. Moreover, when compared PI 20 treated animals with the P90 control group, rIL4 administration revealed improved scores with respect to the control.

After atorvastatin treatment, there was no observable change in the interpodal distance at the end terminal stage (Fig. 8). Taking all of these data together, it would appear that atorvastatin treatment does not offer any beneficial effect for mSODl^G93A mice.

Discussion

These results suggest that the harmful ALS-associated neuro-immune deregulation which involves microglial cells and also cells in systemic lymphoid organs can be balanced to a protective outcome by the administration of IL-4. Whether or not the major sites in which IL-4 acts are immune cells within spinal cord or in the peripheral lymphoid organs still remains to be determined.

The clinical benefits of IL-4 therapy are greater in female S0D1^G93A mice. This is in line with the results of COP-1 immunization and with the much greater immune deficit observed in male ALS mice. In addition, microglial inflammatory gene expression could be sex dependent and alterations in estrogen receptor expression could accompany these changes. An epidemiological analysis of ALS indicated that men are more likely to develop the disease than women.

In conclusion, the inventors have shown that recombinant IL-4 therapy in the

SODl model of ALS demonstrated therapeutic benefits in: slowing weight loss, delaying paralysis and extending survival. This clinical outcome showed a good correlation with an improvement in neuropathological changes in spinal cord. This work provides preliminary preclinical information to further understanding the molecular pathways associated with immune response in ALS and to contribute towards the further development of rIL4 as a therapeutic agent for this disease in humans.

Subcutaneous administration of rIL4 on clinical outcome in S0D1^G93A mice

The goal of this assay was to assess if the route of administration and dose could be relevant in the treatment of SODl mice at advanced stage of the illness (from day 50). After standard intraperitoneal administration in females, the span-life was only slightly delayed (137 days, figure not shown) compared to control (135 days, Figure 9). However, after subcutaneous administration of rIL4, females surprisingly improved their survival (measured as span-life) compared to the control groups: +6 days (50ng, Figure 9) and +17 days (500ng, Figure 11). When no-sex segregation was performed, identical results were achieved (50 ng, Figure 10).

The effects of the treatment with rIL4 were also evident in the delayed apparition of symptoms (measured as paralysis of at least one limb, Figure 12 and Figure 13) and other behavioral parameters (Interpodal distance, Figure 14 and Figure 15). Moreover, clinical evaluation of the state of mice (measured as body weight loss) showed that, after subcutaneous treatment with rIL4, the effects of the body degeneration could be reduced (Figure 16).

Conclusions

In conclusion, the studies carried out with recombinant IL-4 therapy in the SOD1 model of ALS demonstrated a therapeutic benefit in slowing weight loss, delaying paralysis and extending survival, being this clinical outcome in good correlation with an improvement of neuropathological changes in spinal cord.

Claims

1. An interleukin 4 polypeptide, a nucleic acid encoding said interleukin 4 polypeptide or a cell expressing said polypeptide, for use in the prevention and/or treatment of amyotrophic lateral sclerosis.

2. The interleukin 4 polypeptide, nucleic acid or cell for use according to claim 1, wherein the cell does not have endogenous expression of IL-4

3. The interleukin 4 polypeptide, nucleic acid or cell for use according to any of claims 1 or 2, wherein the interleukin 4 polypeptide is a mature polypeptide lacking a signal peptide.

4. The interleukin 4 polypeptide for use according to any of claims 1 to 3, wherein the interleukin 4 polypeptide is a recombinant interleukin 4 polypeptide.

5. The interleukin 4 polypeptide, nucleic acid or cell for use according to any of claims 1 to 4, wherein the interleukin 4 polypeptide is a human interleukin 4 polypeptide and the prevention and/or treatment is in a human subject.

6. The interleukin 4 polypeptide, nucleic acid or cell for use according to any of claims 1 to 5, wherein the interleukin 4 polypeptide comprises the sequence SEQ ID NO: 3.

7. The interleukin 4 polypeptide, nucleic acid or cell for use according to any of claims 1 to 6, wherein said interleukin 4 polypeptide, nucleic acid or cell is administered intraperitoneally, intravenously, subcutaneously, intradermically or intramuscularly.

8. The interleukin 4 polypeptide, nucleic acid or cell for use according to any of claims 1 to 7, wherein the interleukin 4 polypeptide, nucleic acid or cell is administered so that serum levels of interleukin 4 in the subject are between 0.002 ng/mL and 10 ng/mL.

9. The interleukin 4 polypeptide, nucleic acid or cell for use according to any of claims 1 to 8, wherein said interleukin 4 polypeptide, nucleic acid or cell is administered before the clinical manifestation of the disease.

10. The interleukin 4 polypeptide, nucleic acid or cell for use according to any of claims 1 to 9, wherein the amyotrophic lateral sclerosis is a familial amyotrophic lateral sclerosis.

11. The interleukin 4 polypeptide, nucleic acid or cell for use according to claim 10, wherein the familial amyotrophic lateral sclerosis is caused by a mutation in the superoxide dismutase 1 gene.