Intravenous administration of neuroglobin for treating neuropathy

Abstract

Using the Harlequin (Hq) mouse (a model that develops a dysregulated phenotype), the inventors confirmed that neuroglobin-based gene therapy is a very promising means for the treatment of neurological diseases. The Hq mice received either intravenous (injection into the vein of the posterior orbit) or local (neurosurgery in the cerebellar hemisphere) administration of an AAV vector encoding neuroglobin. The inventors showed that very specific intravenous administration of neuroglobin significantly increased the survival rate of treated Hq mice, minimized weight loss in the mice, prevented cerebellar tissue degeneration, and preserved Purkinje cells and the branching of their dendrites, compared to other administration modes. Thus, the inventors demonstrated that very specific intravenous administration of neuroglobin has a significant neuroprotective effect, thereby becoming a very promising therapeutic strategy for treating neuropathy. Accordingly, the present invention relates to neuroglobin for use in the treatment of neuropathy, which is administered intravenously to patients in need thereof.

Description

Technical Field

[0001] The present invention relates to the specific use of neuroglobin administered intravenously for the treatment of neuropathy.

Background Art

[0002] The functional integrity of the central nervous system depends on a complex mechanism in which mitochondria are important factors due to their involvement in a number of bioenergetic and biosynthetic pathways (Thompson et al. J Inherit Metab Dis. 2020;43(1):36-50). Mitochondrial diseases are one of the most prevalent groups of hereditary neuropathies, affecting up to 1 in 5,000 adults (Gorman et al. Ann Neurol. 2015;77(5):753-759). Despite the remarkable progress achieved in the past 34 years in discovering the genetic causes of mitochondrial diseases, therapies that effectively improve the quality of life of patients remain elusive (Thompson et al. 2020; Bottani et al. Pharmaceutics. 2021;12(11)).

[0003] Gene therapy is a promising strategy for treating the tragic conditions caused by nerve cell death. In fact, encouraging results have recently been reported for spinal muscular atrophy (Aslesh et al. Cells. 2022;11(3)) and Leber hereditary optic neuropathy (Zhang et al. Curr Gene Ther 2019;19(2):134-138; Biousse et al. J Neuroophthalmol. 2021;41(3):309-315). However, this requires identifying appropriate gene candidates whose expression is impaired in neuropathy and whose administration effectively reduces / reverses nerve cell death.

[0004] Neuroglobin (encoded by the Ngb gene) was identified as a member of the globin superfamily in 2000 (Burmester et al. Nature. 2000;407(6803):520-523). This 151-amino acid protein is very abundant in the brain and is present in both neurons (Hundahl et al. Brain Res. 2010;1331:58-73) and astrocytes (Chen et al. Neurosci Lett. 2015;606:194-199). The neuroprotective role of neuroglobin has been widely documented in vitro and in vivo (Ascenzi et al. Mol Aspects Med. 2016;52:1-48; Van Acker et al. Mol Neurobiol. 2019;56(3):2101-2122). It is now well accepted that most of neuroglobin is localized in mitochondria, where the protein ensures the functional integrity of the respiratory chain (Lechauve et al. Biochim Biophys Acta. 2012;1823(12):2261-2273; Cwerman-Thibault et al. Neurobiol Dis. 2021;159:105483).

[0005] From the perspective of these properties, neuroglobin is a very interesting candidate for gene therapy. The use of neuroglobin agonists for the prevention / treatment of mitochondrial RCCI and / or RCCIII deficiency has already been shown to be effective (see the international patent application published as International Publication No. WO 2015 / 044462).

[0006] However, there is always a need to develop new and more efficient treatments for neurological disorders.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008] [Non-Patent Document 1] Thompson et al., J Inherit Metab Dis. 2020;43(1):36 - 50 [Non-Patent Document 2] Gorman et al., Ann Neurol. 2015;77(5):753 - 759 [Non-Patent Document 3] Bottani et al., Pharmaceutics. 2021;12(11) [Non-Patent Document 4] Aslesh et al., Cells. 2022;11(3) [Non-Patent Document 5] Zhang et al., Curr Gene Ther 2019;19(2):134 - 138 [Non-Patent Document 6] Biousse et al., J Neuroophthalmol. 2021;41(3):309 - 315 [Non-Patent Document 7] Burmester et al., Nature. 2000;407(6803):520 - 523 [Non-Patent Document 8] Hundahl et al., Brain Res. 2010;1331:58 - 73 [Non-Patent Document 9] Chen et al., Neurosci Lett. 2015;606:194 - 199 [Non-Patent Document 10] Ascenzi et al., Mol Aspects Med. 2016;52:1 - 48 [Non-Patent Document 11] Van Acker et al., Mol Neurobiol. 2019;56(3):2101 - 2122 [Non-Patent Document 12] Lechauve et al., Biochim Biophys Acta. 2012;1823(12):2261 - 2273 [Non-Patent Document 13] Cwerman-Thibault et al., Neurobiol Dis. 2021;159:105483

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Summary of the Invention

Means for Solving the Problems

[0009] The present invention is defined by the claims.

[0010] Using the Harlequin (Hq) mouse (a model that develops a dysregulated phenotype), the inventors confirmed that neuroglobin-based gene therapy is a very promising means for the treatment of neurological diseases. Furthermore, more interestingly, the inventors showed that very specific intravenous administration of neuroglobin significantly increased the survival rate of treated Hq mice, minimized weight loss in the mice, prevented cerebellar tissue degeneration, and maintained the branching of Purkinje cells and their dendrites, compared to other modes of administration.

[0011] Thus, the inventors showed that very specific intravenous administration of neuroglobin results in a strong neuroprotective effect, thereby being a very promising therapeutic strategy for treating neuropathy.

[0012] Accordingly, the present invention relates to neuroglobin for use in the treatment of neuropathy, administered intravenously to patients in need thereof.

[0013] According to a particular embodiment, the neuropathy is associated with a mitochondrial disease, more particularly a mitochondrial disease associated with a respiratory chain complex I (RCCI) deficiency and / or a respiratory chain complex III (RCCIII) deficiency.

[0014] In the context of the present invention, the neuropathy to be treated is preferably ataxia, more particularly hereditary ataxia, such as Friedreich's ataxia, cerebellar ataxia or spinocerebellar ataxia. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

Figure 1

Mode for Carrying Out the Invention

[0016] The inventors have shown that it is possible to obtain a particularly effective neuroprotective effect by intravenous neuroglobin administration by subjecting Harlequin (Hq) mice to neuroglobin gene therapy via various administration routes.

[0017] The Hq mouse develops vision loss and cerebellar ataxia with aging (Klein et al. Nature. 2002;419(6905):367-374). The Hq mouse exhibits a proviral insertion in the first intron of the apoptosis-inducing factor gene (Aifm1), resulting in a nearly complete deletion of the corresponding protein, apoptosis-inducing factor - Aif (Klein et al. 2002). Therefore, with aging, the Hq mouse exhibits progressive degeneration of the retina, optic nerve, cerebellum, and cortical regions, leading to incurable blindness and ataxia. This phenotype is due to severe respiratory chain complex I deficiency that causes neuronal degeneration (Cwerman-Thibault et al. Neurobiol Dis. 2021;159:105483; Vahsen et al. Embo J. 2004;23(23):4679-4689; Bouaita et al. Brain. 2012;135(Pt 1):35-52; Lechauve C, Augustin et al. Mol Ther. 2014;22(6):1096-1109).

[0018] Based on this model, the inventors have shown the following: - Intravenous administration of a vector encoding neuroglobin has no harmful effect on the body weight of Harlequin mice, which normally exhibit growth retardation. The treated Harlequin mice show a significant increase in body weight, indicating that the overall health of the mice is not deteriorated by overexpression of neuroglobin. - The mass, surface, and overall morphology of the cerebellum in Hq mice are better maintained after intravenous administration of a vector encoding neuroglobin compared to administration into the tissue (cerebellar hemisphere) by neurosurgery. - The number and density of Purkinje cells are greater when a vector encoding neuroglobin is administered intravenously compared to administration by stereotactic neurosurgery in the cerebellar hemisphere (Purkinje cell degeneration is one of the most common features of hereditary ataxia), and - Harlequin mice treated with a vector encoding neuroglobin by intravenous administration exhibit both enhanced motor ability and stronger spatial memory compared to mice treated by local administration of the vector.

[0019] Therefore, the present application shows that the specific intravenous administration of neuroglobin brings about a plurality of unexpected and surprising effects compared to other administration routes. Therefore, the present application shows that by specifically administering neuroglobin intravenously, it is possible to efficiently prevent / reverse the neurodegenerative processes observed in neuropathy.

[0020] Accordingly, the present invention relates to neuroglobin for use in the treatment of neuropathy, administered intravenously.

[0021] Neuroglobin or "NGB" is an oxygen-binding protein related to members of the globin family. Neuroglobin is encoded by the Ngb gene, which is highly conserved among other vertebrates. Neuroglobin is expressed in the central and peripheral nervous systems, where it is involved in increasing oxygen availability and providing protection in hypoxic / ischemic conditions. Human neuroglobin has the amino acid sequence accessible as reference number Q9NPG2 in the Uniprot database:

[0022]

Chemical formula

[0023] and is encoded by the human Ngb gene having the following nucleic acid sequence:

[0024]

Chemical formula

[0025] The bold regions correspond to the coding regions of the gene. The first 375 nucleotides correspond to the 5'UTR, and the last 1054 nucleotides correspond to the 3'UTR.

[0026] As described above, neuroglobin has a strong neuroprotective effect. Therefore, neuroglobin can be advantageously used to treat disorders that affect neurons, particularly neuropathies.

[0027] "Neuropathy" encompasses a well-defined group of diseases that affect the nervous system. The World Health Organization defines neuropathy as diseases of the central and peripheral nervous systems that affect the brain, spinal cord, cranial nerves, peripheral nerves, nerve roots, autonomic nervous system, neuromuscular junction, and muscles. These disorders include cerebrovascular diseases such as epilepsy, Alzheimer's disease and other dementias, stroke, migraine and other headache disorders, multiple sclerosis, Parkinson's disease, ataxia, neurological infections, brain tumors, and traumatic disorders of the nervous system due to head injuries.

[0028] As fully described in the international application published as International Publication No. WO 2015 / 044462, neuroglobin can be efficiently used for the treatment or prevention of mitochondrial diseases associated with respiratory chain complex I (RCCI) deficiency and / or respiratory chain complex III (RCCIII) deficiency.

[0029] Therefore, according to a particular embodiment, the present invention relates to the intravenous administration of neuroglobin for treating neuropathies associated with mitochondrial diseases, particularly mitochondrial disorders associated with respiratory chain complex I (RCCI) deficiency and / or respiratory chain complex III (RCCIII) deficiency.

[0030] As used herein, the term "mitochondrial disease" refers to a disorder in which a defect in mitochondrial respiratory chain activity contributes to the pathophysiology of such a disorder in a mammal. Mitochondrial disorders can be caused by acquired or inherited mutations in mitochondrial DNA (mtDNA) or nuclear genes encoding mitochondrial components. Mitochondrial disorders can also be the result of acquired mitochondrial dysfunction due to the harmful effects of drugs, infections, or other (environmental, etc.) causes.

[0031] "Mitochondrial diseases associated with respiratory chain complex I deficiency" or "mitochondrial diseases associated with RCCI deficiency" refer to mitochondrial diseases in which dysregulation, reduction, or disappearance of RCCI complex activity is observed. The term "mitochondrial diseases associated with RCCI deficiency" also refers to mitochondrial diseases that are induced by RCCI deficiency or in which RCCI deficiency increases the risk of developing such mitochondrial diseases. Examples of mitochondrial diseases associated with RCCI deficiency may include Leber's hereditary optic neuropathy (LHON), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), MERRF (myoclonic epilepsy with ragged red fibers), Leigh syndrome, LS (subacute necrotizing encephalomyelopathy is a progressive neurological disease defined by specific neuropathological characteristics with brainstem and basal ganglia lesions), leukodystrophy (white matter brain disease), cardiomyopathy, liver damage with tubular disorders, and fatal infantile multisystem disorder (for confirmation, see Scheffler J Inherit Metab Dis, 2014 DOI 10.1007 / s10545-014-9768-6; Papa and De Rasmo, Trends in Molecular Medicine, 2013, Vol. 19, No. 1: pp. 61-69 and the MITOMAP website).

[0032] As used herein, the term "respiratory chain complex I" or "RCCI" refers to a protein complex located in the inner mitochondrial membrane that forms part of the mitochondrial respiratory chain. RCCI contains approximately 45 various polypeptide subunits including NADH dehydrogenase (ubiquinone), flavin mononucleotide, and several different iron-sulfur clusters containing non-heme iron. The iron undergoes redox between Fe(II) and Fe(III) and catalyzes proton transport associated with the oxidation of NADH by ubiquinone. RCCI is also called NADH:quinone oxidoreductase (E.C. 1.6.5.3). RCCI function or RCCI activity can be measured by: (1) a very accurate and powerful spectrophotometric assay designed for very small biological samples (Benit et al., Clinica Chimica Acta 374 (2006) pages 81-86), (2) biochemical analysis of respiratory chain (oxidative phosphorylation) complexes using blue native (BN) polyacrylamide gel electrophoresis (PAGE) after extraction of enriched mitochondrial membranes from tissues or cells, both the in-gel activity of respiratory chain complexes and the protein composition for each of them could be analyzed (Calvaruso et al., Methods 46 (2008) pages 280-286).

[0033] As used herein, the term "mitochondrial disease associated with respiratory chain complex III deficiency" or "mitochondrial disease associated with RCCIII deficiency" refers to a mitochondrial disease in which dysregulation, reduction or disappearance of RCCIII complex activity is observed. The term "mitochondrial disease associated with RCCIII deficiency" also refers to a mitochondrial disease that is induced by RCCIII deficiency or in which RCCIII deficiency increases the risk of developing such a mitochondrial disease. Examples of mitochondrial diseases associated with RCCIII deficiency may include encephalopathy, liver failure and tubular disorders, Leigh syndrome, GRACILE and GRACILE-like syndromes (growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death), Björnstad syndrome (sensorineural deafness and twisted hair), hypoglycemia, lactic acidosis, LHON, progressive exercise intolerance, degeneration of cerebellar neurons and progressive psychiatric syndromes (see, for confirmation, Benit et al., Biochimica et Biophysica Acta 1793 (2009) pages 181-185; http: / / www.mitomap.org / MITOMAP).

[0034] As used herein, the term "respiratory chain complex III" or "RCCIII" refers to a protein complex located in the inner mitochondrial membrane that forms part of the mitochondrial respiratory chain. RCCIII contains approximately 11 polypeptide subunits including four redox centers: cytochrome b / b6, cytochrome c1 and a 2Fe-2S cluster. The RCCIII function is to catalyze the oxidation of ubiquinol by oxidized cytochrome c1. RCCIII is also called the bc1 complex; ubiquinol cytochrome c reductase (EC 1.10.2.2). The RCCIII function or RCCIII activity can be measured by: (1) a very accurate and powerful spectrophotometric assay designed for very small biological samples (Benit et al., Clinica Chimica Acta 374 (2006) 81-86); (2) biochemical analysis of respiratory chain (oxidative phosphorylation) complexes using blue native (BN) polyacrylamide gel electrophoresis (PAGE) after extraction of enriched mitochondrial membranes from tissues or cells, both the in-gel activity of the respiratory chain complexes and the protein composition for each of them could be analyzed (Calvaruso et al., Methods 46 (2008) 280-286).

[0035] Intravenous administration of neuroglobin prevents neuronal degradation. Thus, this can be used in the treatment of all neurological disorders, particularly those associated with neurodegeneration, i.e., neurodegenerative disorders. Neurodegenerative disorders include Alzheimer's disease, ataxia, Huntington's disease, Parkinson's disease, motor neuron disease, leukodystrophy or multiple system atrophy. In contrast to acute neurological disorders such as stroke, neurodegenerative disorders are progressive disorders that induce long-term and progressive neuronal damage. These progress over several years and are associated with specific mechanisms that lead to neuronal death and permanent brain damage over the course of their progression.

[0036] According to a preferred embodiment, the present invention relates to the intravenous administration of neuroglobin for the treatment of ataxia, more particularly hereditary ataxia such as Friedreich's ataxia, cerebellar ataxia or spinocerebellar ataxia.

[0037] "Ataxia" refers to a group of neurological disorders that affect coordination, balance, and speech. These usually result from damage in the cerebellum. Ataxia can cause difficulties with walking and balance, hand coordination, speech and swallowing, and eye movements.

[0038] In contrast to the related group of acquired neurological disorders due to accidents, trauma, or other external factors, "hereditary ataxia" is genetic. Hereditary ataxia is characterized by degenerative processes in the brain and spinal cord that often lead to abnormal gait associated with poor eye-hand coordination and dysarthria (see Perlman S or Bird T. "Hereditary ataxia overview." GeneReviews® [Internet] (2019) for confirmation). Hereditary ataxia includes, for example, Friedreich's ataxia, cerebellar ataxia, or spinocerebellar ataxia (autosomal dominant ataxia).

[0039] In the context of the present invention, neuroglobin is administered particularly intravenously. Those skilled in the art are familiar with several techniques for administering neuroglobin intravenously in the context of the present invention. Techniques useful in the context of the present invention are used, for example, in the clinical trials referenced as ClinicalTrials identifiers: NCT02122952, NCT03461289, NCT03952637, NCT03362502, NCT05092685, NCT04998396, NCT03955679 or NCT04040049. The corresponding composition may be presented as an ampoule in unit dose form during pre-filled syringe small volume injection, or as a multi-dose container with added preservatives. The composition may be in the form of a suspension, solution, or emulsion in an oily or aqueous medium, for example a solution in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents or media include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate), and may contain formulating agents such as preservatives, wetting agents, emulsifying or suspending agents, stabilizers and / or dispersing agents. Alternatively, the active ingredient may be in the form of a powder obtained by lyophilization from a solution for use in the constitution prior to use with sterile isolated, or preferably, sterile pyrogen-free solid in a suitable medium, such as sterile pyrogen-free water.

[0040] In the context of the present invention, neuroglobin may be administered as a polypeptide by administering the mature NGB protein. However, neuroglobin is preferably administered as gene therapy. Gene therapy can be carried out by replenishing target cells with functional neuroglobin. The production of suitable gene products can be achieved by using recombinant techniques. For example, a suitable vector may be inserted into a host cell and expressed in that cell. Gene therapy is particularly advantageous for the treatment of disorders such as neurodegenerative disorders since it allows for long-term and persistent release of the target gene in target cells. Gene therapy is usually achieved by administering to a patient a polynucleotide encoding the target gene.

[0041] Accordingly, the present invention relates to a polynucleotide encoding neuroglobin for use in the treatment of neuropathy. Therefore, a method for treating neuropathy is also disclosed herein, which includes administering to a patient in need thereof a polynucleotide encoding neuroglobin. The polynucleotide encoding neuroglobin is administered in a therapeutically effective amount.

[0042] In the context of the present invention, a "patient" or "subject" is a mammal (such as a dog, cat, pig, rodent or primate). In a particular embodiment, the patient is a human.

[0043] Generally, the polynucleotide is included in an expression cassette.

[0044] An "expression cassette" refers to a linear or circular nucleic acid molecule. This expression cassette also refers to DNA and RNA sequences that enable the production of a functional nucleotide sequence in a suitable host cell. Generally, an expression cassette includes a polynucleotide encoding neuroglobin operably linked to at least one transcriptional regulatory sequence. Generally, an expression cassette includes a polynucleotide encoding a neuroglobin protein, and the polynucleotide is operably linked to at least one transcriptional regulatory sequence for the expression of the neuroglobin protein in a target cell, and the at least one transcriptional regulatory sequence is a 3'UTR and / or 5'UTR NGB sequence. Generally, when the patient is human, the polynucleotide includes the coding region of the human Ngb gene shown in SEQ ID NO: 2. However, as will be understood by those skilled in the art, this polynucleotide may be modified to optimize its expression or the activity of its transcript. Therefore, such optimized sequence variants can also be used in the context of the present invention. The corresponding mRNA sequences can also be used in the context of the present invention.

[0045] The expression cassette may also include sequences necessary for normal translation of the nucleotide sequence of interest. In addition, the expression cassette may contain a selectable marker gene. Typically, the cassette includes, in the 5' to 3' direction of transcription, a transcription and translation initiation region, a polynucleotide encoding the NGB protein, and a functional transcription and translation termination region in mammalian cells.

[0046] The expression cassette may also include multiple cloning sites. In addition to the components mentioned above, the expression cassette of the present invention may include components necessary for homologous recombination.

[0047] The term "operably linked" refers to the functional relationship of a nucleic acid to another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, the operable linkage of DNA to a transcription control element refers to the physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0048] As used herein, the terms "transcriptional regulatory sequence", "transcription regulatory sequence", or "regulatory sequence" refer to a nucleotide sequence that affects the transcription, RNA processing or stability, or translation of an associated (or functionally linked) nucleotide sequence to be transcribed. Transcriptional regulatory sequences can have various localizations relative to the nucleotide sequence to be transcribed. Transcriptional regulatory sequences can be located upstream (5' non-coding sequence), within the sequence, or downstream (3' non-coding sequence) of the sequence to be transcribed (e.g., a polynucleotide encoding an NGB protein). Transcriptional regulatory nucleotide sequences can be selected from the group consisting of enhancers, promoters, translation leader sequences, introns, 5'-untranslated sequences (5'UTR), 3'-untranslated sequences (3'UTR), and polyadenylation signal sequences. These can include natural and synthetic sequences, as well as sequences that can be combinations of synthetic and natural sequences. As indicated above, the term "transcriptional regulatory sequence" is not limited to a promoter. However, the transcriptional regulatory sequences of the present invention can include at least one promoter sequence (e.g., a sequence localized upstream of the transcription start point of a gene that can induce transcription of a downstream sequence), and / or at least one 3'UTR and / or one 5'UTR. According to a particular embodiment, the transcriptional regulatory nucleotide sequences of the present invention include the promoter sequence of the Ngb gene and / or the native 3'UTR of the Ngb gene and / or the native 5'UTR of the Ngb gene. Furthermore, fragments of the Ngb 3'UTR and / or Ngb 5'UTR are also available. According to another embodiment, the promoter is the promoter of another gene, for example a gene that is strongly expressed in the brain. Advantageously, the promoter of the PGK1 gene is used. Thus, the transcriptional regulatory sequences of the present invention can include the promoter sequence of the PGK1 gene and / or the native 3'UTR of the Ngb gene and / or the native 5'UTR of the Ngb gene. The presence of the native 3'UTR and 5'UTR of the Ngb gene advantageously ensures mRNA stability, translation ability, and efficient delivery of Ngb into the mitochondria.

[0049] As used herein, the term "promoter" or "promoter sequence" refers to a DNA sequence in a gene that is typically upstream (5') of its coding sequence and controls the transcription of a coding sequence, such as a polynucleotide encoding a neuroglobin protein, by bringing about recognition by RNA polymerase and other factors necessary for normal transcription. For example, the promoter may be an Ngb promoter, a variant or fragment thereof, preferably a human Ngb promoter. The promoter may contain a DNA sequence involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental states. Usually, the Ngb promoter may contain two GC boxes to which Sp1 and Sp3 factors bind. According to the present invention, the promoter sequence may also contain enhancer elements. An "enhancer" is a DNA sequence capable of stimulating promoter activity. This may be an inherent element of the promoter or a heterologous element inserted to enhance the level and / or tissue specificity of the promoter. Usually, the promoter sequence of the present invention is a ubiquitous promoter, a tissue-specific promoter or an inducible promoter. A "ubiquitous promoter" refers to a promoter that always directs gene expression in all tissues. The ubiquitous promoter may be a eukaryotic promoter or a viral promoter. In one embodiment, the promoter sequence is a eukaryotic promoter selected from the group consisting of the chicken β-actin promoter (CBA), the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken β-actin promoter), and the human phosphoglycerate kinase 1 (PGK) promoter. According to another embodiment, the promoter sequence is a viral promoter such as the human cytomegalovirus (CMV) promoter. A "tissue-specific promoter" is a promoter that directs gene expression in only a nearly specific tissue, such as a retina-specific promoter or a central nervous system-specific promoter. The promoter may be selected from RGC-specific promoters. Usually, the promoter is an "inducible promoter", i.e., a promoter that directs gene expression in response to external stimuli such as light, heat shock, and chemicals.

[0050] The "untranslated region" or "UTR" refers to either of two regions immediately adjacent to the coding sequence in the mature mRNA strand. When it is seen on the 5'-side, it is called the 5'UTR (or 5'-untranslated region), or when it is seen on the 3'-side, it is called the 3'UTR (or trailer sequence). As used herein, the "3'UTR neuroglobin sequence" refers to the sequence of the 3'UTR of the Ngb gene, e.g., the human Ngb 3'UTR, etc. The human 3'UTR of the Ngb gene is the 3'-end of the sequence of SEQ ID NO: 2 and corresponds to the last 1054 nucleotides of SEQ ID NO: 2. As used herein, the term "5'UTR neuroglobin sequence" refers to the sequence of the 5'UTR of the Ngb gene. The human 5'UTR of the Ngb gene is the 5'-end of the sequence of SEQ ID NO: 2 and corresponds to the first 375 nucleotides of SEQ ID NO: 2. According to a particular embodiment, the polynucleotide encoding neuroglobin comprises the neuroglobin gene coding region and the neuroglobin gene 5'UTR and / or 3'UTR.

[0051] In one embodiment, the expression cassette is contained in an expression vector. Accordingly, the present invention also relates to a polynucleotide encoding neuroglobin for use in the treatment of neuropathy, wherein said polynucleotide is contained in an expression vector.

[0052] The term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid to which a vector sequence is ligated into a cell. The term "expression vector" includes any vector containing a gene construct or expression cassette in a form suitable for expression by a cell. An "expression vector" may be any recombinant vector capable of expressing an NGB protein or a fragment thereof. More specifically, the expression vectors used may be bacterial plasmids, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, such as adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, replicable adenoviral vectors, replication-deficient adenoviral vectors, and gutless adenoviral vectors, herpesvirus vectors, baculoviruses, those blinked as SV40 virus, vaccinia virus, fox pox virus, pseudorabies virus. AAV and lentiviral vectors have emerged as optimal vectors for gene transfer to the central nervous system because they mediate efficient long-term gene expression without obvious toxicity. Furthermore, several clinical trials have shown that direct injection of AAV2 vectors into the brain parenchyma in humans is well tolerated (Bowers et al., Human Molecular Genetics, 2011, Vol. 20, Review Issue 1, pp. R28-R41). The expression cassette may be inserted into the expression vector by methods well known in the art.

[0053] The expression vector may contain a reporter gene. Examples of reporter genes include luciferase, (green / red) fluorescent proteins and their variants, such as eGFP (enhanced green fluorescent protein), hrGFP (humanized recombinant green fluorescent protein), RFP (red fluorescent protein, such as DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), β-galactosidase or chloramphenicol acetyltransferase, and the like. These sequences are selected according to the host cell to be performed.

[0054] According to one embodiment of the present invention, the expression vector is a viral vector. The viral vector of the present invention may be derived from a retrovirus, herpes simplex virus, adenovirus or AAV.

[0055] According to a preferred embodiment, the expression vector is preferably an adeno-associated virus (AAV) vector, preferably an AAV2 vector.

[0056] In one embodiment, the expression vector of the present invention is an AAV vector comprising AAV 5' terminal inverted repeat (ITR5') and 3' terminal inverted repeat (ITR3') sequences at the 5' and 3' ends of the expression cassette, respectively.

[0057] The expression "terminal inverted repeat sequence" or "ITR" means a 145 base pair (bp) terminal inverted repeat sequence of a palindrome in contact with the 5' and 3' AAV vectors. The ITR sequence is essential for the integration, replication and packaging of the viral vector. The AAV ITR can be modified using standard molecular biology techniques. Therefore, the AAV ITR used in the vector of the present invention does not necessarily have a wild-type nucleotide sequence and may be changed, for example, by nucleotide insertion, deletion or substitution. In fact, ITR5' and ITR3' are not necessarily identical but are functional. A "functional ITR sequence" means an ITR sequence capable of vector replication and packaging. In addition, the AAV ITR may be derived from any of several AAV serotypes including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV5, AAV8 or AAV-9.

[0058] The expression cassette may be included in the viral particles. Usually, the expression cassette is inserted into an expression vector, which is then packaged or capsid - formed in the viral particles. As used herein, "viral particle" refers to a packaged or capsid - formed viral vector capable of binding to and entering into the interior of a host cell. Techniques for isolating the viral particles of the present invention from host cell components and, ultimately, other types of viruses that may be present in the host cell (e.g., helper viruses) are known to those of skill in the art and include, for example, centrifugation and affinity chromatography. Usually, the viral particles may be AAV particles.

[0059] "Adeno - associated virus" or "AAV" belongs to the family Parvoviridae and the genus Dependovirus. Wild - type AAV is a low - integrating virus, but it is not lytic and is non - pathogenic to humans. Adeno - associated virus infects a variety of mitotic and quiescent cells, but its replication depends on a helper virus, such as an adenovirus or a herpesvirus.

[0060] As used herein, the term "rAAV" refers to a recombinant AAV nucleic acid molecule containing several AAV sequences, usually at least the ITRs and some foreign or exogenous (i.e., non - AAV) DNA, such as the NGB nucleic acid sequence of the present invention.

[0061] As used herein, the term "serotype" refers to an AAV that is identified by and distinguished from other AAVs based on its capsid protein reactivity with a defined antiserum. For example, AAV serotype 2,9 (AAV2 / 9) is used to refer to an AAV composed of the AAV2 Rep regulatory protein and the AAV9 cap gene. The viral particle serotype determines its affinity. In the context of the present invention, AAV2 / 9 is particularly advantageous due to its marked affinity for neurons (see, for confirmation, Huang et al., Life Sciences 270 (2021): 119142 or Abulimiti et al., Mechanisms of Ageing and Development 199 (2021): 111549). Thus, according to a particular embodiment, a polynucleotide encoding neuroglobin is included in an AAV2 / 9 vector. The AAV2 / 9 vector can transduce cells from several tissues and, in particular, cells from the central nervous system, whose functional integrity is impaired by the progressive and irreversible loss of neurons in patients suffering from neurodegenerative diseases. The AAV2 / 9 vector can naturally bypass the blood-brain barrier (BBB), thereby enabling widespread and long-term ngb expression after a single intravenous injection.

[0062] Typically, the capsid protein of the viral particle may contain at least one tyrosine residue mutated to phenylalanine. For example, the capsid protein may be mutated by substitution of at least three tyrosine residues with phenylalanine residues. The mutation of the capsid protein modifies the viral affinity or increases the transduction efficiency of the rAAV vector and reduces host cell damage. Advantageously, tyrosine 444 of the capsid is substituted with a phenylalanine residue. Typically, the vector is AAV-2 Y444F.

[0063] In another embodiment, the expression vector may be a lentiviral vector that contains sufficient lentiviral genetic information to enable packaging of the RNA genome into viral particles capable of infecting a host cell (e.g., a target cell) in the presence of packaging components.

[0064] As used herein, the term "lentiviral vector" refers to a vector derived from a lentivirus (i.e., sharing nucleotide sequences unique to lentiviruses). The term "lentiviral vector" also refers to a modified lentivirus having a modified proviral RNA genome that includes the NGB polynucleotide sequence. According to the present invention, the lentiviral vector is derived from the human immunodeficiency virus (HIV).

[0065] Alternatively, one method for delivering polynucleotides across cell membranes in vivo may involve direct application of high concentrations of free or naked polynucleotides (usually mRNA or DNA). By "naked DNA (or RNA)" is intended a DNA (RNA) molecule that is not pre-complexed with other chemical moieties. Uptake of naked DNA by animal cells can be increased by co-administering an additive and the nucleic acid to the cells. Such additives are reagents that enhance or increase the penetration of DNA across the cell membrane and thus enhance or increase the delivery of therapeutic agents to the cells. Various additives are described in the art, such as surfactants, e.g., Triton X-100, sodium dodecyl sulfate, Tween 20, and Tween 80, which act by disrupting the osmotic pressure near the cell membrane; bacterial toxins, e.g., streptolysin O, cholera toxin, and recombinant modified labile toxin of Escherichia coli (E. coli); and polysaccharides, e.g., glucose, sucrose, fructose, or maltose, such as surfactants selected from the group consisting of.

[0066] The present invention is now illustrated by the following examples. These examples are not intended to limit the scope of the invention.

[0067] References to methods of treatment or surgical treatment should be construed as references to the compounds of the invention for use in those methods.

Examples

[0068] (Example 1) Comparison of two routes of AAV2 / 9 vector administration: local (neurosurgery in the cerebellar hemisphere) vs. systemic (injection into the vein of the posterior orbit) Method Mice: C57BL / 6J and Harlequin (Hq) mice derived from the B6CBACaAw-J / A-Pdc8Hq / J strain. These have a natural mutation in the Aifm1 gene, which is composed of a 9 kb homologous-directed proviral insertion in intron 1 of the gene (Jackson Laboratory strain No. 000501). This insertion leads to a decrease in gene expression, associated with an approximately 90% reduction in Aifm1 messenger RNA and protein levels in Hq mutant mice compared to wild-type control levels (Klein et al. 2002). The Hq mutation is a proviral insertion in the Aifm1 gene, causing an approximately 90% reduction in Aif expression. The Hq mouse strain exhibits the main characteristics of human neurodegenerative diseases due to RCCI deficiency, such as degeneration in the cerebellum, retina, optic nerve, thalamus, striatum, and cortical regions. All hemizygous (Hq / Y) male mice used in this study had a mixed genetic background and were F2 - F4 mice bred from the original (Hq / X female mice and wild-type male mice) transported from the Jackson Laboratory. Only hemizygous (Hq / Y) mice and their male littermates were evaluated and targeted for gene therapy. The mice were housed in a temperature-controlled environment in a pathogen-free barrier facility with 2 - 4 animals per cage, on a 12-hour light / dark cycle, and with free access to food and water. The animal facility (PHENO-IC Mice) is located at the Paris Brain Institute, ICM (see the ICM website for confirmation). The study was conducted in accordance with European Community Council Directive 2010 / 63 / UR regarding the protection of animals used for scientific purposes. This scientific project has been approved by the internal scientific committees of the Brain Institute (#P128R) and the French Ministry of Research (#2410) regarding the rules for the care and use of animals in research and the use of genetically modified organisms (class 2). It has also been approved by the ethics committees of the University of Paris and INSERM (APAFIS#9423 - 2017032721505008).

[0069] Generation of single-stranded adeno-associated virus vectors: AAV2 / 9-Aifm1 and AAV2 / 9-Ngb vectors were obtained with respect to serotype 2 (Cwerman-Thibault et al.; Bouaita et al. Brain. 2012; 135(Pt1): 35-52; Lechauve et al., 2014). Briefly, the mouse sequences were inserted using the pAAV-IRES-hrGFP vector (Agilent Technologies). The AAV2 / 9-Ngb vector contains the open reading frame (ORF) (453 bp), 5' untranslated region (UTR) (279 bp), and 3' UTR (895 bp) of mouse Ngb mRNA variant 2 (NM022414.2). The AAV2 / 9-Aifm1 vector contains the 5' UTR (87 bp), full ORF (1836 bp), and 176-bp full-length 3' UTR of mouse Aifm1 mRNA (NM012019). The presence of the full UTR sequences in each construct ensures mRNA stability and translational ability (Weis et al., Biochim Biophys Acta. 2013; 1833(2): 260-273; Bae et al., Int J Mol Sci. 2020; 21(10)). The expression cassette flanked by two inverted terminal repeats (ITRs) was packaged into the AAV9 capsid to ensure high-yield neuronal transduction (Huda et al., Mol Ther Methods Clin Dev. 2014; 1: 14032). AAV2 / 9-GFP from Agilent contains the 3' UTR from the mouse Aifm1 gene, and no transgene was inserted into the multiple cloning site (MCS) of the original vector. Thus, this was used as a negative control. A schematic diagram of each vector is illustrated in Figure 1. The vectors were generated by the Translational Vector Core of INSERM UMR1089 Research Unit in Nantes, France.

[0070] Stereotactic neurosurgery: On the day of surgery, mice are under volatile anesthesia (3% isoflurane for induction, then 2% in the mask). Each mouse is placed in a stereotactic frame and maintained on a heating mat. An incision is made in the scalp, and with a mini drill (carbon steel burr, 0.5 mm diameter), two small-diameter holes are drilled in the skull on each side of the cerebellum (6 - 6.5 mm caudal from bregma and 1.75 - 2 mm lateral to the midline; coordinates were specified using the Atlas of Franklin and Paxinos). The vector stock is diluted with 0.09% NaCl solution to a final concentration of 5x10 9 or 7x10 9 vector genomes / μL. At each position corresponding to the right and left cerebellar hemispheres, 2 μL is injected at a depth of 1.1 mm ventral from the dura. The injection rate is 0.35 μL / min, and the vector is administered with a 10 μL-Hamilton microsyringe with a 33-gauge needle. To minimize backflow of the vector solution, the microsyringe is left in place for 3 minutes and then moved upward by 0.3 mm every 45 seconds before complete withdrawal. Each animal receives approximately 1.0 or 1.4x10 10 vector genomes (VG). Since the mice weighed approximately 25 g at the time of treatment, the dose used was approximately 9.5x10 11 per kg. Next, the surgical site is aseptically closed with non-absorbable surgical sutures. Immediately after this procedure, the mice are administered analgesics (buprenorphine 0.05 - 0.1 mg / kg) and 400 μL of physiological serum subcutaneously. Analgesic treatment is given every 8 - 12 hours for 5 days after neurosurgery. The stereotactic neurosurgery protocol was based on the publication by Lin and colleagues (Gene. 2015;571(1):81 - 90). Stereotactic neurosurgery is classified as being of moderate severity, and thus all postoperative care is carried out to minimize pain after the intervention and preserve "animal welfare". To appropriately manage the pain caused by the surgery, postoperative evaluations (twice a day) were very thorough over a period of one week.

[0071] Posterior orbital cavity injection Various steps are described below, according to two publications from 2011 (Yardeni et al., LabAnim (NY) 2011;40(5):155-160) and 2021 (Seldeen et al., Mech Ageing Dev. 2019;180:49-62). 1) The vector is prepared in a microbiological safety station; the injection volume varies from 50 μL to 150 μL depending on the vector title. 2) For vector administration, an insulin syringe with a 29-gauge needle is used. 3) The mice are 24 - 30 days old, and the body weight of the mice varies from 12 - 22 g. 4) The final amount of vector per injection is approximately 2.6x10 13 VG, corresponding to approximately 1.53x10 11 VG / kg (approximately 17 times more than that of stereotactic surgery). 5) The animals are placed in a plexiglass box with 3% isoflurane circulating. 6) When the animal is completely asleep (slow heartbeat, no response to stimuli), it is removed from the box. 7) To maintain the body temperature of the sleeping animal, the mice are placed on a heating mat set at 37 °C. 8) Next, the animal is slightly restrained, and the eyeball is slightly protruded from the eye socket before inserting the needle to avoid eye damage. 9) The needle is inserted into the retro-bulbar sinus at a 45° angle until the experimenter feels a slight resistance indicating that the needle tip has reached the bony eye socket. 10) At this point, the required volume of the vector solution is injected gently and smoothly. 11) The needle is left in place for 3 - 5 seconds and then gently withdrawn to prevent eye injury. Absence of solution leakage and bleeding indicates proper injection. 12) To stop possible bleeding, a wet cloth is applied with light pressure to the injected eye for several seconds. 13) The injection site is inspected for swelling or other visible trauma to the eye. 14) Finally, the mice are returned to their home cages.

[0072] Behavioral evaluation of mice: Control and Hq mice were evaluated together with age-matched untreated counterparts. When stereotactic surgery was selected as the route of vector administration, the cognitive and motor abilities of the mice were examined before gene therapy and 6 months later. When retro-orbital injection was performed, the evaluation was carried out 1 - 2 months after injection and immediately before euthanasia, i.e., 5 months after vector administration. For motor skills, muscle tone, and coordination, mice were judged by subjecting them to the Grip test, Rotarod, and Open Field (Seldeen et al., 2019; Deacon, J Vis Exp. 2013; (75):e2609; Kraeuter et al., Methods Mol Biol. 2019; 1916:99 - 103). The learning and memory abilities were evaluated by using the Novel Object Recognition test (Miedel et al. J Vis Exp. 2017; (123)) and the Y-maze, Forced Alternation (Kraeuter et al. Methods Mol Biol. 2019; 1916:105 - 111). Finally, after subjecting the mice to the Open Field (Kraeuter et al., 2019) and Tail Suspension test (De Sousa et al. Naunyn Schmiedebergs Arch Pharmacol. 2018; 391(8):803 - 817), the mouse response to situations that cause acute stress or anxiety was estimated.

[0073] Morphological and Functional Studies on the Processed Mouse-derived Cerebellum: After euthanasia, the mice were perfused intracardially with a 4% paraformaldehyde solution, the cerebellum was collected, and immunohistochemistry (cell number estimation, tissue size, transduction yield) was performed. Tissues were also collected and maintained at -80 °C and used for biochemical studies (Krebs cycle, respiratory chain) or analysis of the steady-state levels of mRNA and protein by qPCR and Western blot, respectively, as performed for retinal studies (Cwerman-Thibault et al., 2021). Transmission electron microscopy analysis (Gilbert et al. Elife. 2021;10) and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS / MS) analysis (Millan et al., Antioxidants (Basel) 2018;7(12)) were performed on the cerebella from untreated and processed mice.

[0074] Results Survival Rates of Animals Receiving Local or Systemic Vector Administration: Comparing stereotactic surgery within the cerebellar hemisphere and injection into the retro-orbital sinus with respect to mouse survival rates, it is clearly considered that the ROI is better tolerated. For example, Hq mice treated with AAV2 / 9-GFP, AAV2 / 9-Aifm1, or AAV2 / 9-Ngb showed percentages of death before the end of the experiment of 28%, 6.7%, and 9.5% respectively due to the ROI, while after stereotactic surgery, these were 37.4%, 23.9%, and 21.6%.

[0075] The difference in the degree of mortality between Hq and control mice that underwent stereotactic surgery may be due to the extremely low pain tolerance of Hq mice. Therefore, the beneficial advantages of the ROI protocol were shown. Indeed, the ROI protocol is much less invasive, so fewer animals died after vector administration in both the control and Hq groups of mice.

[0076] Body Weight Differences in Mice Receiving Intravenous or Local Delivery of Gene Therapy Vectors: The body weight and cerebellar mass were compared in control and Hq mice that received local or intravenous administration of AAV2 / 9-GFP, AAV2 / 9-Aifm1, or AAV2 / 9-Ngb. Mice that received an ROI were euthanized 5 months after treatment, and mice that received local administration of the vector were euthanized 6 months after vector administration. Overall, in the treatment of control mice, regardless of the administered vector and the route of administration used, their body weight or cerebellar mass did not change. Thus, intravenous administration of AAV2 / 9-Ngb, which leads to systemic overexpression of Aifm1 or Ngb, had no harmful effects on the health status of the mice when considering body weight and cerebellar mass.

[0077] No significant difference in body weight was measured for either route of administration, except for an 11.5% increase (P = 0.0047) in Hq mice treated with AAV2 / 9-Ngb by ROI compared to Hq mice treated with AAV2 / 9-Ngb by stereotactic surgery. Furthermore, when Hq mice treated with AAV2 / 9-GFP were compared to Hq mice treated with AAV2 / 9-Ngb, a significant difference was observed only for intravenous injection. Indeed, Hq mice that received an ROI with the AAV2 / 9-Ngb vector were 16% heavier than their counterparts treated with AAV2 / 9-GFP (P = 0.0002).

[0078] The expected weight gain of the mice from birth to maturity was evaluated before treatment at 2 months of age (surgery) or 1 month of age (ROI) and immediately before euthanasia: 5 months after (ROI) or 6 months after (surgery).

[0079] An increase in body weight was evident only in Hq mice when the AAV2 / 9-Ngb vector was administered by retro-orbital injection (39%) (P < 0.0001), while Hq mice that received cerebellar administration of AAV2 / 9-Ngb had only a 5% increase in body weight after 6 months (P = 0.967).

[0080] Therefore, systemic administration of AAV2 / 9-Ngb not only did not induce harmful effects on the health of Hq mice, but rather, considering body weight, it can be said that overexpression of Ngb was beneficial for the overall development of Hq mice, which are known to exhibit significant growth retardation (Benit et al. PLoS One. 2008;3(9):e3208).

[0081] Cerebellar mass and size differences in mice receiving intravenous or local delivery of gene therapy vectors: Regarding the route of vector administration, when comparing the cerebellar mass of Hq mice at the time of euthanasia, it was found that GFP-treated Hq mice had the smallest mass compared to the tissues of mice overexpressing either Aifm1 or Ngb (data not shown). Aifm1-treated Hq mice showed a 52% or 49% greater mass of the cerebellum compared to GFP-treated Hq mice for local or intravenous injection, respectively. The difference was nearly significant for surgery (P = 0.083) and was significant when comparing the two groups of mice receiving retro-orbital injections (P = 0.0465).

[0082] Interestingly, the cerebellar mass of Ngb-treated Hq mice increased by 36% and 53% compared to the cerebellar mass of GFP-treated Hq mice for local and intravenous vector administration, respectively. The difference did not reach significance for local administration (P = 0.41), but was significant for ROI (P = 0.049).

[0083] After evaluation of cerebellar sections subjected to immunohistochemistry, the total surface area of the cerebellum from Hq mice treated with any one of the vectors was estimated. For this purpose, cerebellar sections were scanned with a NanoZoomer Digital Pathology 2.0 HT scanner, and digital images reconstructed from up to six independent sections corresponding to the central zone (vermis) of the tissue were evaluated. The total surface area of the tissue per section was estimated using the ruler tool of NDP viewer software, in mm 2It was represented by. The average on the surface in control mice treated with the AAV2 / 9-GFP vector by ROI for 4 mice was 7.79 ± 0.25, and this number is very similar to the number seen in 4 control mice treated with the AAV2 / 9-Aifm1 vector surgically: 7.99 ± 0.35. Clearly, in Hq mice, especially since a reduction of about 64% was observed, regardless of the administration route, the size of the cerebellum in GFP-treated Hq mice is significantly smaller than that of one of the control mice. The data collected for Hq mice that received gene therapy were compared for the administration route and the injected vector. When AAV2 / 9-Aifm1 (43.7%) or AAV2 / 9-Ngb (48.5%) was administered by ROI, an increase in the cerebellar area of Hq mice was observed compared to their counterparts injected with AAV2 / 9-GFP, and the difference was significant for Hq mice treated with AAV2 / 9-Ngb (P = 0.042), but this does not apply to Hq mice treated with AAV2 / 9-Aifm1 (P = 0.08).

[0084] In conclusion, the data obtained suggest that treatment with AAV2 / 9-Ngb by intravenous injection led to less severe weight loss and a more mild reduction in the mass of their cerebellum in Hq mice. This may reflect better maintenance of the overall health of Hq mice when Ngb is overexpressed in various tissues. Furthermore, it can be hypothesized that protection against Purkinje cell degeneration increases when the AAV2 / 9-Aifm1 or AAV2 / 9-Ngb vector is injected into the retro-orbital cavity, despite the tissue size remaining smaller than that of the control mice. Indeed, both the mass and surface of the cerebellum from Hq treated with ROI by AAV2 / 2-Ngb were significantly larger than their counterparts treated with AAV2 / 2-GFP by ROI.

[0085] Morphological evaluation of the cerebellum from Harlequin and control mice that received retro-orbital injection: After retro-orbital injection, control and Hq mice administered with AAV2 / 9-Aifm1, AAV2 / 9-Ngb or AAV2 / 9-GFP were subjected to behavioral assessment 5 months later and euthanized upon completion of these tests. The cerebellum was dissected and 40-μm sections were obtained using a cryomicrotome. Next, the floating sections were used for immunohistochemistry with antibodies against GFP, β3-tubulin, Aif or Ngb combined with an antibody against calbindin D-28k, a reliable marker for Purkinje cells (Kim et al., Korean J Physiol Pharmacol. 2009;13(5):373-378; Orduz et al., Front Cell Neurosci. 2014;8:364). The fluorescence intensity of GFP, Aif or Ngb labeling was greater in treated control or Hq mice. Signals corresponding to antibodies recognizing proteins synthesized from each vector were strong in all cell layers, indicating that a population of neuronal groups in the tissue was efficiently transduced. From these results, it is deduced that the overall cerebellar structure of Aifm1- and Ngb-treated Hq mice is better maintained compared to GFP-treated Hq mice when the vector is delivered by intravenous injection. Reconstructed images of tissues from control and Hq mice that underwent stereotactic surgery were compared with images obtained from their counterparts that received intravenous injection. No change in the overall cerebellar morphology was observed when ROI was performed on control mice using any vector. Therefore, delivery of 16-fold more vector by intravenous injection did not lead to harmful results in the cerebellum of these mice. This also holds true for Hq mice, and indeed, these mice exhibited a better-maintained cerebellar morphology. Furthermore, the cerebellum from Aifm1-treated Hq mice and Ngb-treated Hq mice by ROI was larger than that of surgically treated Hq mice, supporting the previous estimation of the cerebellar surface. Therefore, vector administration by ROI may be more protective against tissue degeneration than surgery.

[0086] Purkinje cell number and connectivity in Harlequin mice that received gene therapy either locally or by intravenous injection. Most hereditary ataxias are due to Purkinje cell degeneration (Robinson et al., Front Neurosci. 2020;14:707). These cerebellar neurons are particularly vulnerable because they are probably one of the largest neuron types in the brain, with a widespread dendritic arbor that receives high metabolic activity and widely distributed excitatory inputs (Huang et al., Neurosci Lett. 2019;688:49-57). Notably, Purkinje cells receive more synaptic inputs than any other neuron in the brain, and the number of spines on a single human Purkinje cell can reach 200,000. After sending out collateral branches that affect nearby cortical regions, the axons of Purkinje cells enter the deep cerebellar nuclei, where they each form approximately 1,000 synapses with several types of nuclear cells. Thus, Purkinje cells are the only neurons that send output from the cerebellar cortex (Hirano et al., Cerebellum. 2018;17(6):699-700).

[0087] For all these reasons, as shown in spinocerebellar ataxia type 28, which is caused by mutations in the AFG3L2 gene encoding a mitochondrial metalloprotease involved in the quality control of small organs, Purkinje cells and their connections require robust mitochondria to function (Almajan et al. J Clin Invest. 2012;122(11):4048-4058). In Hq mice, deletion of mitochondrial Aif results in cerebellar degeneration due to granule cell loss and Purkinje cell disappearance. Various estimations of the cell loss dynamics in untreated Hq mice and our experience (cell counting and electron microscopy) indicate that granule cell degeneration in Hq mice begins at 1-2 months of age and Purkinje cells disappear 2 months later. By 12-14 months of age, Hq mice had lost more than 50% of the granule cells in the posterior lobe and substantially all Purkinje cells in lobules VI-VIII (Klein et al.; El Ghouzzi et al. J Neuropathol Exp Neurol. 2007;66(9):838-847; Chung et al. Dis. 2011;41(2):445-457). Thus, Harlequin mutant mice showed severe motor deficits with markedly altered and unstable gait, and further a decrease in muscle tone. These characteristics are easily visible in 4-month-old animals (Preisig et al., Behav Brain Res. 2016;311:340-353).

[0088] Cerebellar slices from Aifm1- and Ngb-treated mice were observed by confocal microscopy after immunolabeling with an antibody against calbindin and compared with cerebellar slices from GFP-treated control mice and GFP-treated Hq mice. The aim was to strengthen the hypothesis that Purkinje cell degeneration is reduced in Hq mice that received intravenous administration of the AAV2 / 9-Aifm1 or AAV2 / 9-Ngb vector with respect to stereotactic surgery by the ROI. The images confirmed that Purkinje cells exhibited a maintained structure in the Aifm1- and Ngb-treated Hq cerebellum and that the branching of their dendrites was more refined than the dendritic branching shown by GFP-treated Hq mice. Harlequin mice that received an intravenous injection of the AAV2 / 9-GFP vector showed that there were few Purkinje cells and they were not sufficiently labeled with the anti-calbindin antibody. In contrast, in the cerebellum from Hq mice treated with AAV2 / 9-Aifm1 or AAV2 / 9-Ngb, Purkinje cells were significantly labeled with the anti-calbindin antibody and several enlarged connections were visible from their cell bodies to the molecular cell layer. In addition, the abundance of Aif or neuroglobin was high in their cell bodies and dendrites. Control mice treated with AAV2 / 9-Aifm1 or AAV2 / 9-Ngb showed a labeling pattern similar to their Hq counterparts except for the dendritic arbors of Purkinje cells that were more refined in control mice than in Hq mice. However, Purkinje cell connectivity was thought to be better maintained in the Hq cerebellum overexpressing Aifm1 or Ngb compared to GFP-treated Hq mice.

[0089] Next, (a) the number of Purkinje cells in the posterior range (lobules VI-X) of each cerebellum; (b) the length (mm) of the Purkinje cell layer within these lobules were estimated to calculate the cell density by normalizing the number of neurons to this measure. In view of this objective, cerebellar slices from the vermis were selected and histochemistry with an antibody against calbindin was performed. The number of calbindin-positive cells was counted in lobules VI-X corresponding to the posterior region of the tissue.

[0090] (1) In Hq mice that received local administration of the vector, the number of Purkinje cells in Hq mice treated with AAV2 / 9-Aifm1 or AAV2 / 9-Ngb increased by 72.4% or 84.4%, respectively, compared to the number of Purkinje cells obtained in Hq mice treated with the AAV2 / 9-GFP vector (P = 0.025 or 0.0075, respectively). When comparing Hq mice that received the ROI, overexpression of Aifm1 or Ngb resulted in a 1.8-fold and 2.06-fold increase in the number of Purkinje cells, and better and significant maintenance of Purkinje cells was observed compared to GFP-treated Hq mice (P = 0.0071 and 0.0003 for AAV2 / 9-Aifm1 or AAV2 / 9-Ngb, respectively). The number of Purkinje cells in Hq mice that received the ROI of AAV2 / 9-Aifm1 or AAV2 / 9-Ngb was 31% and 40% greater than the number of Purkinje cells measured in Hq mice locally treated with the same vector. The observed differences were highly significant (P < 0.0001 for both vectors).

[0091] (2) When comparing the length of the Purkinje cell layer in surgically treated Hq mice, no change was evident. In contrast, when using the intravenous route, this measure increased in Hq treated with AAV2 / 9-Aifm1 or AAV2 / 9-Ngb compared to Hq treated with AAV2 / 9-GFP. Indeed, a 42.4% and 29.2% increase was observed in tissues overexpressing Aifm1 or Ngb compared to GFP-treated Hq mice. These data reached statistical significance (P = 0.0043 for mice treated with either AAV2 / 9-Aifm1 or AAV2 / 9-Ngb).

[0092] (3) The most prominent remission observed was the Purkinje cell density for both routes of vector administration. An increase of 61.5% or 77.4% (P = 0.0022 for both vectors) was observed in Hq mice overexpressing Aifm1 or Ngb compared to the values measured in GFP-treated Hq mice in mice injected into the cerebellum. When comparing Hq mice treated by ROI, increases of 22% and 52.5% were observed in the Hq cerebellum expressing Aifm1 (P = 0.823) or Ngb (P = 0.017) compared to Hq mice treated with the AAV2 / 9-GFP vector.

[0093] There is no doubt that when the Aifm1 or Ngb vector was administered by retro-orbital injection, the number of Purkinje cells in the posterior part of the tissue was greater than when the administration was performed by stereotactic surgery in the cerebellar hemisphere.

[0094] This difference may be due to the higher transduction efficiency of cerebellar neurons when the vector is delivered by intravenous injection instead of direct injection into the cerebellum, which should ultimately lead to better prevention of Purkinje cell degeneration. Cerebellar injection with AAV2 / 9-Aifm1 or AAV2 / 9-Ngb led to 57.6% or 56.7% of transduced Purkinje cells, while when AAV2 / 9-Aifm1 or AAV2 / 9-Ngb was delivered by ROI, 71.1% or 80.3% of Purkinje cells were transduced.

[0095] The gene therapy protocol by vector injection into the retro-orbital sinus did not produce harmful effects on the health of Hq mice, despite using 17 times more of each vector compared to the dose used in the operated mice. Furthermore, improvements in various histochemical parameters were observed: a) the tissue mass and surface were better maintained; b) the morphological deterioration of the cerebellum was less prominent; (c) remission was observed in the number and connectivity of Purkinje cells.

[0096] Determination of mitochondrial function in the cerebellum of Harlequin mice receiving gene therapy by retro-orbital injection To evaluate the enzyme activities of respiratory chain complexes I and IV, and further citrate synthase (a component of the Krebs cycle), cerebellar homogenates from mice euthanized 5 months after receiving ROI were isolated and subjected to spectrophotometry as previously described for mouse retina (Cwerman-Thibault et al. (2021); Bouaita et al. (2012); Lechauve et al. (2014)). Post hoc analysis aimed to confirm whether recovery of the energy state was observed after systemic delivery of the vector, as indicated by stereotactic surgery.

[0097] In Hq mice injected with the AAV2 / 9-Ngb vector, a significant enhancement of complex I (CI), complex IV (CIV) and citrate synthase (CS) activities was observed compared to the activities measured in control mice treated with the same vector, and this was observed independently of the route used to deliver the vector. When gene therapy was performed by surgery or intravenous injection, a 49% or 44% decrease was observed relative to the activities in GFP-treated control mice (P < 0.0001 or P = 0.0055), indicating that Harlequin mice treated with the AAV2 / 9-GFP vector exhibited a significant defect in complex I activity. The cerebellum of Ngb-treated Hq mice reached 83.3% and 98.2% of the values measured in Ngb-treated controls when the vector was delivered by surgery and ROI, respectively (P = 0.62 and > 0.99, respectively). For local or intravenous administration of the vector, complex I activity in Ngb-treated Hq mice increased by 50.6% or 67.1% compared to the measured values in the cerebellum of GFP-treated Hq mice. These values were significantly different: P = 0.026 and 0.014 for surgery and intravenous injection, respectively.

[0098] When the vector was delivered by surgery or into the ROI, the activity of complex IV was also significantly reduced in the cerebellum of GFP-treated Hq mice, corresponding to 58.5% or 51.2% of the values measured in GFP-treated control mice (P = 0.0022 or 0.0013, respectively). When the values for the cerebellum from Ngb-treated Hq mice were compared to Ngb-treated controls, it was observed that in Hq mice, when the treatment was performed by surgery or intravenous injection, the values reached 92.4% and 110.7% of the values measured in control mice (P = 0.92 or >0.99, respectively). The complex IV activity in Ngb-treated Hq mice increased by 63.5% or 79.0% compared to the cerebellum from GFP-treated Hq mice for local or intravenous administration of the vector. These values were significantly different: P = 0.0048 and 0.0008 for surgery and intravenous injection, respectively.

[0099] The determination of citrate synthase activity confirmed the impairment of the bioenergetic state in the cerebellum of Hq mice. Indeed, in GFP-treated Hq mouse cerebellar homogenates, when comparing the surgery for vector administration and the ROI pathway, significant decreases of 28.8% and 30.7% of this activity were observed relative to GFP-treated control mice (P = 0.0004 and <0.0001, respectively). Since citrate synthase activity is considered an accurate measurement of both mitochondrial content and functionality (Van Bergen et al. Mitochondrion. 2014;15:24 - 33), the decrease observed in Hq mice could have been due to either an inefficient enzyme or a decrease in the overall mitochondrial content in the tissue. Next, it was observed that citrate synthase activity in the cerebellum from Ngb-treated Hq mice was very similar to the activity measured in Ngb-treated controls when the treatment was performed either by surgery or intravenous injection: 107.0% and 113.2% (P = 0.86 and 0.022 for both routes of vector delivery). When comparing between Hq mice treated with AAV2 / 9-Ngb and Hq mice treated with AAV2 / 9-GFP, enhancements of 50.2% and 53.4% were observed for surgery and ROI. Therefore, there were significant differences between these mice: P = 0.0005 and <0.0001 for surgery and intravenous injection.

[0100] Overall, these data suggest that the cerebellum from Hq mice exhibited severe defects in CI and CIV activities. Administration of the AAV2 / 9-Ngb vector was beneficial for both complex activities and this was independent of the route of administration used for vector delivery. Furthermore, in GFP-treated Hq mice, CS activity was also reduced relative to their control counterparts and administration of AAV2 / 9-Ngb resulted in a near-complete recovery of enzyme activity regardless of how the vector was delivered. This was particularly true for Hq mice administered AAV2 / 9-Ngb by ROI, indeed, a significant difference was calculated between GFP-treated Hq mice and Ngb-treated Hq mice (P = 0.022). Therefore, the AAV2 / 9-Ngb vector at 17-fold higher (2.6x1011 Hq mice that received ROI by vector genome (VG) did not exhibit a harmful effect on the bioenergetic state of the cerebellum compared to their counterparts that received vector (1.3 x 10 10 VG) administered to the cerebellar hemisphere.

[0101] It is also worth mentioning that when Hq mice received an intravenous injection of AAV2 / 9-Ngb, the recovery of CI and CIV activities relative to Hq mice treated with AAV2 / 9-GFP was significantly greater for direct administration into the cerebellum, suggesting that this protocol may lead to better protection of mitochondrial homeostasis.

[0102] Evaluation of Cognitive and Motor Abilities of Harlequin and Control Mice Receiving Gene Therapy by Retro-orbital Injection The following tests were performed: Grip test (determination of muscle strength), tail suspension test (determination of anxiety-like behavior), Y-maze, forced alternation test (evaluation of spatial reference memory), and open field (measure of total locomotor activity). The abilities of Ngb-treated Hq mice were significantly improved compared to those of GFP-treated Hq mice.

[0103] Grip test: GFP-treated Hq mice exhibited a reduction of approximately 50% in their abilities compared to GFP-treated control mice, showing a significant decrease in muscle strength of all four limbs for both routes of AAV2 / 9-GFP vector administration (P < 0.0001). When comparing the abilities of Ngb-treated Hq mice to Ngb-treated controls, the muscle strength of Ngb-treated Hq mice reached 73.1% and 78.0% of that of Ngb-treated control mice for local surgery and intravenous injection, respectively, indicating improvement for both routes of vector administration. The difference between GFP-treated Hq mice and Ngb-treated Hq mice was significant for both routes of vector delivery (P < 0.0001). Overexpression of Ngb in the cerebellum of Hq mice resulted in an improvement in mouse muscle strength compared to GFP-treated Hq mice, which was greater with ROI delivery (60.3%) than with local injection (43.6%).

[0104] Tail suspension test (TST): The assay consists of suspending the mouse by its tail for 6 minutes at the end of the shelf. Immobility correlates with the "depressed state" and is calculated during the last 4 minutes of the test (Can et al. J Vis Exp. 2012;(59):e3769). GFP-treated Hq mice showed an increase of 60.08% and 53.5% in immobility time compared to GFP-treated controls when the vector was administered locally or by ROI, respectively, and each was used for vector delivery. This increase indicated that Hq mice had experimentally enhanced depressive-like behavior compared to control mice. In contrast, Ngb-treated Hq mice responded to the test similarly to Ngb-treated controls (P = 0.41 or >0.99 for surgery or ROI vector delivery, respectively).

[0105] As a result, there was a significant difference in the ability of Ngb-treated Hq mice compared to GFP-treated Hq animals, regardless of where the vector was administered (P = 0.0018 and 0.0002 for local or ROI, respectively). Interestingly, the improvement in response for Ngb-treated Hq mice compared to GFP-treated Hq animals was greater when the mice received ROI compared to local vector delivery: 47% and 33%, respectively.

[0106] Y-maze (forced alternation): This test can determine short-term spatial memory (Kraeuter et al. (2019)); GFP-treated Hq mice showed severe spatial memory disruption compared to GFP-treated control mice. In fact, the responses of GFP-treated Hq mice showed only 23.1% or 36.6% of the values measured in GFP-treated controls (P < 0.0001 for both local or ROI delivery). Treatment of Hq mice with AAV2 / 9-Ngb led to a significant increase in the responses of these mice compared to GFP-treated Hq (P = 0.0081 or 0.0009 for local or ROI, respectively). In Ngb-treated Hq mice, the ability to explore new branches reached 79.9% and 93.7% of the values measured in their control counterparts treated with AAV2 / 9-Ngb by surgery and intravenous injection. Therefore, there was no significant difference between Ngb-treated Hq mice when either surgery or ROI was used (P = 0.64 or >0.99, respectively). Interestingly, when comparing Ngb-treated Hq mice to their counterparts treated with AAV2 / 9-GFP, it was shown that spatial memory was enhanced 2.4-fold when the mice received local vector injection and 2.7-fold after ROI. Finally, when the vector was delivered by either local surgery or intravenous injection, significantly improved short-term spatial memory and better responses for ROI were observed in Hq mice treated with AAV2 / 9-Ngb.

[0107] Open field: The open field test is used to evaluate motor activity and emotional responses (Kraeuter et al. (2019); Seibenhener et al. J Vis Exp. 2015; (96): e52434, 54). Analysis of the total walking activity of treated mice allowed confirmation of differences between the two routes of vector administration. The motor function of Hq mice was impaired, and indeed, when the vector was delivered either by local surgery or intravenous injection, the distance traveled by GFP-treated Hq mice reached only 27.4% or 43.9% of the values obtained from their control counterparts. The difference between groups was significant (P < 0.0001). The walking activity of Ngb-treated Hq mice that underwent local surgery increased by 75% compared to Ngb-treated control mice (P = 0.0095). Furthermore, Ngb-treated Hq mice by ROI exhibited 55.6% greater motor activity than GFP-treated Hq mice, and this ability was statistically significant (P = 0.0009). Interestingly, the rescue of motor activity for Ngb-treated Hq mice compared to Ngb-treated controls was 60.4% for local vector administration and 79.2% for the vector delivered by ROI (P < 0.0001 and 0.49, respectively). As a result, the difference between Ngb-treated Hq and Ngb-treated controls remained significant for local administration of the vector (P = 0.0095), while for ROI, the difference was not statistically significant (P = 0.067). Thus, similar to the Y-maze test, the ability of Hq mice treated with AAV2-Ngb by ROI was very similar to one of their control counterparts. Notably, the motor activity in Ngb-treated Hq was significantly improved after ROI compared to local surgery.

[0108] Conclusion The ultimate goal of this research project is to develop a gene therapy protocol leading to Ngb overexpression with a broad spectrum of action, aimed at improving the living conditions of a group of patients suffering from neurological difficulties. This includes not only primary mitochondrial diseases caused by mutations in genes encoding mitochondrial proteins, but also brain disorders where symptoms worsen following severe mitochondrial impairment (Zhou et al. Cells. 2018;7(12)), such as spinocerebellar ataxia (Sullivan et al. J Neurol. 2018;266(2):533 - 544) and leukodystrophy (Van Der Knaap et al. Lancet Neurol. 2019;18(10):962 - 972). In fact, the working hypothesis is that mitochondrial impairment, if untreated in such patients, renders therapies designed to target primary genetic defects insufficient.

[0109] The use of gene therapy by stereotactic surgery targeting cerebellar neurons in Hq mice has revealed limitations. Retro-orbital injection (ROI) brings about several improvements: (a) the survival rate of treated Hq mice increased significantly after ROI compared to stereotactic vector administration to the cerebellum; (b) the weight loss of Hq mice that received ROI was lower than that of one of the Hq mice after stereotactic surgery; (c) for mice that received an injection into the tissue, the reduction in the mass and dimensions of the cerebellum in Hq mice administered AAV2 / 2-Ngb by ROI was not as large; (d) the overall morphology of the cerebellum was better maintained in Hq mice that received ROI than when the tissue was directly targeted; (e) it is thought that the protection of Purkinje cells and the branching of their dendrites was enhanced in Hq mice that received ROI with the AAV2 / 9-Ngb vector compared to mice that received the same vector directly into the cerebellum. From the perspective of histochemical research, it was concluded that the integrity of the cerebellum in Hq mice was better maintained when gene therapy with either AAV2 / 9-Aifm1 or AAV2 / 9-Ngb was achieved by intravenous injection instead of local delivery.

[0110] On the one hand, the data obtained on the bioenergetic state of the cerebellar homogenate show a significant improvement in the activities of complex I (CI) and IV (CIV), and furthermore of citrate synthase (CS), with respect to the values obtained from samples derived from untreated or GFP-treated Hq mice. The significant improvement in mitochondrial functionality was comparable for the two tested vector delivery routes: stereotactic surgery in the cerebellar hemisphere and vector injection into the vein of the retro-orbital cavity. However, when compared to injection into the tissue, a slight improvement was evident in the cerebellum of Hq mice administered AAV2 / 9-Ngb by ROI. For example, when AAV2 / 9-Ngb was delivered by ROI, the improvement in CI and CIV activities was slightly better than when it was injected into the cerebellum. Furthermore, CS activity recovered better in the cerebellum administered AAV2 / 9-Ngb by ROI compared to local injection.

[0111] Finally, a comparison of the ataxia phenotypes between the two administration routes by subjecting the mice to behavioral tests needs to be further completed. However, the improvement in muscle strength, locomotor activity, anxiety state, and spatial memory of Hq mice treated with the AAV2 / 2-Ngb vector by ROI was factual and significant when compared to the abilities of untreated or GFP-treated Hq mice. More importantly, Hq mice treated with Ngb by ROI functioned better than Ngb-treated Hq mice administered the vector into the cerebellum. Therefore, a thorough analysis of the data collected from the Grip test, open field, tail suspension test, and Y-maze - forced alternation test shows that Hq mice treated with the AAV2 / 9-Ngb vector by ROI exhibited enhanced locomotor ability, maintained muscle strength, and stronger spatial memory compared to Hq mice administered the vector locally.

[0112] Demonstrating that Ngb-mediated gene therapy can permanently maintain mitochondrial robustness in neurons would be a pioneering means to alleviate the living conditions of patients suffering from a wide range of neurological difficulties, and would stimulate the initiation of clinical trials with neuroglobin for neurological diseases such as cerebellar ataxia and leukodystrophy (Van Der Knaap et al. (2019); Ghanekar et al. Expert Rev Neurother. 2022;22(2):101-114).

Claims

1. Neuroglobin for use in the treatment of neuropathy, administered intravenously.

2. Neuroglobin for use according to Claim 1, which is a polynucleotide encoding neuroglobin.

3. Neuroglobin for use according to Claim 2, wherein the polynucleotide is contained in an expression vector.

4. Neuroglobin for use according to Claim 3, wherein the vector is an AAV vector.

5. Neuroglobin for use according to Claim 4, wherein the vector is an AAV2 / 9 vector.

6. Neuroglobin for use according to any one of Claims 2 to 5, wherein the polynucleotide comprises a neuroglobin gene coding region and a neuroglobin gene 5'UTR and / or 3'UTR.

7. Neuroglobin for use according to any one of Claims 1 to 6, wherein the neuropathy is related to a mitochondrial disease associated with respiratory chain complex I (RCCI) deficiency and / or respiratory chain complex III (RCCIII) deficiency.

8. Neuroglobin for use according to any one of Claims 1 to 7, wherein the neuropathy is a hereditary ataxia.

9. Neuroglobin for use according to Claim 8, wherein the hereditary ataxia is selected from Friedreich's ataxia, cerebellar ataxia, and spinocerebellar ataxia.

10. Neuroglobin for use according to any one of Claims 1 to 9, wherein the patient to be treated is a human.

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