C-terminal MANF fragment for use in the treatment of a demyelinating disease

Abstract

The present invention provides a C-terminal MANF fragment with the sequence having length of 34-63 amino acids or a sequence which has at least 80 % sequence identity with the sequence, wherein said fragment is a cell membrane penetrating peptide, for use in the treatment of a demyelinating disease such as multiple sclerosis, wherein said fragment is preferably administered subcutaneously.

Description

[0001] C-terminal MANF fragment for use in the treatment of a demyelinating disease

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the fields of bioactive protein fragments and cell membranepenetrating peptides and also to the field of neurotrophic factors and endoplasmic reticulum (ER) located proteins, and more particularly to the field of treating multiple sclerosis.

[0004] BACKGROUND OF THE INVENTION

[0005] Multiple sclerosis (MS) patients suffer from focal demyelination and neuroinflammation in their CNS, followed by impaired remyelination that is unaddressed by current therapeutic options. Inflammation leads to chronic activation of a cellular stress mechanism, the unfolded protein response (UPR), which is thought to both exacerbate neuroinflammation, and prevent regenerative tissue responses such as remyelination. A UPR-modulating protein, MANF, has shown great promise at attenuating chronic UPR activation and enhancing tissue regeneration in various disease models, but does not reach the CNS when given peripherally.

[0006] SUMMARY OF THE INVENTION

[0007] We utilized C-MANF, a C-terminal fragment of MANF, and showed that subcutaneous administration of C-MANF promotes motor function recovery and tissue regeneration in a mouse model of autoimmune demyelination. We demonstrate that C-MANF suppresses neuroinflammatory activation in astrocytes and microglia, and facilitates recovery of oligodendrocyte counts after demyelination, while reducing long-term activation of the UPR. Furthermore, we show that C-MANF mediated promotion of remyelination in cerebellar organotypic slice cultures is dependent on UPR-modulation, and that exogenously applied C-MANF suppresses all three UPR pathways in oligodendroglia. Finally, we show that demyelination without the presence of endogenous MANF leads to extensive neuroinflammation and CNS degeneration, implicating UPR-modulation by MANF as a key component of remyelination. Altogether, we show that UPR modulation with C-MANF is a promising new therapeutic method for treating neuroinflammatory demyelination.

[0008] Therefore, an aspect of the present invention is to provide a C-terminal MANF fragment with the length of 34-63 amino acids, comprising or consisting of at least the consecutive amino acid residues at positions 6-35 or 18-53 of the sequence as set forth in SEQ ID NO: 1 :

[0009] KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 80 % sequence identity with the sequence of positions 6-35 or 18-53 in SEQ ID NO: 1 and the sequence flanking said consecutive amino acid residues preferably has at least 80 % sequence identity with the sequence at corresponding positions in SEQ ID NO: 1, wherein said fragment is a cell membrane penetrating peptide, for use in the treatment of a demyelinating disease, such as multiple sclerosis, wherein said fragment is preferably administered subcutaneously.

[0010] In an embodiment, the present invention provides a C-terminal MANF fragment having the sequence of SEQ ID NO: 1 :

[0011] KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 90 % sequence identity with the sequence of SEQ ID NO: 1 for use in the treatment of a demyelinating disease, such as multiple sclerosis, wherein said fragment is preferably administered subcutaneously.

[0012] The aforementioned and other advantages and benefits of the present invention are achieved in the manner described as characteristics in the accompanying claims.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGURE 1. Therapeutically administered s.c. C-MANF has neurorestorative effects on EAE progression. (A) The structure of C-MANF and relationship with hMANF. NMR structure of C-MANF from Hellman et al () (PDB ID: 2KVE, image created with ChimeraX 1.5). (B) Experimental outline and groups for testing of C-MANF in EAE. (C) Clinical scores were studied daily, and daily subcutaneous injections C-MANF (1 pg / g (n = 8) or 4 pg / g (n = 7)) or vehicle (n = 7) were administered to individual mice after they reached a score of 1. (D) AUC (area under curve) of group mean scores and (E) individual scores at the predetermined endpoint of day 28 after EAE induction. (F) Individual mice were weighed daily, and group average weights are reported as the % change in body weight relative to the day of EAE induction. (G) Motor coordination was tested using the rotarod- apparatus before EAE induction and on days 14, 21 and 28 after EAE induction. Only mice experiencing EAE symptoms & rolled into treatment groups were included at postinduction timepoints. Lines and bar graphs represent group means ± SEM, scatterplot represents individual myelinated axons with a simple linear regression ± 95% confidence bands, * / # P < 0.05, ** P < 0.01, and *** P < 0.001. (C & F) Matched two-way ANOVA, (D-E) one-way ANOVA, (G) matched mixed effects (REML) model, all followed by Dunnet’s post hoc test. FIGURE 2. Neuroprotective effects of C-MANF in the EAE spinal cord. (A) 20x confocal micrographs were taken from the analyzed area in the lumbar spinal cords of EAE animals sacrificed at 18 or 28 days after EAE induction and stained for MBP, NF -200 and DAPI. Images were taken at three different sites in the ventral white matter, and used to analyze (A) the MBP+ area fraction and (B) the NF -200+ area fraction in vehicle-treated and 1 pg / g C-MANF -treated EAE spinal cords, reported as % of the average of the healthy control group (pooled from dl 8 & d28, n = 4). (C) Representative TEM images of lumbar ventral spinal cord white matter of EAE mice treated with vehicle or 1 pg / g C-MANF. Scale bar = 1 pm. (D) Visualized G-ratio to axon diameter relationship, each dot representing a single myelinated axon (EAE + veh n = 237, EAE + 1 pg / g C-MANF n = 300). (E) Mean thickness of myelin sheaths per EAE mouse spinal cord. Bar graphs represent group means ± SEM, * P < 0.05, ** P < 0.01, and *** P < 0.001. (A-B) One-way ANOVA followed by Sidak’s post hoc test. (E) Unpaired 2-tailed t test.

[0015] FIGURE 3. Treatment with C-MANF correlates with improved oligodendrocyte recovery and reduced neuroinflammation and astrogliosis in EAE. 40x IF confocal micrographs were taken of lumbar spinal cord white matter taken from healthy control mice and EAE mice, stained for TPPP (mature oligodendrocytes), GFAP (astrocyte marker), Ibal (microglia marker) & CHOP (chronic proapoptotic UPR marker. Images were taken at three different sites in the ventral white matter, and used to analyze: (A) counts of TPPP+ oligodendrocytes and CHOP+TPPP+ oligodendrocytes normalized to analyzed spinal cord surface area; (B) Ibal+ & Ibal+CHOP+ surface area fraction, reported as % of average of healthy control group; (C) GFAP+ & GFAP+CHOP+ surface area fraction, reported as % of average of healthy control group. Bar graphs represent group means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (A) 1st graph one-way ANOVA followed by Sidak’s post hoc test, 2nd graph Kruskal-Wallis test followed by Dunn’s post hoc test, (B & C) one-way ANOVA followed by Sidak’s post hoc test.

[0016] FIGURE 4. Treatment with C-MANF correlates with suppressed gene expression of all major UPR pathways as well as proinflammatory pathways. mRNA extracted from EAE spinal cords was analyzed by qPCR for expression of (A) Grp78, (B) Xbpls, (C) Atf4, (D) Atf6, (E) Gfap, (F) Cdllb and (G) Tnfa gene expression and reported as fold change in expression relative to timepoint-matched healthy controls Bar graphs represent group means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (A-G) two-way ANOVA followed by Holm-Sidak’s post hoc test. FIGURE 5. The effect of C-MANF on peripheral immune cell populations and CNS T cell infiltration. (A) Cellular abundance of gated populations from spleens of timepoint-matched controls and EAE mice. (B) Analyzed CD3+ T cell counts from lumbar spinal cord white matter stained for CD3+, normalized to analyzed spinal cord surface. Bar graphs represent group means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. (A) Two- way ANOVA followed by Holm-Sidak’s post hoc test. (B) One-way ANOVA followed by Sidak’s post hoc test.

[0017] FIGURE 6. Dose-dependent acceleration in remyelination in C-MANF-treated demyelinated organotypic cerebellar brain slices. (A) Analyzed fraction of NF-200+ axonal fibers covered by myelin in cerebellar slices, each dot corresponding to a single well (average of 2 slices / well, 2 images / slice) and reported as % of average of naive untreated control group. (B) Densitometric analysis of MBP-expression from western blots, normalized to total protein and reported as % of average of naive untreated control. Bar graphs represent group means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. (A) One-way ANOVA and (B) two-way ANOVA, both followed by Sidak’s post hoc test.

[0018] FIGURE 7. Sequence alignment and comparison of C-CDNF and C-MANF. The C- terminal structure of both neurotrophic factors comprises three alpha-helix motifs (helix 1, 2 and 3). Human C-CDNF: KYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQE LAPKYAATHPKTEL (SEQ ID NO:4).

[0019] FIGURE 8. Full-length MANF and 48 aa C-MANF rescue ER stressed SCG neurons. 7 days old SCG neurons were injected with full-length MANF or 48 aa C-MANF (SEQ ID NO:5) consisting of positions 6-53 of SEQ ID NO:1. On the next day, 2 pM tunicamycin was added to trigger ER-stress-induced cell death. After 3 days, living fluorescent neurons were counted, and results were disclosed as a percentage of initial neurons. n=3,*p<0.05.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention is related to neurotrophic factor protein MANF. Particularly important MANF polypeptides are the full-length human MANF with a signal peptide having the total length of 179 amino acids and the mature human MANF without the signal peptide having the total length of 158 amino acids.

[0022] As used herein, the term "C-terminal fragment" as applied to a MANF polypeptide, may ordinarily comprise or consist of 34-63 contiguous or consecutive amino acids, typically, at least about 48 contiguous or consecutive amino acids, more typically, at least about 57 or 60 contiguous or consecutive amino acids located in the C-terminal SAP -like domain. Said C- terminal fragment comprises the CKGC -motif at positions 32-35 of SEQ ID NO: 1 and two or three alpha helix motifs (helix 1, 2 and 3) present between positions 5 and 54 of SEQ ID NO: 1 (see Figure 7). In an embodiment, said C-terminal fragment comprises at least the sequences of helix 1, helix 2 and the CKGC -motif of the MANF sequence as defined in Figure 7, or at least the sequences of helix 2, CKGC -motif, and helix 3 of the MANF sequence as defined in Figure 7. The C-terminal fragment can also be longer than 63 contiguous or consecutive amino acids in length, preferably the C-terminal fragment consists of the SEQ ID NO: 1. In an embodiment, the C-terminal MANF fragment may ordinarily comprise or consist of 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 63 contiguous or consecutive amino acids of SEQ ID NO: 1. These C-terminal fragments are “functional fragments” retaining at least partly biological activity of the intact MANF polypeptide and may even have properties the intact MANF polypeptide does not have.

[0023] In addition to naturally occurring allelic variants of MANF, changes can be introduced by mutation into MANF nucleic acid sequences that incur alterations such as elongations, insertions and deletions in the amino acid sequences of the encoded MANF polypeptide or C-terminal fragment thereof. Nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of a MANF polypeptide and the C-terminal domain thereof.

[0024] A "non-essential" amino acid residue is a residue that can be modified in the wild-type sequences of MANF without altering its biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the MANF molecules of the invention are predicted to be essential and particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well known in the art.

[0025] Each amino acid can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and / or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrolysine. Non-natural amino acids can also be the D-isomer of the natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloisoleucine, arginine, asparagine, aspartic acid, cysteine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, glutamine, glutamic acid, glycine, histidine, homoproline, isoleucine, leucine, lysine, methionine, naphthylalanine, norleucine, phenylalanine, phenylglycine, pipecolic acid, proline, pyroglutamic acid, sarcosine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof.

[0026] Certain embodiments of the invention include C-terminal MANF fragments, wherein at least one, two, three, four or more consecutive amino acids have alternating chirality. As used herein, chirality refers to the "D" and "L" isomers of amino acids. In particular embodiments of the invention, at least one, two, three, four or more consecutive amino acids have alternating chirality and the remaining amino acids are L-amino acids.

[0027] As used herein, the term “demyelinating disease” refers to any disease affecting the nervous system where the myelin sheath surrounding neurons is damaged. This damage disrupts the transmission of signals through the affected nerves, resulting in a decrease in their conduction ability. Consequently, this reduction in conduction can lead to deficiencies in sensation, movement, cognition, or other functions depending on the nerves affected. Demyelinating diseases are traditionally classified into two types: demyelinating myelinoclastic diseases and demyelinating leukodystrophic diseases. In the first group, a healthy and normal myelin is destroyed by toxic substances, chemicals, or autoimmune reactions. In the second group, the myelin is inherently abnormal and undergoes degeneration. Common demyelinating diseases that affect central nervous system (brain and spinal cord) include: multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), transverse myelitis (TM), acute disseminated encephalomyelitis (ADEM), progressive multifocal leukoencephalopathy (PML), myelopathy, leukodystrophy, neuropathy caused by vitamin Bn deficiency, and central pontine myelinolysis (osmotic demyelination syndrome). Common demyelinating diseases that affect peripheral nervous system (nerves outside of brain and spinal cord) include: Guillain-Barre syndrome (GBS), Charcot-Mari e-Tooth disease (CMT), hereditary neuropathy with liability to pressure palsy (HNPP), chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, myelopathy caused by copper-deficiency, neuropathy caused by copper- deficiency, and progressive inflammatory neuropathy.

[0028] Accordingly, the present invention provides a C-terminal MANF fragment with the length of 34-63 amino acids, comprising or consisting of at least the consecutive amino acid residues at positions 6-35 or 18-53 of the sequence as set forth in SEQ ID NO: 1 : KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP

[0029] KYAPKAASAR TDL or a sequence which has at least 80 % sequence identity with the sequence of positions 6-35 or 18-53 in SEQ ID NO: 1 and the sequence flanking said consecutive amino acid residues preferably has at least 80 % sequence identity with the sequence at corresponding positions in SEQ ID NO: 1, wherein said fragment is a cell membrane penetrating peptide, for use in the treatment of a demyelinating disease such as multiple sclerosis, wherein said fragment is preferably administered subcutaneously.

[0030] In a preferred embodiment, the present invention provides a C-terminal MANF fragment having the sequence of SEQ ID NO: 1 :

[0031] KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 90 % sequence identity with the sequence of SEQ ID NO: 1 for use in the treatment of a demyelinating disease such as multiple sclerosis, wherein said fragment is preferably administered subcutaneously.

[0032] As used herein in the specification and in the claims section below, the term "fragment" includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and modified peptides, which may have, for example, modifications rendering the peptides more stable or less immunogenic. Such modifications include, but are not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, backbone modification and residue modification. The fragment may also comprise further elongations, deletions or insertions.

[0033] In the embodiments of the invention, the length of the fragment is in the range of 34-63, 35- 63, 36-63, 37-63, 38-63, 39-63, 40-63, 41-63, 42-63, 43-63, 44-63, 45-63, 46-63, 47-63, 48- 63, 49-63, 50-63, 51-63, 52-63, 53-63, 54-63, 55-63, 56-63, 57-63, 58-63, 59-63, 60-63, 61- 63, 62-63 amino acids. For example, the C-terminal MANF fragment can consist of at least 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 amino acids. In an embodiment, the C-terminal MANF fragment consists of 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 63 amino acids. The fragments may comprise any of the naturally occurring amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine as well as non- conventional or modified amino acids. Preferably, the fragment has at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% sequence identity with the sequence of the C-terminal domain in the human MANF protein. More preferably, the fragment has at least 80% sequence identity with the sequence of the C-terminal domain in the human MANF protein. “Sequence identity” as used herein refers to sequence similarity between a reference sequence and at least a fragment of a second sequence. As described below, BLAST will compare sequences based upon percent identity and similarity.

[0034] The terms “identical” or percent “identity,” in the context of two or more amino acid sequences, refers to two or more sequences or subsequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues that are the same (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 10 amino acids in length, or more preferably over a region that is 10, 15, 20, 25, 30 or more amino acids in length.

[0035] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity.

[0036] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art such as ClustalW or FASTA. Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22): 10915-10919] alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For short amino acid sequences, PAM30 scoring matrix can be applied.

[0037] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc Natl Acad Sci USA 90(12): 5873 - 5877). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two amino acid sequences would occur by chance.

[0038] The present invention also shows that the C-terminal fragment may be conjugated to a detectable chemical or biochemical moiety such as a FITC-label. As used herein, a "detectable chemical or biochemical moiety" means a tag that exhibits an amino acid sequence or a detectable chemical or biochemical moiety for the purpose of facilitating detection of the peptide; such as a detectable molecule selected from among: a visible, fluorescent, chemiluminescent, or other detectable dye; an enzyme that is detectable in the presence of a substrate, e.g., an alkaline phosphatase with NBT plus BCIP or a peroxidase with a suitable substrate; a detectable protein, e.g., a green fluorescent protein. Preferably, the tag does not prevent or hinder the penetration of the fragment into a target cell.

[0039] N- and / or C-terminal modifications of the C-terminal MANF fragments to increase the stability and / or cell permeability of the fragments are also preferred. Acetylation - amidation of the termini of the MANF fragment (i.e. N-terminal acetylation and C-terminal amidation) is one of the options known in the art (see e.g. Marino et al. 2015, ACS Chem. Biol. 10: 1754-1764).

[0040] Our results shown in Figures 1-7 confirm that the C-terminal MANF fragment is effective in the treatment of demyelinating diseases such as multiple sclerosis. In a method of treatment of a demyelinating disease, a pharmaceutically effective amount of the C-terminal MANF fragment is administered to a patient. In other words, the MANF fragment as described in the present invention is for use in the treatment of a demyelinating disease such as multiple sclerosis. The actual dosage amount of the C-terminal fragment of MANF (e.g., an effective amount) that is administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration can determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0041] In one embodiment of the present invention, the C-terminal MANF fragment can be incorporated into pharmaceutical compositions. Such compositions of the invention are prepared for storage by mixing the peptide having the desired degree of purity with optional physiologically acceptable carriers (such as nanocarriers), excipients, or stabilizers (Remington's Pharmaceutical Sciences, 22nd edition, Allen, Loyd V., Jr, Ed., (2012)), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium; and / or non-ionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

[0042] The fragment may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0043] In an embodiment, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose of a pharmaceutical composition or formulation can comprise from about 1 ng / kg / body weight of C-terminal MANF fragment, about 5 ng / kg / body weight, about 10 ng / kg / body weight, about 50 ng / kg / body weight, about 100 ng / kg / body weight, about 200 ng / kg / body weight, about 350 ng / kg / body weight, about 500 ng / kg / body weight, 1 pg / kg / body weight, about 5 pg / kg / body weight, about 10 pg / kg / body weight, about 50 pg / kg / body weight, about 100 pg / kg / body weight, about 200 pg / kg / body weight, about 350 pg / kg / body weight, about 500 pg / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight of C-terminal MANF fragment or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 pg / kg / body weight to about 500 milligram / kg / body weight of C-terminal MANF fragment, etc., can be administered, based on the numbers described above.

[0044] In another embodiment, the pharmaceutical composition comprises a therapeutically effective amount of recombinant vectors comprising a nucleotide sequence that encodes the C-terminal MANF fragment as defined above, recombinant viral vectors comprising a nucleotide sequence that encodes the C-terminal MANF fragment as defined above, or a host cell expressing the C-terminal MANF fragment as defined above. Said viral vector is preferably selected from the group consisting of an adenovirus, an adeno-associated virus, a retrovirus such as a lentivirus, herpes virus, and papillomavirus comprising a polynucleotide encoding the C-terminal MANF fragment as defined above. Typically, the recombinant vectors and recombinant viral vectors include expression control sequences like tissue- or cell-type specific promoters that direct the expression of the polynucleotide of the invention in various systems, both in vitro and in vivo. Vectors can also be hybrid vectors that contain regulatory elements necessary for expression in more than one system. Vectors containing these various regulatory systems are commercially available and one skilled in the art will readily be able to clone the C-terminal MANF fragment as defined herein into such vectors. Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the C-terminal MANF fragment into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310. The route of administration is in accord with known methods as well as the general routes of injection or infusion by intravenous or peripheral administration, intraperitoneal, subcutaneous, intrathecal, intracerebroventricular, intranasal, transdermal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional means, or sustained release systems as noted below. The C-terminal MANF fragment or a pharmaceutical composition comprising said fragment can be administered continuously by infusion or by bolus injection. Generally, where the disorder permits, one should formulate and dose the fragment for site-specific delivery. Administration can be continuous or periodic. Administration can be accomplished by a constant- or programmable-flow implantable pump or by periodic injections. Peripheral, systemic or subcutaneous administration is preferred as it has been showed that C-terminal MANF fragments are capable of penetrating neuronal cell membrane as well as the blood-brain-barrier. Other preferred administration routes are intrathecal, intracerebroventricular, intranasal, or transdermal administration. In the Figures, the effect of a subcutaneous injection of C-MANF protein is shown in rats having an induced multiple sclerosis.

[0045] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the fragment, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels as described by Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982) or polyvinylalcohol, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), or non-degradable ethylene-vinyl acetate (Langer et al., supra).

[0046] Gene therapy vectors can be delivered to a subject using corresponding administration modes as defined above for the peptide fragment, preferably by, for example, intravenous injection, or by intraperitoneal, subcutaneous, intrathecal, or intracerebroventricular administration. The pharmaceutical preparation of a gene therapy vector can include an acceptable diluent, or can comprise a slow-release matrix in which the gene delivery vehicle is embedded.

[0047] The publications and other materials used herein to illuminate the background of the invention, and in particular, to provide additional details with respect to its practice, are incorporated herein by reference. The present invention is further described in the following examples, which are not intended to limit the scope of the invention. EXPERIMENTAL SECTION

[0048] Materials and methods

[0049] MANF sequences

[0050] Mature human MANF :

[0051] LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFCREARGKENRLCYYIGATD DAATKIINEVSKPLAHHIPVEKICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKEL KKILDDWGETCKGCAEKSDYIRKINELMPKYAPKAASARTDL (SEQ ID NO:2)

[0052] Human N-MANF

[0053] LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFCREARGKENRLCYYIGATD DAATKIINEVSKPLAHHIPVEKICEKLKKKDSQICEL (SEQ ID NO:3)

[0054] Human C-MANF

[0055] KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAPKA ASARTDL (SEQ ID NO: 1)

[0056] C-MANF was ordered as a 63 aa chemically synthesized peptide from Apeptide (Shanghai, China), with the amino-acid sequence of SEQ ID NO:1. C-MANF was stored at -80 °C and diluted in PBS for experiments.

[0057] EAE animal model

[0058] EAE induction: Female 6-week old C57BL / 6IRccHsd mice from Envigo were habituated to animal facilities for two weeks prior to EAE-induction. EAE was induced using the Hooke Laboratories EK-2110 EAE induction kit, according to manufacturer’s instructions. In addition to two s.c. injections of MOG35-55 / CFA emulsion, mice received 120 ng PTX (pertussis toxin) i.p. at 2 and 24 hour-timepoints. For healthy control animals, MOG / CFA was replaced by an equal volume of PBS.

[0059] Scoring and treatment: Mice were weighed and scored daily according to Hooke’s EAE scoring guide, in which 0 = no symptoms, 1 = limp tail, 2 = weak hind limbs, 3 = completely paralyzed hind limbs, 4 = partial front leg paralysis and 5 = mouse found dead due to paralysis (scores between integers, i.e. 2.5, indicate a clinical picture between the two numbered scores). Individual mice were rolled into blinded treatment groups balanced by score and body weight upon showing EAE symptoms corresponding to a clinical score of 1; mice which failed to show symptoms of EAE were excluded (2 mice in each set) by the end of the experiment. Mice in EAE treatment groups received 1 pg / g C-MANF (i.e. a 20g mouse received 20 pg of C-MANF in a single injection), 4 pg / g C-MANF (only in the 1stset of EAE experiments) or vehicle (PBS), as daily s.c. bolus injections of 5 pl / g. Researchers performing animal experiments were blinded to EAE treatment groups, but not the healthy control group. Analysis of motor function

[0060] Behavioral testing was carried out at the Mouse Behavioural Phenotyping Facility (MB PF) supported by Biocenter Finland and HiLIFE. Rotarod (Ugo Basile) and open field (ENV- 520, Med Associates Inc.) tests were performed to evaluate motor function deficit.

[0061] Rotarod: Mice were trained once a day for 2 days prior to EAE-induction on an accelerating rotating platform (from 4 to 40 rpm for a maximum of 240 s). Once a mouse fell off the rotating rod, the latency to fall (time it takes for the mouse to drop from the rod) was recorded. During the experiment, mice were tested at days 14, 21 & 28 after induction.

[0062] Open field: Spontaneous locomotor activity was tested at the chronic EAE timepoint of day 28. Mice were placed in an enclosed, lit arena, and their ambulation speed, time and counts as well as vertical rearing counts recorded for 10 minutes.

[0063] Sacrifice & tissue processing

[0064] At the endpoint (18 or 28 days) of EAE experiments, all mice were sedated with terminal anaesthesia using sodium pentobarbital (90 mg / kg, i.p.; Orion Pharma).

[0065] TEM sample preparation: EAE mice were perfusion fixed with 2% glutaraldehyde (EM- grade) and 2% formaldehyde (EM-grade) in 0.1 M sodium phosphate buffer, pH 7.4. Lumbar spinal cords were dissected and cut into ca. 200 pm vibratome sections in ice-cold phosphate buffer. Tissue sections were further fixed with 2% glutaraldehyde and 2% formaldehyde in phosphate buffer for 4 h in fridge. Prior embedding, samples were stored in 2% formaldehyde in phosphate buffer in fridge over weekend. Tissue sections were then washed with phosphate buffer, prior to osmication with 1% reduced osmium tetroxide in 0.1 M phosphate buffer for 2 hours on ice. Phosphate buffer was exchanged to distilled water prior uranyl acetate en-block staining with 2% aqueous solution in fridge for 1 hour, followed with washes with distilled water, gradual dehydration with 20%, 30%, 50%, 70%, 90%, 96% and two times 100% ethanol (each step for 8 minutes), two incubations in acetone (a 8 minutes) and gradual infiltration into Hard Epoxy (TAAB) over two days and polymerization at 60 °C for a day.

[0066] Semithick sections were cut and stained with toluidine blue for light microscopy to guide the trimming of the target area, where ultrathin 60 nm sections were cut using Ultracut UCT7 ultramicrotome (Leica Mikrosysteme GmbH), picked on Pioloform-coated single-slot copper grids, and poststained with uranyl acetate (SPI Supplies) and lead citrate (Leica Ultrostain 2).

[0067] RNA & Protein extraction: Mice were transcardially perfused with cold PBS for 5 minutes, until the liver was clear of blood. The spine was resected on ice, and immediately snap frozen in dry ice and stored at -80 C. RNA and protein purification was performed using a NucleoSpin RNA / Protein purification kit (Macherey-Nagel) following the manufacturers recommended protocol. The lumbar spine L2-L6 was resected from the spinal column of the frozen samples, and immediately placed into complete lysis buffer supplemented with 2- Mercaptoethanol. The tissue was homogenized by passing through a G20 needle 5 times, then passing through a G25 needle 5 times, before finally being centrifuged in the manufacturer provided filtration column. Following the manufacturers protocol, eluted RNA was screened for purity and concentration by nanodrop, and protein samples were assayed using a protein quantification assay (Macherey-Nagel) before being stored at -80 °C. Immunofluorescence: EAE mice were transcardially perfused with cold PBS for 5 minutes, followed by resection of the lumbar spine into cold 4% paraformaldehyde (PF A). After postfixation at 4 °C over a weekend, the lumbar spinal cord L2-L6 was resected, and processed for paraffin embedding with a Leica ASP300S. 16 pm slices were sectioned with a microtome and transferred onto glass slides for staining. LPC-injected mice were transcardially perfused with cold PBS for 5 minutes, then with cold 4% PFA for another 5 minutes, after which brains were collected. After post-fixation at 4 °C over a weekend, brains were processed for paraffin embedding with a Leica ASP300S. 10 pm slices were sectioned with a microtome and transferred onto glass slides for staining.

[0068] Flow cytometry. Spleens were harvested from mice under terminal anesthesia prior to perfusion and placed in cold HBSS. To produce single-cell suspensions, spleens were pushed through 70 pm cell strainers (VWR), which were then washed with 5 mL PBS. Suspensions were then centrifuged at 650g for 5 minutes, then cell pellets were resuspended in 500 pl RBC lysis buffer (BD Biosciences) and incubated for 10 minutes in room temperature. Suspensions were then washed by adding 500 pl PBS and centrifuging at 650g for 5 minutes twice, after which they were resuspended in 100 pl cell staining buffer (BD Biosciences).

[0069] Organotypic cerebellar brain slice model

[0070] To make organotypic cerebellar brain slices, P9-12 C57BL / 6JRccHsd pups of both sexes were sacrificed by CO2-inhalation, and dissected cerebella were cut into 350 pm slices using a McIlwain Tissue Chopper. Samples were placed on Millicell cell culture inserts (30mm, 0.4 pm pores) in 6-well plates with serum-supplemented media (SSM) consisting of 50% MEM, 25% HBSS and 25 % horse serum, supplemented with 1% antibiotic / antimycotic, 0.5% L-glutamine and 0.5% glucose (all SSM reagents from Gibco). Slices from each pup were divided equally to wells, with the number of slices / well in separate experiments indicated in figure legends. Media was replaced in each well 24h after preparation, and every 48h subsequently. Demyelination was induced on the 5thday after slice preparation by replacing media with SSM containing LPC (L-a-Lysophosphatidylcholine from Glycine max) at 0.5 mg / ml for 17h. LPC was diluted in HBSS, and sonicated for 30 minutes before addition to media. After toxin-treatment, media was replaced with SSM with possible additions of C-MANF or UPR modulators. 4p8c, GSK2606414 (both from MedChemExpress) and ISRIB (Sigma) were dissolved in DMSO and diluted by 1 :500 in SSM for use. SSM for C-MANF and vehicle-groups contained a corresponding dilution of DMSO.

[0071] After 5 or 6 days of remyelination, for IF analysis of myelination, slices were fixed with 4% PF A for Ih at room temperature, and then washed with PBS before immunofluorescence staining. RNA and protein purification was performed using a NucleoSpin RNA / Protein purification kit (Macherey -Nagel) following the manufacturers recommended protocol, as described earlier.

[0072] Oli-Neu cell line model

[0073] Oli-Neu cells (a gift from Professor liris Hovatta’s lab with permission by Professor Jacqueline Trotter) were maintained in a simple growth media containing DMEM, 10% FBS, 2mM L-Glutamine, lx PENSTREP, 0.1% Sodium Pyruvate and 15mM HEPES (all growth media reagents from Gibco). For analysis of UPR activation in differentiating Oli- Neu, cells were plated in 6-well plates at 250 000 cells / well in a modified DMEM-SATO Base Growth Medium (1) containing DMEM, BSA (0.1 mg / ml, Sigma), Putrescine (0.016 mg / ml, Sigma), progesterone (60 ng / ml, Sigma), L-Glutamine (2 mM, Gibco), lx PENSTREP (Gibco), Sodium Pyruvate (1 mM, Gibco), lx Insulin-Transferrin-Selenium (ITS -G, Gibco), N-acetyl-L-cysteine (5 pg / ml, Sigma), lx B-27 (Gibco), lx Trace Elements B (Corning), d-Biotin (10 ng / mL, Sigma), PDGF (20 ng / mL, Peprotech) and NT-3 (1 ng / mL, Peprotech). After 24h of OPC-like proliferation in 6-well plates, growth media was replaced by DMEM-SATO Differentiation Media, which had the following modifications from the DMEM-SATO Growth Media: B-27 concentration lowered to O.lx, PDGF and NT- 3 replaced with PD 174,265 (1 nM, EGFR tyrosine kinase inhibitor from Cayman Chemicals) and 3,3',5-Triiodo-L-thyronine (T3, 10 ng / mL, Sigma). After 48h in differentiation media, toxin-treated groups were supplemented with 50 nM Thapsigargin and drug-treated groups with 5 pg / mL C-MANF. Total RNA was isolated from differentiated Oli-Neu cells at 5h and 24h after beginning of treatment with thapsigargin, C-MANF and / or corresponding vehicle controls using the TRIzol Reagent solution (Thermo Fisher Scientific). TEM image acquisition and image analysis

[0074] TEM micrographs were acquired using Hitachi HT7800 microscope (Hitachi High- Technologies) operated at 100 kV, and equipped with a Rio9 CMOS-camera (AMETEK Gatan Inc.). Micrographs were taken at 2000x magnification from 5 randomly selected areas in the ventral white matter of the sections. Myelinated axons were analyzed using the G-ratio plugin for Image J software. Summarized, the plugin selects random areas in a micrograph, which when located in an axon leads to the researcher drawing the outline of both the axon and the myelin sheath. Approximately 60-90 myelinated axons were quantified per micrograph, and the software calculated g-ratios as the ratio of axon perimeter divided by myelin sheath perimeter. Myelin thickness was calculated for each sheath as myelin sheath diameter subtracted by axon inner diameter, with an assumption of circularity.

[0075] Immunofluorescence

[0076] Mounted section staining: Glass-mounted spinal cord or brain sections were deparaffinated using a series of Xylene and Ethanol, after which antigen retrieval was performed by heating slides in a sodium citrate buffer (10 mM Sodium citrate, pH 6.0, 0.05% Tween-20). Sections were washed in TBS, permeabilized in two changes of TBS-T (TBS with 0.1% Tween-20), and then blocked with 5% BSA (bovine serum albumin) and 1% horse serum in TBS-T for Ih. Sections were then incubated with primary antibodies diluted in TBS-T with 5% BSA overnight at 4°C (see table below for used antibodies & concentrations). After primary antibody incubation, sections were washed three times with TBS-T, and then incubated with secondary antibodies diluted in TBS-T with 5% BSA for Ih at room temperature. Afterwards, sections were possibly incubated with DAPI diluted in TBS, washed in TBS three times, and mounted with coverslips using Immu-Mount (Epredia).

[0077] Whole-mount staining: Fixed organotypic brain slices were cut out of inserts with a scalpel, and transferred to 24 well plates with blocking solution containing 0.5% Triton-X, 5% horse serum and 1% BSA in PBS for Ih. Blocking solution was then replaced with primary antibodies diluted in more blocking buffer, and samples were incubated with primary antibodies overnight at 4°C. Samples were then washed three times with blocking solution, and incubated with secondary antibodies diluted in blocking solution for 2h at room temperature. Following a single wash with PBS, samples were incubated with DAPI diluted in PBS, and then washed three times with PBS. Finally, samples were transferred onto glass histology slides, and gently mounted with coverslips using Immu-Mount.

[0078] Imaging: Laser scanning confocal micrographs of the fluorescently labeled samples were acquired with an SP8 Stellaris Stellaris 8 FALCON (Leica) confocal microscope using a HC PL APO 20x / 0.75 CS2 air objective. For higher magnification analysis of CHOP colocalization with glial cell markers in spinal cord samples, an HC PL APO 40x / 1.25 motCORR glycerol ojective was used. Z-stacks of 10 images at 1 pm intervals (2 pm for organotypic brain slices) were taken from the stained area and combined into a maximum intensity projection using the imaging software Las X (version 4.4.0, Leica).

[0079] Image analysis: All confocal micrographs were analyzed using Fiji Image J Software (version 1.53). For analysis of EAE spinal cords, three Z-stack images were taken from sequential spinal cord sections at fixed locations in the ventral are of the spinal cord. For quantification of MBP, NF200, Ibal & GFAP area fraction from 20x micrographs, the fraction of positively stained pixels within a region-of-interest (ventral white matter) was calculated, the results averaged for each spinal cord, and represented as a percentage of the mean of the healthy control group. For counting TPPP+ and CD3+ cells, Fiji automated cell counting was used to count particles over the size of 10 pm2in the ventral white matter. To quantification of Ibal+CHOP+ and GFAP+CHOP+ area fraction in 40x micrographs, object-based-overlap analysis was performed: the Fiji image calculator was used to create an image containing only pixels positive for both Ibal and CHOP, from which the area fraction in the ventral white matter was calculated. The number of CHOP+ oligodendrocytes was counted manually from 40x micrographs as the number of TPPP+ cells that were clearly positive for CHOP.

[0080] RNA analysis with RT-qPCR cDNA synthesis: Synthesis of cDNA for qPCR was performed using the previously purified RNA using the commercially available Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufactures protocol. The samples were synthesized using the same concentration of RNA template, resulting in 50 ng / pl final concentration (for RNA isolated from organotypic brain slices, due to smaller output, a final concentration of 12.5 ng / pl was used for cDNA synthesis).

[0081] RT-qPCR: Prepared cDNA samples were diluted 1 : 10 in sterile water and combined with LightCycler® 480 SYBR Green I Master mix (Roche) and primers (sequences in supplemental table 2) in 384-well plates (10 pl total volume / well). Plates were centrifuged briefly and placed in a LightCycler 480 System (Roche) for RT-qPCR. Results were acquired using the Basic Relative Quantification-function in the LightCycler® 480 Real- Time PCR System software and analyzed using the AACt method. All samples were performed in triplicates. Outlier analysis (Grubb's test at an alpha of 0.05) was performed on all RT-qPCR results, and outliers with an RNA purity (260 / 230 or 260 / 280 as measured by nanodrop) below 1.8 were excluded from the final analysis. Protein analysis with western blot

[0082] Equal amounts of protein (20 pg from EAE spinal cords, 10 pg from organotypic brain slices) were separated on 4-20% Mini-PROTEAN TGX Stain-Free gels (BioRad). After electrophoresis, gels were activated with UV in an ChemiDoc MP imaging system (Biorad), and protein transferred to 0.2 pm PVDF using a Transblot Turbo (Biorad). Total protein images were taken from PVDF membranes for normalization using the ChemiDoc MP, after which the membranes were blocked in TBS with 5% BSA for 1 h at room temperature. Primary incubation with antibodies diluted in PBS-T with 5% BSA was performed at 4°C overnight, then membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Stained proteins were visualized using Pierce ECL western blotting substrate (Thermo Fisher Scientific) on the ChemiDoc MP. Semi quantitative analysis of protein quantity relative to total protein stain was performed using Fiji Image J, and results were expressed as percentage of mean results of healthy control groups.

[0083] Results

[0084] Subcutaneously administered C-MANF is therapeutic and increases myelination in a model of experimental autoimmunity

[0085] The EAE (experimental autoimmune encephalomyelitis) model recapitulates chronic autoimmune-mediated demyelination in rodents. We previously evaluated the efficacy of full-length human MANF on EAE in Nam el al. (2), finding that with peripheral delivery MANF had a limited capacity of preserving motor function in an early stage of the disease. To test the hypothesis that C-MANF has improved pharmacological properties, we performed another set of EAE experiments. C57Bl / 6JRcc mice were induced with EAE via using the Hooke EK-2110 kit. After individual mice began to show the early symptoms of EAE, an ascending paralysis starting with a limp tail, rolling enrollment was used to assign mice to balanced treatment groups. Mice received either C-MANF at either 1 pg / g or 4 pg / g, or vehicle (PBS) as daily subcutaneous injections (Fig. 1 A). Motor coordination was analyzed with the rotarod test before EAE induction, then weekly starting at day 14 after EAE induction, and exploratory locomotion was assayed with the open field test before euthanasia at the endpoint of day 28.

[0086] Mice that received C-MANF featured a significantly less severe disease course than vehicle- treated mice, as calculated by mean area under curve of the clinical score (Fig. IB; Vehicle vs 1 pg / g C-MANF - P < 0.001; Vehicle vs 4 pg / g C-MANF - P < 0.01), and the group receiving 1 pg / g C-MANF had a significantly lower average score at the endpoint of 28 days (Fig. 1C; P < 0.05). The reduction in clinical score was reflected in reduced body weight loss, as treatment with 1 pg / g C-MANF led to a significant recovery in body weight by day 28, reported as % change from starting body weight (Fig. ID; Vehicle vs 1 pg / g C-MANF - P < 0.001). While the performance of all EAE-groups in the rotarod test was very poor after EAE onset at week 2, the group receiving 1 pg / g C-MANF showed a significant improvement in motor coordination compared to the vehicle-treated group at weeks 3 & 4 (Fig. IE; P < 0.05 at both timepoints). Open field test performed at day 28 indicated that treatment with 1 pg / g C-MANF caused a nonsignificant trend towards increased ambulation. Ultrastructural morphometry analysis was performed using a plugin for the ImageJ software (3). 1 pg / g C-MANF -treated EAE spinal cords showed an increased presence of myelinated axons compared to vehicle-treated samples (Fig. IF). Analysis of the G-ratio across axonal sizes showed an increase in myelination (Fig. 1G), and a significant increase in average myelin sheath thickness in C-MANF treated spinal cords (Fig. 1H; P < 0.01), leading us to suspect that the efficacy of C-MANF was linked to an increase in remyelination capacity.

[0087] C-MANF enhances remyelination and prevents axon loss during chronic EAE

[0088] To further analyze the neuroprotective effects of C-MANF on EAE mice, we sacrificed mice treated with 1 pg / g C-MANF or vehicle at day 18 (peak phase) and 28 (chronic phase) after EAE induction. Immunohistochemical analyses of white matter in the lumbar spinal cord showed that while treatment with C-MANF did not inhibit myelin loss at the peak of the disease, myelination recovered and was significantly improved compared to vehicle-treated groups by the end of the experiment (Fig. 2A-B; EAE + C-MANF dl 8 vs d28 p<0.05; d28 EAE + C-MANF vs EAE + Vehicle p<0.001), supporting the hypothesis that C-MANF increases remyelination in EAE mice.

[0089] Chronic EAE is known to feature progressive loss of axons in the spinal cord, which correlates with chronic disability, despite intermittent relapses and remissions in clinical score. This resembles the chronic axonal degeneration seen in MS patients, especially those suffering from progressive MS (4). We observed that treatment with C-MANF was able to prevent the progressive axon loss in the lumbar spinal cord (Fig. 2C; d28 EAE + C-MANF vs EAE + Vehicle p<0.05), as determined by staining for neurofilament heavy chain (NF- 200).

[0090] Next, to verify the effects on myelination on the transcriptomic level, we evaluated MBP gene expression. We observed that while MBP was downregulated in the entire lumbar spinal cord at peak EAE (Fig. 2D; Healthy Control vs EAE + Vehicle dl 8 p<0.01), treatment with C-MANF led to an increase in MBP expression (Fig. 2D; EAE + Vehicle vs EAE + C-MANF dl 8 p<0.05). By day 28, MBP-expression in vehicle-treated samples had caught up and was no longer significantly lower than in healthy control or C-MANF treated EAE-spinal cords. Interestingly, C-MANF did not prevent the significant decrease in expression of OPC surface antigen NG2 at day 18, but significantly reduced overexpression of NG2 at day 28 (Fig. 2E; Healthy Control vs EAE + Vehicle day28 p<0.01; EAE + Vehicle vs EAE + C-MANF day28 p<0.001).

[0091] C-MANF reduces neuroinflammation and promotes oligodendrocyte recovery by regulating chronic UPR

[0092] Both EAE and MS feature the strong activation of neuroinflammatory microglia and astrocytes expressing CHOP, a pro-apoptotic and pro-inflammatory UPR component upregulated by chronic activation of the IREla, PERK and ATF6 pathways (5, 6, 7, 8). Myelinating oligodendrocytes also feature CHOP-activation in EAE and acute MS, which likely contributes to demyelination via oligodendrocyte apoptosis. We performed a set of immunofluorescence analyses of CHOP expression in TPPP+ myelinating oligodendrocytes, Ibal+ microglia and GFAP+ astrocytes. We observed that the lumbar white matter of healthy control mice showed healthy numbers of oligodendrocytes, and low levels GFAP+ and Ibal+ expression with minimal CHOP-expression (Fig. 3A). In contrast, EAE spinal cords had reduced oligodendrocyte counts and intense microgliosis and astrogliosis, with CHOP-expression apparent in all three glial cell types (Fig. 3B, D & F). As observed with staining of MBP earlier, treatment with C-MANF did not prevent oligodendrocyte loss at the peak of EAE but lead to a recovery in cell counts by day 28 (Fig. 3C; EAE + Veh vs EAE + C-MANF d28 p<0.01; EAE + C-MANF dl 8 vs d28 p<0.05;). Simultaneously, treatment with C-MANF drastically reduced the number of CHOP+ oligodendrocytes in the lumbar white matter (Fig 3C; dl 8 EAE + C-MANF vs dl 8 EAE + Veh p<0.01; d28 EAE + C- MANF vs d28 EAE + Veh p<0.05). Treatment with C-MANF reduced Ibal+ microgliosis at both timepoints (Fig. 3E; dl 8 EAE + C-MANF vs EAE + Veh p<0.05; d28 EAE + C-MANF vs d28 EAE + Veh p<0.001), while also leading to a strong decrease in the fraction of CHOP+ microglia (Fig. 4E; EAE + Veh vs EAE + C-MANF dl 8 p<0.0001; d28 EAE + C- MANF vs d28 EAE + Veh p<0.05). Treatment with C-MANF also reduced GFAP+ astrogliosis at both timepoints (Fig. 3G; EAE + Veh vs EAE + C-MANF dl 8 p<0.01; EAE + Veh vs EAE + C-MANF d28 p<0.05), while reducing the expression of CHOP in astrocytes at day 18 ((Fig. 3G; EAE + Veh vs EAE + C-MANF dl 8 p<0.05).

[0093] Next, we performed qPCR to analyze changes in gene expression for UPR and neuroinflammation. EAE significantly increased the expression of UPR genes GRP78 (primary sensor of ER stress), Xbpls (IRE la-pathway-activated transcription factor) and ATF4 (PERK-pathway activated transcription factor) at day 28, but not at day 18 of EAE (Fig. 3H-J; Healthy Control vs EAE + Veh d28 GRP78 p<0.01, XBPls p<0.0001, ATF4 p<0.05). This chronic UPR activation was significantly reduced by treatment C-MANF (Fig. 3H-J; EAE + Veh vs EAE + C-MANF d28 GRP78 p<0.05, Xbpls p<0.001, ATF4 p<0.05), supporting the theory that C-MANF promotes recovery in EAE by reducing the chronic activation of UPR. Interestingly, we did not observe an upregulation of the ATF6 pathway of UPR, but treatment with C-MANF still lead to a decrease in ATF6-expression at both day 18 & day 28 (Fig. 3K; EAE + Veh vs EAE + C-MANF dl 8 & d28 p<0.05). Compared to vehicle-treated EAE mice, C-MANF significantly reduced the mRNA levels of GFAP, proinflammatory microglial marker CD1 lb & inflammatory cytokine TNFa at day 28 (Fig. 3L-N; EAE + Veh vs EAE + C-MANF d28 GFAP p<0.001, CD1 lb p<0.05, TNFa p<0.05), but not at day 18. The observed differences in effects of C-MANF on neuroinflammation on day 18 between immunofluorescent analyses and qPCR may be due specificity of the tissue analyzed - while the immunofluorescent analysis was performed on lumbar white matter, the area most affected by immune infiltration in EAE, qPCR analysis was performed on RNA isolated from the entire lumbar spinal cord, which may drown out localized white matter effects from treatment with C-MANF.

[0094] C-MANF does not induce peripheral immune suppression, but can delay initial immune cell infiltration

[0095] The UPR plays an important function in regulating peripheral immune responses and contributes to chronic autoimmunity (9). As EAE is a T cell-driven autoimmune disease model, and T cell differentiation and expansion is known to be affected by UPR activation (10, 11), it stands to reason that UPR modulation by C-MANF may affect peripheral immune responses in EAE. In order to ascertain whether the therapeutic effect we observed from C-MANF administration in EAE mice was caused by peripheral immune suppression, we used flow cytometry to measure the levels of CD45+ leukocytes, CD4+ / CD8+ T cells and CD1 lb+ myeloid cells in the spleens of EAE mice at peak (day 18) and chronic (day 28) phases of EAE (Fig. 4A). At peak EAE, vehicle-treated mice featured a significant increase in CD1 lb+ myeloid cells (Fig. 4Bp<0.0001), and a significant decrease in CD45+ lymphocytes (P < 0.05), CD4+ T cells (P < 0.01) and CD8a+ T cells (P < 0.001) when compared against healthy control mice (Fig. 4B). By day 28 of EAE, vehicle-treated mice still featured a significant increase in CD1 lb+ myeloid cells (P < 0.0001) but did not feature significant changes in counts of lymphocytes or CD4+ / CD8+ T cells. C-MANF -treated EAE mice did not differ from vehicle-treated EAE mice in any cell population counts at either timepoint, indicating there were no effects from C-MANF on peripheral immune cell populations (Fig. 4B).

[0096] Next, we stained EAE spinal cords with CD3+, a pan-T cell surface marker, in order to determine whether C-MANF influenced infiltrating T cell counts. While we could not detect any CD3+ T cell infiltration in healthy control spinal cords, CD3+ cells were present in the white matter of all EAE spinal cords (Fig. 4C). Using automated cell counting, we determined that the level of CD3+ T cell infiltration was significantly decreased at day 18 by treatment with C-MANF (P < 0.05), whereas there was no difference between vehicle and C- MANF -treated mice at day 28 (Fig. 4D). Together, these data suggest that while the administration of C-MANF in EAE mice did not affect peripheral immune cell populations, it could reduce levels of T cell infiltration during the initial peak of the disease course.

[0097] C-MANF enhances remyelination via UPR modulation

[0098] In order to observe the direct effects of C-MANF on CNS remyelination, we utilized a commonly used ex vivo model of LPC-induced demyelination (12). Organotypic cerebellar slices were derived from the brains of postnatal day 9-12 mouse pups and cultured on semiporous membranes. After a limited recovery period, samples were treated with 0.1% LPC for 17 hours, followed by removal of LPC and treatment with either C-MANF or vehicle for 5 days (Fig. 5 A). Analysis of immunostained slices showed that treatment with a high dose of C-MANF had no effect on myelination in naive slices (Fig. 5B). Treatment with LPC induced a consistent demyelination in slices compared to naive controls that was still apparent at day 5 after LPC-removal (P < 0.0001). While low doses of C-MANF did not improve recovery, treatment with 5 pg / ml and 10 pg / ml of C-MANF led to a significant improvement in the ratio of myelinated axonal fibers in the slices (Fig. 5C and D; LPC + Vehicle vs LPC + 5 pg / ml C-MANF p<0.0001; LPC + Vehicle vs LPC + 10 pg / ml C- MANF p<0.01). By analyzing protein extracted from LPC-treated slices, we observed no effect from C-MANF on the expression of MBP in the slices at day 1 post-LPC-removal (Fig. 5E). In contrast, C-MANF administration for 5 days increased MBP-expression significantly (Fig. 5E; P < 0.001). Thus, we found that direct treatment with C-MANF did not prevent toxin-induced demyelination, but significantly improved remyelination in a dose-dependent manner. Moreover, qPCR analysis of extracted RNA showed that C-MANF caused a significant reduction in Xbpls and TNFa mRNA expression, as well as a non- significant trend in reducing mRNA expression of other UPR and neuroinflammatory pathways.

[0099] Next, we utilized pharmacological compounds capable of inhibiting specific UPR pathways. We observed in EAE that treatment with C-MANF suppresses chronic activation of the IREla and PERK-pathways, so we tested the effect of inhibiting one of these pathways on C-MANF -induced remyelination. 4p8c inhibits the endonuclease activity of IREla (13) and GSK2606414 is a potent direct inhibitor of PERK (14), whereas ISRIB inhibits the PERK pathway further downstream by dephosphorylating eIF2a (15). Both 4p8c and GSK2606414 have been observed to inhibit the neuroprotective properties of full-length MANF in mouse primary neuron cultures (16), but this has not been reported in more complex tissue models before. By using the similar experiment timeline as in Fig. 4, we showed that C-MANF once again significantly improved remyelination at 5 days after removal of LPC (Fig. 6A-B; C- MANF vs LPC p<0.001). None of the used UPR-inhibitors affected remyelination at day 5 when given after LPC-withdrawal, but when given together with C-MANF, inhibition of the IREla and PERK-pathways both abrogated the ability of C-MANF to enhance remyelination (Fig. 6A-B; C-MANF vs C-MANF + 4p8c p<0.05; C-MANF vs C-MANF + GSK2606414 p<0.0001). Inhibition downstream of PERK using ISRIB had no effect on C-MANF -induced remyelination. Together, this data highlights that the effect of C-MANF on the UPR goes beyond simple inhibition, and that for its neuroprotective properties it requires functionality of both the IREla & PERK pathway sensors, while downstream modulation of the PERK pathway seemed negligible in this modulation (visualized in Fig. 6C).

[0100] One of the suggested protective forms of UPR-modulation by MANF is promoting acute, adaptive UPR-activation while inhibiting chronic UPR-activation, which is supported by the observable chronic UPR visible in MANF -deficient tissues with high protein-loads (17). As the effects of MANF or C-MANF on acute and chronic UPR in oligodendroglia specifically have not been published before, we tested simultaneous treatment with C-MANF and the UPR-inducing toxin thapsigargin in differentiated Oli-Neu cells (Fig. 6D). Oli-Neu are an immortalized mouse oligodendroglial cell line capable of limited differentiation into myelinproducing oligodendrocyte-like cells (1, 18). While C-MANF had no effects on UPR- pathway activation in naive cells or cells treated with thapsigargin for 5 hours, after 24 hours of toxin-exposure C-MANF downregulated the mRNA expression of all three UPR pathways, as well as Chop (Fig. 6E; Thapsigargin vs Thapsigargin + C-MANF Xbpls p<0.05, Atf4 p<0.05, Atf6 p<0.05 & Chop p<0.0001). This supports the theory that C-MANF can directly modulate the UPR in oligodendroglia by restricting the chronic activation of all three UPR pathways, leading to lowered expression of the pro-apoptotic transcription factor CHOP.

[0101] CNS tissue lacking endogenous MANF is unable to resolve demyelination

[0102] As tissue lacking endogenous MANF tends to be unable to resolve chronic UPR, we wanted to see the effects of demyelination in MANF -deficient tissue. To achieve this, we prepared cerebellar organotypic brain slices from MANF-knockout mouse pups (17). Slices were genotyped after preparation, and maintained for 6 days post-LPC withdrawal. Analysis of whole-mount-stained slices showed that while MANF-knockout brain slices featured normal morphology in the absence of treatment with LPC, all homozygote knockouts were completely altered after LPC-treatment, featuring shrinking, near-total axonal degeneration and dysmyelination. While Manf + / + and + / - slices showed no axonal degeneration and a quantifiable reduction in myelination at 6 days after LPC, myelination was not analyzable in LPC-treated -I- slices due to the lack of usual cerebellar morphology. When fixed and stained at days 1 and 3 post-LPC, MANF-knockout slices seemed as viable as heterozygous knockouts at day 1, but at day 3 slices from half of the -I- mice were degenerating considerably, indicating that lack of endogenous MANF leads to a gradual progressive degeneration of cerebellar slices. Finally, to determine whether this demyelination-induced degeneration was specific to the slice model, we injected LPC into the corpus callosum of MANF-knockout mice (n=2 for MANF KO and control group). Brains were fixed and collected at day 6 post-injection and analyzed using immunofluorescence. Observation of the limited number of samples showed that while WT mice featured some astrocytic activation and microglial migration to the injection area, Manf -I- brains showed extensive microgliosis across the entire corpus callosum, with no surviving mature oligodendrocytes within the formed glial scar, supporting the hypothesis that chronic UPR caused by lack of endogenous MANF drives ongoing inflammation.

[0103] In conclusion, we show that subcutaneously administered C-MANF enhances regeneration in the CNS of mice suffering from MS-like autoimmune demyelination. Our data shows that C-MANF achieves this by reducing neuroinflammation via UPR-modulation in glial cells and increasing the production of remyelinating oligodendrocytes. REFERENCES

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Claims

CLAIMS1. A C-terminal MANF fragment with the length of 34-63 amino acids, comprising or consisting of at least the consecutive amino acid residues at positions 6-35 or 18-53 of the sequence as set forth in SEQ ID NO: 1 :KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 80 % sequence identity with the sequence of positions 6-35 or 18-53 in SEQ ID NO: 1 and the sequence flanking said consecutive amino acid residues preferably has at least 80 % sequence identity with the sequence at corresponding positions in SEQ ID NO: 1, wherein said fragment is a cell membrane penetrating peptide, for use in the treatment of a demyelinating disease, wherein said demyelinating disease is preferably selected from the group consisting of multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), transverse myelitis (TM), acute disseminated encephalomyelitis (ADEM), progressive multifocal leukoencephalopathy (PML), myelopathy, leukodystrophy, neuropathy caused by vitamin Bn deficiency, central pontine myelinolysis (osmotic demyelination syndrome), Guillain-Barre syndrome (GBS), Charcot-Mari e-Tooth disease (CMT), hereditary neuropathy with liability to pressure palsy (HNPP), chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, myelopathy caused by copper-deficiency, neuropathy caused by copper-deficiency, and progressive inflammatory neuropathy, and wherein said fragment optionally comprises N- and / or C- terminal modifications to increase the stability of the fragment.

2. The fragment for use according to claim 1, wherein said fragment is administered subcutaneously, intravenously, intraperitoneally, intrathecally, intracerebroventricularly, intranasally, transdermally, intracerebrally, intramuscularly, intraocularly, or intraarterially.

3. The fragment for use according to claim 1 or 2, wherein said fragment is administered subcutaneously.

4. The fragment for use according to any one claims 1-3, said C-terminal MANF fragment comprises or consists of the sequence of SEQ ID NO: 1 :KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 90 % sequence identity with the sequence of SEQ ID NO: 1.

5. The fragment for use according to any one of claims 1-4, wherein said demyelinating disease is multiple sclerosis (MS).

6. The fragment for use according to any one of claims 1-4, wherein said demyelinating disease is selected from the group consisting of neuromyelitis optica spectrum disorder (NMOSD), transverse myelitis (TM), acute disseminated encephalomyelitis (ADEM), progressive multifocal leukoencephalopathy (PML), myelopathy, leukodystrophy, neuropathy caused by vitamin Bn deficiency, central pontine myelinolysis (osmotic demyelination syndrome), Guillain-Barre syndrome (GBS), Charcot-Mari e-Tooth disease (CMT), hereditary neuropathy with liability to pressure palsy (HNPP), chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, myelopathy caused by copper-deficiency, neuropathy caused by copper-deficiency, and progressive inflammatory neuropathy.

7. The fragment for use according to any one of claims 1-6, wherein the C-terminal MANF fragment comprises modifications protecting the fragment from enzymatic degradation selected from the group consisting of: cyclization of the fragment, amidation of the C- terminus of the fragment, and acetylation of the N-terminus of the fragment.

8. A nucleic acid vector encoding a C-terminal MANF fragment with the length of 34-63 amino acids, comprising or consisting of at least the consecutive amino acid residues at positions 6-35 or 18-53 of the sequence as set forth in SEQ ID NO: 1 :KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 80 % sequence identity with the sequence of positions 6-35 or 18-53 in SEQ ID NO: 1 and the sequence flanking said consecutive amino acid residues preferably has at least 80 % sequence identity with the sequence at corresponding positions in SEQ ID NO: 1, wherein said fragment is a cell membrane penetrating peptide, for use in the treatment of a demyelinating disease, wherein said demyelinating disease is preferably selected from the group consisting of multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), transverse myelitis (TM), acute disseminated encephalomyelitis (ADEM), progressive multifocal leukoencephalopathy (PML), myelopathy, leukodystrophy, neuropathy caused by vitamin Bn deficiency, central pontine myelinolysis (osmotic demyelination syndrome), Guillain-Barre syndrome (GBS), Charcot-Mari e-Tooth disease(CMT), hereditary neuropathy with liability to pressure palsy (HNPP), chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, myelopathy caused by copper-deficiency, neuropathy caused by copper-deficiency, and progressive inflammatory neuropathy.

9. A pharmaceutical composition comprising the C-terminal MANF fragment as defined in any one of claims 1-6 or the nucleic acid vector as defined in claim 7 and at least one of the following: physiologically acceptable carrier, buffer, excipient, preservative and stabilizer, for use in the treatment of a demyelinating disease, wherein said demyelinating disease is preferably selected from the group consisting of multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), transverse myelitis (TM), acute disseminated encephalomyelitis (ADEM), progressive multifocal leukoencephalopathy (PML), myelopathy, leukodystrophy, neuropathy caused by vitamin Bn deficiency, central pontine myelinolysis (osmotic demyelination syndrome), Guillain-Barre syndrome (GBS), Charcot- Marie-Tooth disease (CMT), hereditary neuropathy with liability to pressure palsy (HNPP), chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, myelopathy caused by copper-deficiency, neuropathy caused by copper- deficiency, and progressive inflammatory neuropathy.

10. The pharmaceutical composition for use according to claim 9, wherein said composition is administered by subcutaneous, intravenous, intraperitoneal, intrathecal, intracerebroventricular, intranasal, transdermal, intracerebral, intramuscular, intraocular, or intraarterial administration.

11. The pharmaceutical composition for use according to claim 9 or 10, wherein said composition is administered by subcutaneous administration.

12. A method of treating a demyelinating disease comprising administering to a patient an effective amount of a C-terminal MANF fragment with the length of 34-63 amino acids, comprising or consisting of at least the consecutive amino acid residues at positions 6-35 or 18-53 of the sequence as set forth in SEQ ID NO: 1 :KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 80 % sequence identity with the sequence of positions 6-35 or 18-53 in SEQ ID NO: 1 and the sequence flanking said consecutive amino acid residues preferably has at least 80 % sequence identity with the sequence at corresponding positionsin SEQ ID NO: 1, wherein said fragment is a cell membrane penetrating peptide, wherein said demyelinating disease is preferably selected from the group consisting of multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), transverse myelitis (TM), acute disseminated encephalomyelitis (ADEM), progressive multifocal leukoencephalopathy (PML), myelopathy, leukodystrophy, neuropathy caused by vitamin Bn deficiency, central pontine myelinolysis (osmotic demyelination syndrome), Guillain-Barre syndrome (GBS), Charcot-Mari e-Tooth disease (CMT), hereditary neuropathy with liability to pressure palsy (HNPP), chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, myelopathy caused by copper-deficiency, neuropathy caused by copper- deficiency, and progressive inflammatory neuropathy.

13. The method according to claim 12, wherein said C-terminal MANF fragment is administered by subcutaneous, intravenous, intraperitoneal, intrathecal, intracerebroventricular, intranasal, transdermal, intracerebral, intramuscular, intraocular, or intraarterial administration, preferably said C-terminal MANF fragment is administered by subcutaneous administration.

14. The method according to claim 12, wherein said C-terminal MANF fragment is administered in a pharmaceutical composition comprising at least one of the following: physiologically acceptable carrier, buffer, excipient, preservative and stabilizer.

15. The method according to claim 12, wherein said C-terminal MANF fragment comprises or consists of the sequence of SEQ ID NO: 1 :KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 90 % sequence identity with the sequence of SEQ ID NO: 1.

16. A method of treating a demyelinating disease comprising administering to a patient an effective amount of a nucleic acid vector encoding a C-terminal MANF fragment with the length of 34-63 amino acids, comprising or consisting of at least the consecutive amino acid residues at positions 6-35 or 18-53 of the sequence as set forth in SEQ ID NO: 1 :KYDKQIDLST VDLKKLRVKE LKKILDDWGE TCKGCAEKSD YIRKINELMP KYAPKAASAR TDL or a sequence which has at least 80 % sequence identity with the sequence of positions 6-35 or 18-53 in SEQ ID NO: 1 and the sequence flanking said consecutive amino acid residues preferably has at least 80 % sequence identity with the sequence at corresponding positionsin SEQ ID NO: 1, wherein said fragment is a cell membrane penetrating peptide, wherein said demyelinating disease is preferably selected from the group consisting of multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), transverse myelitis (TM), acute disseminated encephalomyelitis (ADEM), progressive multifocal leukoencephalopathy (PML), myelopathy, leukodystrophy, neuropathy caused by vitamin B 12 deficiency, central pontine myelinolysis (osmotic demyelination syndrome), Guillain-Barre syndrome (GBS), Charcot-Mari e-Tooth disease (CMT), hereditary neuropathy with liability to pressure palsy (HNPP), chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, myelopathy caused by copper-deficiency, neuropathy caused by copper- deficiency, and progressive inflammatory neuropathy.

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