Cholesterol 24-hydrolase expression vector for the treatment of amyotrophic lateral sclerosis (ALS)

Abstract

To provide an expression vector for cholesterol 24-hydrolase in the therapy of amyotrophic lateral sclerosis.SOLUTION: The present invention relates to a vector for use in the treatment of amyotrophic lateral sclerosis, with or without association with frontotemporal dementia and associated motor neuron disorders, which vector expresses a nucleic acid encoding cholesterol 24-hydroxylase. In one embodiment, the vector is selected from the group of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors, preferably AAV vectors, more preferably AAV9, AAV10 (AAVrh.10) or AAVPHP.eB vectors, even more preferably AAVPHP.eB.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to the treatment of amyotrophic lateral sclerosis (ALS). [Background technology]

[0002] Background of the Invention Amyotrophic lateral sclerosis (ALS) is a rare neurological disorder belonging to a group of motor neuron disorders that primarily affect neurons responsible for controlling voluntary muscle movements such as chewing, walking, and speaking (Zarei S et al. 2015). ALS is characterized by the gradual decline and death of motor neurons extending from the brain to the spinal cord and to the muscles throughout the body. ALS involves both upper motor neurons (which send messages from motor neurons to the brain via the spinal cord) and lower motor neurons (motor nuclei in the brain), which degenerate and, as a result, stop sending messages to the muscles, causing the muscles to gradually weaken, begin to spasm, and atrophy (Rowland LP et al. 2001).

[0003] Most people with ALS die within 3 to 5 years of the onset of their first symptoms, usually due to respiratory failure. Only about 10% of people with ALS survive more than 10 years after the onset of symptoms.

[0004] Several potential risk factors are associated with ALS: age (symptoms typically appear between the ages of 55 and 75), sex (ALS is slightly more prevalent in men), and race and ethnicity (ALS is most likely to occur in Caucasians who are not Hispanic).

[0005] However, the majority of ALS cases (90%) are sporadic. In contrast, approximately 10% of ALS cases are familial (FALS). Several genes have been linked to these familial cases: C9ORF72, SOD1, FUS, TARDBP, and more recently, SMCR8 (Greenway et al. 2006; Kabashi et al. 2008; Kaur et al. 2016; Turner MR et al. 2017; Ticozzi et al. 2011; Valdmanis PN et al. 2008).

[0006] Regarding the pathophysiology of ALS, several aspects are currently under investigation. One of the principal theories is based on the finding that it is related to RNA processing, and in particular, that TDP-43 is involved in most ALS cases, as well as genes such as TARDBP and FUS as genetic causes of ALS. Both of these genes are thought to be involved in pre-mRNA splicing, as well as RNA transport and translation. Pre-mRNAs containing C9orf72 repeats can sequester nuclear RNA-binding proteins, thus rendering them unavailable for the precise splicing of other mRNAs. Another well-recognized aspect of ALS, which can be observed in both ALS patients and animal models, is protein aggregation involving SOD1, TDP43, and FUS. It is proposed that these aggregates disrupt normal protein homeostasis and induce cellular stress. Furthermore, protein aggregates can sequester RNA or other proteins essential for normal cellular function.

[0007] To date, there is no known treatment for ALS. Only two drugs have been approved by the FDA for ALS: riluzole and edaravone. Riluzole is thought to work by reducing damage to motor neurons and decreasing glutamate levels. Riluzole extends survival by several months but does not improve damage already present in neurons (Zoccolella et al. 2007). Edaravone has only been shown to reduce the clinical assessment of daily disability (Brooks et al. 2018). Therefore, developing new treatment strategies for ALS is urgently needed. [Overview of the project] [Means for solving the problem]

[0008] Summary of the Invention The inventors hereby propose combating ALS by regulating the cholesterol metabolic pathway, more specifically by using a vector containing a nucleic acid encoding cholesterol 24-hydroxylase, which expresses cholesterol 24-hydroxylase in target cells.

[0009] Accordingly, an object of the present invention is to provide a vector for use in the treatment of amyotrophic lateral sclerosis, wherein the vector comprises a nucleic acid encoding cholesterol 24-hydroxylase.

[0010] In one embodiment, the vector includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2. Alternatively, the vector includes the nucleic acid sequence of SEQ ID NO: 1.

[0011] In one embodiment, the vector is selected from the group consisting of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors, preferably AAV vectors, more preferably AAV9, AAV10 (AAVrh.10) or AAVPHP.eB vectors, and even more preferably AAVPHP.eB.

[0012] In one embodiment, the vector is administered directly to the brain and / or spinal cord of the patient, preferably to the spinal cord and / or motor cortex.

[0013] Another object of the present invention is to provide a pharmaceutical composition for use in the treatment of amyotrophic lateral sclerosis, the pharmaceutical composition comprising a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase. In certain embodiments, for example, the following items are provided: (Item 1) A vector for use in the treatment of amyotrophic lateral sclerosis (ALS), the vector comprising a nucleic acid encoding cholesterol 24-hydroxylase. (Item 2) The vector for use in the treatment of ALS according to item 1, wherein the ALS is associated with at least one motor neuron-related disorder. (Item 3) The vector for use in the treatment of ALS according to item 2, wherein the ALS is associated with frontotemporal dementia. (Item 4) The vector for use in the treatment of ALS according to any one of items 1 to 3, comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2. (Item 5) The vector for use in the treatment of ALS according to any one of items 1 to 4, comprising the nucleic acid sequence of SEQ ID NO: 1. (Item 6) The vector for use in the treatment of ALS according to any one of items 1 to 5, selected from the group consisting of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors. (Item 7) The vector for use in the treatment of ALS according to any one of items 1 to 6, which is an AAV vector. (Item 8) Vectors for use in the treatment of ALS according to item 7, which are AAV9, AAV10 vectors, such as AAVrh.10, or AAVPHP.eB, preferably AAVPHP.eB. (Item 9) Vectors for use in the treatment of ALS according to any one of items 1 to 8, which are administered intravenously or directly into the brain of a patient. (Item 10) Vectors for use in the treatment of ALS according to item 9, which are administered to the spinal cord and / or motor cortex. (Item 11) Vectors for use in the treatment of ALS according to item 10, which are administered to motor neurons. (Item 12) Vectors for use in the treatment of ALS according to any one of items 1 to 11, which are administered by intravascular, intravenous, intranasal, intraventricular or intrathecal injection. (Item 13) A pharmaceutical composition for use in the treatment of amyotrophic lateral sclerosis, wherein the pharmaceutical composition comprises a therapeutically effective amount of a vector as defined in any one of items 1 to 12.

Brief Description of Drawings

[0014] [Figure 1] Figure 1 - mRNA levels of genes involved in cholesterol metabolism in the lumbar spinal cord of WT and SOD1G93A animals at 8 weeks and levels of 24-OH cholesterol in the spinal cord at 15 weeks. (A) mRNA was extracted from the lumbar spinal cord of 8-week-old WT and SOD1G93A mice. mRNA levels were normalized to the actin housekeeping gene. Data are presented as mean ± SEM (n = 4 - 5 / group). (B) Lumbar spinal cord 24S-hydroxycholesterol content in SOD1G93A mice evaluated by UPLC-HRMS. Data are presented as mean ± SEM (n = 7 - 9 / group). Statistical analysis: Student's t-test. *p < 0.05, **p < 0.01.

[0015] [Figure 2] Figure 2 - Evaluation of CYP46A1-HA expression in WT animals after intravenous delivery of AAVPHP.eB-CYP46A1-HA at 8 weeks, and analysis at 3 weeks after low, medium, or high dose injection. HA staining of cervical, thoracic, and lumbar sections of the spinal cord.

[0016] [Figure 3] Figure 3 - Evaluation of inflammation after intravenous delivery of AAVPHP.eB-CYP46A1-HA in WT animals at 8 weeks and analysis at 3 weeks after low, medium, or high dose injection. GFAP staining of lumbar spinal cord sections. NI: Not injected.

[0017] [Figure 4] Figure 4 - Behavioral evaluation of SOD1 mice after intravenous administration of AAVPHP.eB-CYP46A1 during the prevention stage (3 weeks). (A) Weight tracking, (B) Clasping test, and (C) Inverted test. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Black asterisks represent p-values ​​relative to WT animals, and gray asterisks represent p-values ​​relative to SOD1 animals.

[0018] [Figure 5] Figure 5 - Biodistribution of AAVPHP.eB-CYP46A1 after prophylactic treatment in SOD1 animals and evaluation of target engagement by quantitative determination of 24OH cholesterol levels at 15 weeks of age after prophylactic treatment. (A) Biodistribution in the central nervous system and (B) peripheral organs, and (C) 24S-hydroxycholesterol content in the lumbar spinal cord in SOD1G93A mice evaluated by UPLC-HRMS. Results are expressed as mean ± SEM.

[0019] [Figure 6]Figure 6 - Evaluation of CYP46A1-HA expression at 15 weeks of age and histological analysis of the spinal cord after intravenous delivery of AAVPHP.eB-CYP46A1 as a preventive measure. (A) HA staining of cervical, thoracic, and lumbar sections of the spinal cord of AAV-treated SOD1 animals; (B) Co-staining for HA and VachT on cervical, thoracic, and lumbar sections of the spinal cord of WT, SOD1, and SOD1 AAV animals; (C) Quantification of motor neuron count; (D) Luxor staining of lumbar sections of the spinal cord; and (E) Evaluation of myelin percentage. Results are expressed as mean ± SEM. *p<0.05, **p<0.01. Black * corresponds to p-values ​​for WT animals and gray corresponds to p-values ​​for SOD1 animals.

[0020] [Figure 7] Figure 7 - Improvement of muscle phenotype in AAV-treated SOD1 animals after preventive treatment. (A) Hematoxylin-eosin colorimetric analysis for analyzing fiber size in muscle. Shows mean fiber area in tibialis (B), gastrocnemius (D), and quadriceps (F), and reallocation of fiber percentage according to cross-sectional size in tibialis (C), gastrocnemius (E), and quadriceps (G). Results are expressed as mean ± SEM. *p<0.05, **p<0.01.

[0021] [Figure 8] Figure 8 - Preservation of neuromuscular junctions in AAV-treated SOD1 animals after prophylactic treatment. (A) Co-staining of nerve fibers (NF) and bungarotoxin (BTX) in 15-week-old WT, SOD1, and SOD1-treated animals after intravenous delivery of AAVPHP.eB-CYP46A1. (B-C) Scoring of NMJs according to their innervation status and integrity at 15 weeks of age. (D) Evaluation of NMJ formation at 3 weeks in WT and SOD1 animals using triple staining for pan-NF, bungarotoxin, and VachT. (E) Quantification of NMJs based on their status as mature M1-M4 at 3 weeks of age.

[0022] [Figure 9]Figure 9 - Molecular analysis of NMJs at 15 weeks in preventive treatment. Analysis of Musk expression in tibialis muscle (A) and gastrocnemius muscle (B), and quantification of nAchR expression in tibialis muscle (C) and gastrocnemius muscle (D).

[0023] [Figure 10] Figure 10 - Behavioral evaluation of SOD1 mice after intravenous administration of AAVPHP.eB-CYP46A1 at the healing stage (8 weeks). (A) Weight tracking, (B) Grasp test, (C) Inversion test, and (D) Survival. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Black asterisks correspond to p-values ​​for WT animals, and gray asterisks correspond to p-values ​​for SOD1 animals.

[0024] [Figure 11] Figure 11 - Biodistribution of AAVPHP.eB-CYP46A1 in SOD1 animals after prophylactic treatment, and evaluation of target binding by quantitative determination of 24OH cholesterol levels at 15 weeks of age after curative treatment. (A) Biodistribution in the central nervous system and (B) peripheral organs, and (C) 24S-hydroxycholesterol content in the lumbar spinal cord of SOD1G93A mice as assessed by UPLC-HRMS. Results are expressed as mean ± SEM.

[0025] [Figure 12] Figure 12 - Evaluation and histological analysis of CYP46A1-HA expression in the spinal cord at 15 weeks of age after intravenous delivery of AAVPHP.eB-CYP46A1 as a curative treatment. (A) Co-staining for HA and VachT on lumbar sections of the spinal cord of WT, SOD1, and SOD1 AAV animals. (B) Quantification of motor neuron count on lumbar spinal cord sections, and (C) Luxor staining on lumbar sections of the spinal cord. Results are expressed as mean ± SEM. *p<0.05, **p<0.01. Black * corresponds to p-values ​​for WT animals, and gray corresponds to p-values ​​for SOD1 animals.

[0026] [Figure 13]Figure 13 - Partial correction of muscle phenotype in AAV-treated SOD1 animals after curative treatment. Shows mean fiber area in the tibialis (A), gastrocnemius (C), and quadriceps (E), and reallocation of fiber percentage according to their cross-sectional size in the tibialis (B), gastrocnemius (D), and quadriceps (F). Results are expressed as mean ± SEM. *p<0.05, **p<0.01.

[0027] [Figure 14] Figure 14 - Preservation of neuromuscular junctions in AAV-treated SOD1 animals after curative treatment. (A) Co-staining of nerve fibers (NF) and bungarotoxin (BTX) in 15-week-old WT animals treated with SOD1 after curative intravenous delivery of SOD1 and AAVPHP.eB-CYP46A1. (B-C) Scoring of NMJs according to their innervation status and integrity at 15 weeks of age.

[0028] [Figure 15] Figure 15 - Molecular analysis of NMJs at 15 weeks in curative treatment. Analysis of Musk expression in the tibialis muscle (A) and gastrocnemius muscle (B), and quantification of nAchR expression in the tibialis muscle (C) and gastrocnemius muscle (D).

[0029] [Figure 16] Figure 16 - Behavioral evaluation of SOD1 mice after intravenous administration of AAVPHP.eB-CYP46A1 during the therapeutic stage (8 weeks) at high doses. Grasp test. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Black asterisks correspond to p-values ​​for WT animals, and gray asterisks correspond to p-values ​​for SOD1 animals.

[0030] [Figure 17]Figure 17 - Behavioral evaluation of C9 ORF72 mice after intravenous administration of AAVPHP.eB-CYP46A1 during the high-dose prevention stage (4 weeks). (A) Weight tracking and (B) Notched bar results. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Black asterisks correspond to p-values ​​for WT animals, and gray asterisks correspond to p-values ​​for C9ORF72 animals.

[0031] [Figure 18] Figure 18 - (A) Evaluation of p62 aggregates in cells after overexpression of SMCR8 WT or mutant and CYP46A1. (B) Quantification of the total number of transduced cells (HA) positive, reflecting neuronal survival under different conditions. [Modes for carrying out the invention]

[0032] Detailed description of the invention The inventors have shown that intravenous delivery of a vector expressing the CYP46A1 gene in a mouse model of ALS can prevent or correct the development of motor impairment. More specifically, the inventors have shown that delivery of a plasmid expressing the CYP46A1 gene to primary striatal neurons modeling amyotrophic lateral sclerosis pathology results in a significant reduction in p62 aggregates, which are characteristic of SMCR8 dysfunction and involved in ALS through impaired autophagy, and improves neuronal survival.

[0033] Based on this, the inventors provide a viral vector for the treatment of ALS, the vector expressing CYP46A1 in cells of the central nervous system, particularly motor neurons and neurons in the motor cortex. This strategy is useful in treating any motor neuron disorder linked to certain forms of ALS, primarily through common mutant genes. In particular, the inventors provide a viral vector for the treatment of frontotemporal dementia (FTD) associated with ALS.

[0034] Amyotrophic lateral sclerosis (ALS) The present invention specifically relates to the treatment of amyotrophic lateral sclerosis (ALS). Preferably, the present invention relates to the treatment of ALS caused by sporadic factors and / or in which disease-associated proteins (i.e., SOD1, FUS, C9ORF72, TDP43…) acquire functional mutations. In one embodiment, the present invention relates to the treatment of ALS associated with at least one motor neuron-associated disorder. Such associated motor neuron disorders are preferably selected from lower motor neuron disorders or upper motor neuron disorders, including spastic paraplegia. In a particular embodiment, the present invention relates to the treatment of ALS associated with frontotemporal dementia (FTD). FTD is a fatal neurodegenerative disease characterized by neuronal degeneration in the frontal and temporal lobes, causing a progressive deterioration of language, personality, and behavior. At least 15–20% of ALS patients are simultaneously diagnosed with FTD (Prudencio et al. 2015). In another particular embodiment, the present invention relates to the treatment of ALS without frontotemporal dementia.

[0035] In the context of the present invention, the terms “treatment,” “to treat,” or “to treat” are used herein to characterize treatments or processes that aim to (1) slow down or halt the progression, aggravation, or worsening of the symptoms of the disease state or condition in which such terms apply; (2) improve or bring about improvement of the symptoms of the disease state or condition in which such terms apply; and / or (3) improve or cure the disease state or condition in which such terms apply.

[0036] As used herein, the terms “subject” or “patient” refer to animals, preferably mammals, and more preferably humans (including adults and children). However, the term “subject” may also refer to non-human animals, mammals such as mice, and non-human primates.

[0037] CYP46A1 sequence A first object of the present invention relates to a vector for use in the treatment of amyotrophic lateral sclerosis, wherein the vector comprises the entire sequence of a nucleic acid encoding cholesterol 24-hydroxylase.

[0038] As used herein, the term “gene” refers to a polynucleotide containing at least one open reading frame that, after transcription or translation, can encode a particular polypeptide or protein.

[0039] As used herein, the terms “coding sequence” or “sequence encoding a particular protein” refer to a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed in vitro or in vivo (in the case of DNA) and translated into polypeptides (in the case of mRNA). The boundaries of such coding sequences are determined by a start codon at the 5' (amino) terminus and a translation termination codon at the 3' (carboxy) terminus. Coding sequences may include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0040] The CYP46A1 gene encodes cholesterol 24-hydroxylase. This enzyme is a member of the cytochrome P450 superfamily of enzymes. This enzyme converts cholesterol into 24S-hydroxycholesterol (24S-OH-Chol), which can dynamically cross the blood-brain barrier (BBB), achieve peripheral circulation, and is excreted from the body (Bjork). Hem et al. 1998), therefore, cholesterol homeostasis is maintained. The cDNA sequence of CYP46A1 is disclosed in Genbank accession number AF094480 (SEQ ID NO: 1). Its amino acid sequence is shown in SEQ ID NO: 2.

[0041] The present invention utilizes nucleic acid constructs comprising the sequence of Sequence ID No. 1 or variants thereof for the treatment of amyotrophic lateral sclerosis and, if necessary, related motor neuron disorders (e.g., FTD).

[0042] The above-mentioned variants include naturally occurring variants resulting, for example, from inter-individual allele variations (e.g., polymorphism), alternative splicing morphologies, etc. The term variant also includes CYP46A1 gene sequences derived from other sources or organisms. The variants are preferably substantially homologous to SEQ ID NO: 1, i.e., exhibiting at least about 75%, preferably at least about 85%, more preferably at least about 90%, and more preferably at least about 95% nucleotide sequence identity with SEQ ID NO: 1. The CYP46A1 gene variants also include nucleic acid sequences that hybridize with the sequence (or its complementary strand) as defined above under stringent hybridization conditions. Typical stringent hybridization conditions include a temperature above 30°C, preferably above 35°C, more preferably above 42°C, and / or a salinity below about 500 mM, preferably below 200 mM. Hybridization conditions can be adjusted by those skilled in the art by modifying the temperature, salinity, and / or the concentration of other reagents (e.g., SDS, SSC, etc.).

[0043] Nonviral vectors In one embodiment, the vector for use according to the present invention is a non-viral vector. Typically, the non-viral vector may be a plasmid encoding CYP46A1. This plasmid may be administered directly or by liposomes, exosomes, or nanoparticles.

[0044] Viral vector Gene delivery viral vectors useful in carrying out the present invention can be constructed using methodologies well known in the field of molecular biology. Typically, a viral vector containing a transgene is assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements necessary for the production of a viral protein that mediates cell transduction.

[0045] The terms “gene transfer” or “gene delivery” refer to methods or systems for the reliable insertion of foreign DNA into a host cell. Such methods may result in transient expression of the transferred DNA that is not integrated, extrachromosomal replication and expression of the transferred replicon (e.g., episome), or integration of the transferred genetic material into the host cell’s genomic DNA.

[0046] Examples of viral vectors include adenovirus, lentivirus, retrovirus, herpesvirus, and adeno-associated virus (AAV) vectors.

[0047] Such recombinant viruses can be generated by techniques known in the field (e.g., by transfecting packaging cells or by transient transfection with helper plasmids or viruses). Representative examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for generating such replication-deficient recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056, and WO94 / 19478.

[0048] In a preferred embodiment, an adeno-associated virus (AAV) vector is used.

[0049] "AAV vector" means a vector derived from an adeno-associated virus serotype (including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10 (e.g., AAVrh.10, AAVPHP.eB, etc.)). An AAV vector may have one or more of the AAV wild-type genes (preferably rep and / or cap genes) deleted whole or partially, but may retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication, and packaging of AAV virions. Accordingly, AAV vectors are specified herein to contain at least those sequences (e.g., functional ITRs) required in cis for the replication and packaging of the above-mentioned viruses. The above-mentioned ITRs do not need to be wild-type nucleotide sequences and may be modified, for example, by nucleotide insertions, deletions, or substitutions, insofar as the sequences provide functional rescue, replication, and packaging. The AAV expression vector is constructed using known techniques to provide at least a regulatory element containing a transcription start region, the DNA of interest (i.e., the CYP46A1 gene), and a transcription termination region as components operably linked in the direction of transcription. Two copies of the DNA of interest may be included as a self-complementary construct.

[0050] In a more preferred embodiment, the AAV vector is an AAV9, AAV10, preferably AAVrh.10, AAVPHP.B, or AAVPHP.eB vector, or a vector derived from one of these serotypes. In the most preferred embodiment, the AAV vector is an AAVPHP.eB vector, which is an evolved AAV-PHP.B variant that efficiently transduces CNS neurons (Chan KY et al. 2017; WO2017100671). Other vectors (e.g., those described in WO2015038958 and WO2015191508) may also be used.

[0051] The regulatory elements are selected to be functional in mammalian cells. The resulting construct, including operably linked components, borders the functional AAV ITR sequence (at the 5' and 3' ends). "Adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to the region recognized in that region, found at each end of the AAV genome, that functions together cis-wise as a DNA replication origin and as a viral packaging signal. AAV ITRs, together with the AAV rep coding region, provide efficient excision and rescue from nucleotide sequences sandwiched between two adjacent ITRs, as well as their incorporation into the mammalian cell genome. The nucleotide sequences of AAV ITR regions are publicly known (e.g., Kotin, for the AAV-2 sequence). 1994; Berns, KI “Parvoviridae and their Replication” Fundamental Virology, 2nd edition, (See BN Fields and DM Knipe, eds.). As used herein, “AAV ITR” does not necessarily have to contain a wild-type sequence and may be modified, for example, by nucleotide insertions, deletions, or substitutions. Furthermore, AAV ITRs may originate from any of several AAV serotypes (including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc.). Moreover, the 5' and 3' ITRs adjacent to the selected nucleotide sequence in an AAV vector do not necessarily have to be identical or originate from the same AAV serotype or isolate, insofar as they function as intended, namely, to enable excision and rescue of the desired sequence from the host cell genome or vector, and to enable the incorporation of the heterologous sequence into the recipient cell genome if the AAV Rep gene product is present in the cell.

[0052] Vectors derived from AAV serotypes that exhibit tropism and transduction efficiency to cells of the mammalian central nervous system (CNS), particularly neurons, are especially preferred. A study and comparison of transduction efficiencies of various serotypes is provided in Davidson et al., 2000. In one preferred example, AAV2-based vectors have been shown to direct the long-term expression of the transgene in the CNS, preferably in the transduced neurons. Other non-limiting examples of preferred vectors include those derived from AAV4 and AAV5 serotypes, which have also been shown to transduce CNS cells (Davidson et al., cited above). In particular, these vectors may be AAV vectors containing a genome derived from AAV5 (specifically, the ITR is AAV5 ITR) and a capsid derived from AAV5.

[0053] In certain embodiments of the present invention, the vector is a pseudotyped AAV vector. Specifically, the pseudotyped AAV vector comprises an AAV genome derived from a first AAV serotype and a capsid derived from a second AAV serotype. Preferably, the genome of the AAV vector is derived from AAV2. Furthermore, the capsid is preferably derived from AAV5. Specific non-limiting examples of pseudotyped AAV vectors include AAV vectors containing a genome derived from AAV2 within a capsid derived from AAV5, and AAV vectors containing a genome derived from AAV2 within a capsid derived from AAVrh.10.

[0054] The selected nucleotide sequences are operably ligated to control elements that direct their transcription or expression in vivo in the subject. Such regulatory elements may typically include regulatory sequences associated with the selected gene. In particular, such regulatory elements may include promoters of the CYP46A1 gene, especially the human CYP46A1 gene (Ohyama Y et al., 2006).

[0055] Alternatively, heterologous control sequences may be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PGK) promoter, the SV40 early promoter, the mouse mammary tumor virus LTR promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter (e.g., the CMV very early promoter region (CMVIE)); the Roussarcoma virus (RSV) promoter; synthetic promoters; and hybrid promoters. Furthermore, sequences derived from non-viral genes (e.g., the mouse metallothionein gene) are also found to be useful herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA). For the purposes of the present invention, both heterologous promoters and other control elements (e.g., CNS-specific and inducible promoters, enhancers, etc.) are particularly useful.

[0056] An example of a heterologous promoter is the CMV promoter. Examples of CNS-specific promoters include those isolated from myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), synapsin (e.g., the human synapsin 1 gene promoter), and neuron-specific enolase (NSE) genes.

[0057] Examples of inductive promoters include the DNA-responsive elements ecdysone, tetracycline, hypoxia, and aufin.

[0058] AAV expression vectors containing the target DNA molecule bounded by AAV ITRs can be constructed by directly inserting selected sequences into the AAV genome excised from a major AAV open reading frame ("ORF"). Other parts of the AAV genome can also be deleted, insofar as a sufficient portion of the ITR remains capable of replication and packaging. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patents 5,173,414 and 5,139,941; International Publication Numbers WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992; Muzyczka, 1992; Kotin, 1994; Shelling and Smith, 1994; and Zhou et al., 1994. Alternatively, the AAV ITR may be excised from the viral genome or from an AAV vector containing the viral genome and fused to the 5' and 3' ends of a selected nucleic acid construct present in another vector using standard ligation techniques. AAV vectors containing the ITR are described, for example, in U.S. Patent No. 5,139,941. In particular, several AAV vectors are described therein, and these are available from the American Type Culture Collection ("ATCC") under accession numbers 53222, 53223, 53224, 53225, and 53226. Furthermore, chimeric genes may be synthesized to contain AAV ITR sequences positioned at the 5' and 3' ends of one or more selected nucleic acid sequences. Codons suitable for the expression of chimeric genes in mammalian CNS cells may be used. Complete chimeric sequences are assembled from duplicate oligonucleotides prepared by standard methods (see, e.g., Edge, 1981; Nambair et al., 1984; Jay et al., 1984). To generate rAAV virions, AAV expression vectors are introduced into suitable host cells using known techniques, for example, by transfection.Many transfection techniques are generally known in this field (e.g., Graham et al., 1973; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York). See Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981). Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome-mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Felgner et al., 1987), and nucleic acid delivery using fast microlaunchers (Klein et al., 1987).

[0059] For example, a preferred viral vector (e.g., AAVPHP.eB) includes, in addition to the nucleic acid sequence encoding cholesterol 24-hydroxylase, an AAV vector backbone having an ITR derived from AAV-2, a promoter (e.g., a cytomegalovirus / β-actin hybrid promoter (CAG) consisting of an enhancer derived from the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus early gene, a promoter derived from the chicken β-actin gene, splice donors and introns, and a splice acceptor derived from rabbit β-globulin), or any neuron promoter (e.g., a promoter for the dopamine-1 receptor or dopamine-2 receptor with or without the wild-type or mutant form of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)).

[0060] Vector delivery A method for treating amyotrophic lateral sclerosis is disclosed, the method comprising the step of administering a vector containing a nucleic acid encoding cholesterol 24-hydroxylase to a patient in need. The vector may be delivered directly to the brain and / or spinal cord of the subject, or by intravascular, intravenous, intranasal, intraventricular, or intrathecal injection. In certain embodiments, the vector is AAVrh10 or AAVPHP.eB and is delivered by intravenous injection.

[0061] In a particular embodiment, a method for treating amyotrophic lateral sclerosis (ALS) in a subject, wherein the method is (a) the step of providing a vector as defined above, comprising a nucleic acid encoding cholesterol 24-hydroxylase; and (b) The vector is delivered to the brain and / or spinal cord of the subject, thereby the vector transduces cells in the brain and / or spinal cord, thereby cholesterol 24-hydroxylase is expressed in the transduced cells at a therapeutically effective level. A method for including this is provided.

[0062] Advantageously, the above vector is a viral vector; more advantageously, it is an AAV vector; and even more advantageously, it is an AAV vector selected from the group consisting of AAV9, AAV10, or AAVPHP.eB.

[0063] In certain embodiments, the vector is delivered to the brain, particularly the motor cortex, and / or the spinal cord. In certain embodiments, the vector is delivered exclusively to the spinal cord.

[0064] In another specific embodiment, the vector is administered by intravenous injection.

[0065] Methods for delivering or administering viral vectors to neurons and / or astrocytes and / or oligodendrocytes and / or microglia generally include any method suitable for delivering the vector to the selected synaptic cell populations, either directly or through hematopoietic cell transduction, such that at least a portion of the cells of the synaptic cell population are transduced. The vector may be delivered to any cells of the central nervous system, cells of the peripheral nervous system, or both. Preferably, the vector is delivered to cells of the brain and / or spinal cord. Generally, the vector is delivered to cells of the brain (e.g., including cells of the motor cortex), cells of the spinal cord, or a combination thereof, or any suitable subpopulation thereof.

[0066] To deliver the above vector specifically to a particular region and to a specific population of cells in the brain or spinal cord, the vector may be administered by stereotactic microinjection. For example, the patient is fixed in place (screwed to the skull) with a stereotactic frame base. The brain and the stereotactic frame base (MRI of reference markers and compatibility) are imaged using high-resolution MRI. The MRI images are then transferred to a computer running stereotactic software. A series of coronal, sagittal, and axial images are used to determine the target (vector injection site) and trajectory. The software directly converts the trajectory into three-dimensional coordinates suitable for the stereotactic frame. A burr hole is drilled above the entry site, and the stereotactic device is positioned by embedding the needle to a predetermined depth. The vector is then injected into the target site, ultimately mixed with a contrast agent. Since the above vector is incorporated into target cells rather than generating viral particles, its subsequent spread is limited and is primarily a function of passive diffusion from the injection site and, naturally, of the desired, pre-incorporation transsynaptic transport. The degree of diffusion can be controlled by adjusting the vector-to-fluid carrier ratio.

[0067] Further administration routes may also include local application of the vector under direct visualization, e.g., application to the cortical surface, intranasal application, or other non-stereotactic application.

[0068] The target cells of the vector of the present invention are cells of the spinal cord (motor neurons) and cells of the brain (motor cortex) of subjects suffering from ALS, preferably nerve cells.

[0069] Preferably, the subject is human, generally an adult, but may be a child or an infant. However, the present invention encompasses the delivery of the vector to a biological model of the disease. In this case, the biological model may be any mammal at any developmental stage at the time of delivery (e.g., embryo, fetus, infant, juvenile or adult), and preferably an adult. Furthermore, the target cells may essentially originate from any source, particularly non-human primates and other rodent mammals (mice, rats, rabbits, hamsters), carnivores (cats, dogs), and artiodactyls (cattle, pigs, hogfish, goats, horses), as well as any other non-human lineage (e.g., zebrafish model lineage).

[0070] Preferably, the method of the present invention includes intracerebral administration by stereotactic injection. However, other known delivery methods may also be adapted according to the present invention. For example, with regard to a broader distribution of the vector across the brain, it may be injected into the cerebrospinal fluid by lumbar puncture, cisterna magna, or ventriculotomy. To direct the vector to the brain, it may be injected (subcutaneously or intramuscularly) into the spinal cord, or into peripheral ganglia, or into the muscle tissue of the body part of interest. In certain circumstances (using AAVPHP.eB here), the vector may be administered via an intravascular approach. For example, the vector may be administered intraarterially (into the carotid artery) in situations where the blood-brain barrier is obstructed. Furthermore, with regard to a more overall delivery, the vector may be administered during the "opening" of the blood-brain barrier, achieved by injection of a hypertonic solution containing mannitol or by local ultrasound delivery.

[0071] The vectors used herein may be formulated in any suitable delivery vehicle. For example, they may be encapsulated in pharmaceutically acceptable suspensions, solutions, or emulsions. Suitable media include saline and liposome preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions (including saline and buffering media). Intravenous vehicles include fluid and nutritional replacement fluids, and electrolyte replacement fluids (e.g., those based on Ringer's dextrose).

[0072] For example, preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, and inactivating gases may also be present.

[0073] Colloidal dispersions can also be used for targeted gene delivery. Colloidal dispersions include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems (such as oil-in-water emulsions, micelles, mixed micelles, and liposomes or exosomes).

[0074] The preferred dose and regimen may be determined by the physician and may depend on the subject's age, sex, weight, and stage of the disease. For example, with regard to the delivery of cholesterol 24-hydroxylase using a viral expression vector, each unit dose of the cholesterol 24-hydroxylase expression vector contains the viral expression vector in a pharmaceutically acceptable fluid and is 10 per 1 ml of composition. 10 ~10 15 The composition may contain 2.5 to 100 μl of a composition that provides cholesterol 24-hydroxylase-expressing virus particles up to a certain level.

[0075] The above vectors may be used in curative and / or preventive treatments.

[0076] Therefore, an object of the present invention is to provide a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase for use in preventive treatment of ALS.

[0077] Another object of the present invention is to provide a vector comprising a nucleic acid encoding cholesterol 24-hydroxylase for use in the curative treatment of ALS.

[0078] Pharmaceutical composition A further object of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a vector according to the present invention for use in the treatment of ALS.

[0079] "Therapeutally effective amount" means a sufficient amount of the vector of the present invention to treat ALS, which is applicable to any medical treatment and has a reasonable benefit / risk ratio.

[0080] It is understood that the total daily dose of the compounds and compositions of the present invention is determined by the attending physician within reasonable medical judgment. The specific therapeutically effective dose level for any particular patient depends on a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the patient's age, weight, general health, sex, and diet; the time of administration, the route of administration, and the elimination rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific polypeptide used; and similar factors well known in the medical field. For example, it is well within the art of the art to start with a dose of a compound at a level lower than what is required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the above products can vary over a wide range per adult per day. The therapeutically effective amount of the vector according to the present invention to be administered, and the dose for treating a pathological condition in the number of viral or nonviral particles and / or pharmaceutical compositions of the present invention, depends on many factors, including the patient's age and condition, the severity of the disease (disturbance) or disorder, the method and frequency of administration, and the specific peptide to be used.

[0081] The presentation of pharmaceutical compositions containing vectors according to the present invention may be in any form suitable for intramuscular, intracerebral, intranasal, intrathecal, intraventricular, or intravenous administration. In the pharmaceutical compositions of the present invention for intramuscular, intranasal, intravenous, intracerebral, intrathecal, or intraventricular administration, the active ingredient may be administered to animals and humans alone or in combination with another active ingredient in unit dose forms or as a mixture with conventional pharmaceutical support substances.

[0082] Preferably, the above-mentioned pharmaceutical composition comprises a pharmaceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic sterile saline solutions (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or, optionally, dry, especially lyophilized, compositions that enable the composition of a sterile injectable solution upon addition of sterile water or physiological saline.

[0083] Once formulated, the liquid formulation is administered in a manner compatible with the administration preparation and in a therapeutically effective amount. While the above formulation can be readily administered in various forms (e.g., injectable liquid formulations), drug-releasing capsules and other formulations may also be used.

[0084] Numerous doses may also be administered.

[0085] It is also possible to associate therapeutic approaches with small molecules that can activate CYP46A1 (e.g., efavirenz (Mast N et al. 2013; Mast N et al. 2017; Mast N et al. 204; Mast N et al. 2017)) or, conversely, antifungal drugs that can inhibit CYP46A1 and, therefore, if necessary, can halt gene therapy approaches (Mast N et al. 2013; Fourgeux et al. 2014)).

[0086] The present invention is further illustrated by the following embodiments. However, these embodiments and accompanying drawings should not be construed in any way as limiting the scope of the present invention. [Examples]

[0087] In this experiment, an in vitro model of ALS was generated based on the overexpression of SMCR8 D928V (a mutant of SMCR8), which forms a complex with C9ORF72 and WDR1 and acts as a GDP / GTP exchange factor for RAB8 and RAB39b, thereby regulating the autophagy flux (Sellier et al. 2016). Either WT SMCR8 or mutant SMCR8 (which alters autophagy as an siRNA expression for SMCR8) was overexpressed.

[0088] material and method animal Two male B6SJL-Tg(SOD1*G93A)1Gur / J were introduced by Jackson These mice were obtained from Laboratories (stock 002726) and crossbred with C57BL / 6 mice to generate colonies.

[0089] Three male FVB / NJ-Tg (C9orf72) 500Lpwr / J mice were obtained from Jackson Laboratories (stock 029099) and crossed with FVB / NJ mice to generate colonies.

[0090] Mice were housed in a temperature-controlled room and maintained on a 12-hour light / dark cycle. They were given free access to feed and water. The experiment was conducted in accordance with the Council Directive of the European Community (2010 / 63 / EU) on the management and use of laboratory animals.

[0091] AAV plasmid design and vector generation AAV vectors, Atlantic Gene Therapies (INSERM) The vector was generated and purified by U1089 (Nantes, France). Vector generation is described elsewhere (Hudry E et al. 2010). The viral construct AAVPHP.eB-CYP46A1-HA contained an expression cassette consisting of the human Cyp46a1 gene driven by the CMV early enhancer / chicken β-actin (CAG) synthesis promoter (CAG) surrounded by the AAV2 reverse terminal repeat (ITR) sequence. The final titer of the batch was 1.5–2.10 13 It was between vg / ml.

[0092] Genotype determination The mice were genotyped according to the Jackson lab procedure (https: / / www2.jax.org / protocolsdb / f?p=116:5:0::NO:5:P5_MASTER_PROTOCOL_ID,P5_JRS_CODE:24173,002726) for the SOD1G93A mouse strain, and according to https: / / www2.jax.org / protocolsdb / f?p=116:5:0::NO:5:P5_MASTER_PROTOCOL_ID,P5_JRS_CODE:30889,029099 for the C9ORF72 500r strain.

[0093] Intravenous injection To determine the dose of AAVPHP.eB-CYP46A1-HA to be injected in the ALS mouse model, three doses were administered to C57BL6 animals at 8 weeks of age, totaling 2.5.10. 11 vg (low dose), total 5.10 11 vg (medium dose) and total 1.10 12 VG (high dose) (n=3 / group) was injected.

[0094] Regarding preventive measures, SOD1 in 3-week-old staghorn follicles G93A Alternatively, 4-week-old C9ORF72 500r mice were anesthetized with 4% isoflurane and then maintained at 2% using 80% air and 20% oxygen. SOD1 animals were administered intravenously by retroorbital injection, totaling 2.5.10 11 vg (low dose) or total 5.1011 Received an injection (100 μl volume) of vg (medium dose). WT and non-injected SOD1 animals received an injection of 100 μl of saline solution.

[0095] Details of the prevention group: - SOD1 study: n = 10 WT; n = 12 SOD1 and n = 14 SOD1 AAV (total 2.5×10 11 vg) - C9 study: n = 16 WT; n = 16 C9 and n = 17 C9 AAV (total 5×10 11 vg)

[0096] For curative treatment, 8-week-old SOD1G93A or 16-week-old C9ORF72 500r mice were anesthetized with 4% isoflurane and then maintained at 2% using 80% air and 20% oxygen. SOD1 animals received an intravenous injection via retro-orbital injection of a total of 2.5×10 11 vg (low dose) or a total of 5×10 11 vg (medium dose) (100 μl volume). WT and non-injected SOD1 animals received an injection of 100 μl of saline solution.

[0097] Details of the cure group: - SOD1 study: · n = 24 WT; n = 27 SOD1 and n = 22 SOD1 AAV (total 2.5×10 11 vg) · n = 5 WT; n = 5 SOD1 and n = 7 SOD1 AAV (total 5×10 11 vg) - C9 study: n = 14 WT; n = 13 C9 and n = 12 C9 AAV (total 5×10 11 vg)

[0098] Behavioral tests Body weight tracking All animals were weighed before injection, and SOD1 animals were weighed weekly and C9ORF72 animals were weighed every two weeks.

[0099] Gripping test The grasping test assesses coordination and is a classic assessment for ALS. Animals were scored before injection and then every two weeks from 6 or 8 weeks to 15 weeks. The animals were supported by the tail, with a score of 1 if the mouse was spasmodic, and then 1 point was added to the score for grasping each hind limb. Results are presented for each test as mean ± SEM for each group, and t-test analysis was performed.

[0100] Inverted test The inversion test assesses strength and is a typical ALS assessment. Animals were scored before injection, and then every two weeks from 6 or 8 weeks to 15 weeks. Animals were placed on a grid, the grid was inverted, and the remaining paws attached to the grid were immediately scored (short-time inversion). Results were presented for each test as mean ± SEM for each group, and t-test analysis was performed.

[0101] For the C9 model, mice were further challenged to an inverted test consisting of three 5-minute trials, with a 15-minute recovery period between each trial, in order to obtain more robust results. For each trial, the time the objects remained attached was scored, and the average was calculated for each animal.

[0102] Bar with cutouts Coordination was assessed using the notched bar test (scoring the number of times the upper or hind limbs fell), as previously described (Piguet F et al. 2018).

[0103] Survival assessment The mice were observed daily, and were euthanized immediately if they met the endpoint criteria (dragging of the hind limbs or loss of ≥20% of body weight).

[0104] Organizational collection Mice were anesthetized with pentobarbital (Euthasol 180 mg / kg) solution and perfused through the heart with phosphate-buffered saline (PBS). The brain, spinal cord, and sciatic nerve, as well as peripheral organs (liver, heart, lungs, kidneys, spleen), were collected, post-fixed with 4% PFA, and then paraffin-mounted for histology (cut into 6-10 μm sections with a microtome) or immediately frozen in liquid nitrogen for biomolecular analysis.

[0105] Limb muscles were dissected and fixed overnight in 4% PFA / PBS at 4°C. The samples were rinsed three times in PBS and chilled in 20% sucrose / PBS for 48 hours. The tissue was embedded in cryomatrix (Thermoscientific) and sectioned (20 μm) using a cryostat (Leica CM3050S). Frozen sections were dried at room temperature and stored at -20°C.

[0106] The gastrocnemius, tibialis, quadriceps muscles, and a portion of the spinal cord were dissected and frozen in liquid nitrogen.

[0107] Various tissues were ground / pulverized in liquid nitrogen and isolated for RNA or DNA expression analysis.

[0108] Primary striatal cell culture and transfection Primary striatal neurons were previously described (Charvin D et al. 2005; As in Deyts et al. (2009), embryos from pregnant Swiss mice (Janvier) at day 14 were dissected. Transient transfection of striatal cells was performed in vitro at day 7, using Lipofectamine. TMThe procedure was performed using 2000 (Invitrogen). At this stage, a very small number of glial cells were observed (55%; data not shown). Cells were transfected according to five conditions (CYP46A1-HA; SMWR8 WT-HA; SMCR8 D928V-HA, SMWR8 WT-HA + CYP46A1-HA; SMCR8 D928V-HA + CYP46A1HA), and untransfected cells were used as controls. N=6 replications were performed for each condition. Transfection with 100 ng of DNA per plasmid in an 8-well Labtek chamber for 16 hours with Lipofectamine, followed by cell fixation.

[0109] Primary antibody The antibodies used in the immunohistochemistry (IHC) analysis are listed in Table 1 below. Table 1: Antibodies used in immunofluorescence and immunohistochemistry (IHC) analysis [Table 1]

[0110] immunostaining

[0111] against cells The immunofluorescence procedure was initiated by fixing the cells in PFA for 15 minutes. After three washes, the cells were permeabilized in PBS / 0.3% Triton X-100 and then blocked in PBS / 0.1% Triton X-100 containing 5% normal goat serum (NGS, Gibco) at room temperature for 1 hour. The cells were then incubated overnight at 4°C with their respective primary antibodies. After three washes, the cells were incubated at room temperature for 1 hour with the corresponding secondary antibody (1:1000; Vector Laboratories Inc., CA, USA) diluted in PBS / 0.1% Triton X-100. After three washes, the cells were dried and mounted in a mounting medium containing DAPI.

[0112] For paraffin sections Muscle: Frozen muscle sections were treated with 0.1 M glycine in PBS for 30 minutes before processing. After washing in PBS, the sections were permeabilized in PBS / 0.3% TritonX-100 for 10 minutes and saturated in PBS / 0.3% Triton / 10% BSA for 45 minutes. The primary antibody was diluted in saturated solution and incubated overnight at 4°C. After washing in PBS / 0.1% Triton, the secondary antibody and α-bungarotoxin were diluted in PBS / 0.1% Triton / 10% BSA and added at room temperature for 1 hour. After washing in PBS / 0.1% Triton, the slides were mounted using fluorescent aqueous mounting medium (F4680, Sigma).

[0113] The primary antibodies used for immunofluorescence were mouse anti-panneuronal fiber (1:1000; Biolegend, 837904) and rabbit anti-vesicular acetylcholine transporter (VAChT; 1:1000; sigma, 2000559). The secondary antibodies were donkey anti-rabbit / AlexaFluor 594 and anti-mouse / AlexaFluor Cy3 (Life Technologies, Carlsbad, CA, USA), diluted 1:1000.

[0114] α-Bungarotoxin-Alexa594 (1:2000; Life Technologies, B13422) was incubated with the above secondary antibody. Photographs were taken using a confocal SP8 Leica DLS inverted microscope (Carl Zeiss, Zaventem, Belgium). Brightness and contrast of all images were adjusted post-acquisition using ImageJ software to suit observation.

[0115] Spinal cord: For immunofluorescence, spinal cord was treated with 10 mM Tris / 1 mM EDTA / 0.1% Tween pH 8.75 at 95°C for 45 minutes. After washing in PBS, sections were incubated with permeabilization solution (PBS / 0.3% TritonX-100) for 15 minutes, and then with saturated solution (PBS / 0.1% TritonX-100 / 10% horse serum) for 1 hour. Primary antibodies were diluted in 10% horse serum / PBS / 0.1% Triton X-100 and incubated overnight on tissue sections at 4°C. After washing in PBS / 0.1% TritonX-100, sections were incubated with secondary antibodies (donkey anti-rabbit Alexa 594 and donkey anti-mouse Alexa 488, 1:1000, Life Technologies).

[0116] Immunohistochemical labeling was performed using the ABC method. Briefly, tissue sections were treated with peroxide for 30 minutes to inhibit endogenous peroxidase. After washing in PBS, sections were treated with 10 mM Tris / 1 mM EDTA / 0.1% Tween pH 8.75 at 95°C for 45 minutes (for anti-HA only). After washing in PBS, sections were incubated with blocking solution (10% goat serum or goat serum in PBS / 0.3% TritonX-100) for 1 hour. Primary antibodies were diluted in blocking solution and incubated overnight on tissue sections at 4°C. After washing in PBS, sections were sequentially incubated with biotin-conjugated goat anti-rabbit or goat anti-mouse antibody (Vector Laboratories) for 30 minutes at room temperature, followed by sequential incubation with ABC complex (Vector Laboratories). After washing in PBS, peroxidase activity was detected using diaminobenzidine as the chromogen (Dako, Carpinteria, CA). In some cases, the slides were counterstained with hematoxylin. The slides were mounted using Depex (VWR International).

[0117] Luxor dyeing Myelin in spinal cord sections was stained and visualized using classical Luxor staining.

[0118] Image acquisition Immunofluorescence slide images were acquired at room temperature using a Leica DM 5000B microscope equipped with a Leica DFC310FX digital camera, a macroscope (Leica), and LAS V3.8 (Leica) software. Comparative images were taken under identical image acquisition conditions, and all brightness and contrast adjustments were uniformly applied to all images used for cell culture analysis.

[0119] For muscle tissue, VachT images were acquired using ZEN 2.6 software or an axioscan (Zeiss) with nano-zoomer. For all images, brightness and contrast were adjusted post-acquisition using ImageJ software to match the observed values.

[0120] For all IHC and colorization, slices were acquired using a Hamamatsu slide scanner.

[0121] muscle fiber analysis The tibialis anterior (TA), gastrocnemius (gastro), and quadriceps (quadri) muscles were dissected at 15 weeks. They were formalin-fixed, paraffin-embedded, cut (10 mm), and stained with hematoxylin / eosin. Photographs were acquired using axioscan (Zeiss) with ZEN 2.6 software. Muscle fiber cross-sectional area (CSA) was measured for at least 150 fibers for each animal (n=4-13) for the TA, gastro, and quadri muscles using ImageJ software.

[0122] Neuromuscular junction analysis NMJ morphology was quantified for at least 100 TA junctions in three different animals. Endplate distribution was classified into six categories: (1) normal endplates, (2) modified endplates, (3) fragmented endplates, (4) decomposed endplates, (5) poreless endplates, and (6) ectopic endplates. At least 100 junctions were analyzed for each animal (n=3-4). Motor endplate maturation was evaluated in P21 mice based on previously described criteria (Audouard et al.).

[0123] Motor neuron quantification The number of motor neurons in cervical, thoracic, and lumbar spinal cord sections was quantified after immunohistochemistry with VAChT. The number of transduced motor neurons was quantified after immunohistochemistry with HA. Motor neuron counting was performed on the left and right ventral sides of three spinal cord sections for each mouse (n=3).

[0124] Luxor quantitative analysis The total myelin area was measured for the lumbar spinal cord using Fiji software and normalized by the total spinal cord area. The myelin percentage was then reported as 100% in the wild-type (WT) animals, and the results were reported in comparison to WT animals.

[0125] DNA extraction DNA was extracted from the brain, spinal cord, and peripheral organs using a chloroform / phenol protocol.

[0126] RNA extraction Total RNA was extracted from the lumbar spinal cord, gastrocnemius muscle, tibialis muscle, and quadriceps muscle of mice, as well as from the lumbar spinal cord of patients, using Trizol or TriReagent (Sigma). 1 μg of total RNA was transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions.

[0127] q-PCR cDNA was amplified using SyberGreen (Roche). The primers for RT-qPCR were as shown in the table above. Amplification protocols for all primers, hot start (95°C, 5 minutes), 45 amplification cycles (95°C for 15 seconds, 60°C for 1 minute), and melting curve analysis were performed. Data were analyzed using Lightcycler 480 software with efficiency factors for each gene and normalized to actin. [Table 2]

[0128] Vector genome copy number was measured by qPCR using a Light Cycler 480 SYBR Green I Master (Roche, France) on genomic DNA extracted from DRG, spinal cord (cervical, thoracic, and lumbar levels), brain, cerebellum, and peripheral organs. The results (vector genome copy number / cell) were expressed as the n-fold difference in transgene sequence copy number relative to the Adck3 gene copy (viral genome copy number for 2N genome) as an internal standard.

[0129] Cholesterol and oxysterol measurement Cholesterol and oxysterol analysis uses the "optimal standard" method to minimize the formation of auto-oxidation artifacts. 25The procedure followed the same method. Briefly, mouse striatum tissue samples were weighed and homogenized in a 500 μl solution containing butylated hydroxytoluene (BHT, 50 μg / ml) and EDTA (0.5 M) using a Tissue Lyser II instrument (Qiagen). At this point, an internal standard mix [epicoprostanol, 2H7-7-latosterol, 2H6-desmosterol, 2H6-lanosterol, and 2H7-24(R / S)-hydroxycholesterol] (Avanti Polar Lipids) was added. Alkaline hydrolysis was performed at room temperature for 2 hours using 0.35 M ethanolic KOH under Ar. After neutralizing the solution with phosphoric acid, the sterols were extracted in chloroform. The lower layer was collected and dried under a stream of nitrogen, and the residue was dissolved in toluene. Next, oxysterols were separated from cholesterol and its precursors on a 100 mg isolute silica cartridge (Biotage); cholesterol was eluted in 0.5% propan-2-ol in hexane, and subsequently, oxysterols were eluted in 30% propan-2-ol in hexane. The above sterol and oxysterol fractions were then used as previously described. 26 As such, Regisil (registered trademark) The sterols were independently silylated with 10% TMCS [bis(trimethylsilyl)trifluoroacetamide + 10% trimethylchlorosilane] (Regis technologies). Trimethylsilyl ether derivatives of sterols and oxysterols were separated by gas chromatography (Hewlett-Packard 6890 series) in a moderately polar capillary column RTX-65 (65% diphenyl 35% dimethylpolysiloxane, 30 m length, 0.32 mm diameter, 0.25 μm film thickness; Restesk). A mass spectrometer (Agilent 5975 inert XL) was set up in series with the gas chromatograph for cation detection. The ions were generated in electron collision mode at 70 eV. They were identified by fragmentogram in scan mode and compared with appropriate internal and external standards. After normalization and calibration, specific ions were quantified by selective monitoring: [epicoprostanol m / z 370, 2H7-7-latosterol m / z 465, 2H6-desmosterol m / z 358, 2H6-lanosterol m / z 504, 2H7-24(R / S)-hydroxycholesterol m / z 553, cholesterol]. m / z 329, 7-latosterol m / z, 7-dehydrocholesterol m / z 325, 8-dehydrocholesterol m / z 325, desmosterol m / z [343, lanosterol m / z 393, and 24(R / S)-hydroxycholesterol m / z 413].

[0130] Immunofluorescence quantitative analysis of neuronal survival Neuronal survival was evaluated by direct quantification of HA positivity in cells under various conditions, using a Leica microscope at 10× magnification in 5 wells for each condition.

[0131] statistical analysis Statistical analysis was performed using independent student t-tests. Results are expressed as mean ± SEM. Significant thresholds were set to P<0.05, P<0.01, and P<0.001, as defined in the text. All analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, USA).

[0132] result Basic evaluation of the cholesterol pathway in SOD1-containing animals The expression levels of several genes in the cholesterol pathway were measured in 8-week-old SOD1 G93A These were quantified in the lumbar spinal cord of WT animals (Figure 1). Significant decreases in CYP46A1 and ApoE were observed (Figure 1A), as well as a trend for SREBP1, SREBP2, and HMGCR (Figure 1A). Furthermore, 24-hydroxycholesterol was measured at 15 weeks of age, and SOD1 was also measured. G93A Furthermore, when performed on the lumbar spinal cord of WT animals, SOD1 was found to be more effective compared to WT animals.G93A A significant decrease in its content was observed (approximately a 60% decrease) (Figure 1B). These results confirm the hypothesis that CYP46A1 is regulated in the ALS model to rescue the ALS phenotype.

[0133] Verification of AAVPHP.eB as a good vector for ALS The inventors first investigated whether AAVPHP.eB is a good vector for ALS. For this purpose, low doses (2.5.10) were administered to 8-week-old WT animals. 11 vg), medium dose (5.10 11 vg) or high dose (1.10 12 AAVPHP.eB, which encodes CYP46A1-HA (vg), was injected. There was no significant difference between low and high doses, and the three doses highlighted good targeting of motor neurons with a transduction rate of 50-60% (Figure 2) (Figure 2).

[0134] This significant transduction, as assessed by GFAP (Figure 3) and Iba1 (data not shown) staining evaluations, was not associated with any immune response in the spinal cord, and did not result in any microgliosis or astrogliosis, even in the high-dose group (Figure 3).

[0135] These results motivated further research into low-dose vectors.

[0136] Prevention of ALS phenotype in the SOD1G93A mouse model of ALS Overexpression of CYP46A1 is a preventative measure that prevents behavioral changes in a mouse model of ALS.

[0137] The inventors investigated whether upregulation of the cholesterol metabolic pathway, through increased levels of CYP46A1, could improve motor changes in an in vivo model of ALS: the SOD1G93A model. Overexpression of CYP46A1 by prophylactic intravenous administration of AAVPHP.eB-CYP46A1-HA in 3-week-old mice clearly prevented motor changes in the mouse model. Firstly, AAV-treated SOD1G93A mice had improved growth curves (Figure 4A). Prophylactic AAVPHP.eB-CYP46A1-HA delivery significantly corrected motor impairment measured by the grasp test (Figure 4B) and completely prevented changes measured by the inversion test (Figure 4C).

[0138] Biodistribution studies revealed approximately one vector genome copy (VGC) at all levels of the spinal cord, between two and four copies in the brain (Figure 5A), and very low transduction in peripheral tissues, with a maximum of 0.4 VGCs in the heart and less than 0.1 VGCs in other peripheral organs (Figure 5B). Target binding was verified by quantitative analysis of 24-hydroxycholesterol in the lumbar spinal cord of animals, revealing a three-fold increase in treated animals compared to wild-type animals (Figure 5C).

[0139] This increase in 24OH cholesterol correlated perfectly with high CYP46A1-HA expression at all levels of the spinal cord, particularly in motor neurons (MNs) (Figure 6A), and confirmed previously obtained results in wild-type animals (Figure 2).

[0140] Partial preservation of MN was observed at the lumbar level (approximately 60% compared to WT), assessed by quantitative co-staining with VachT and HA (Figures 6B and C), while MN counts were similar to WT in thoracic sections, and no difference was observed at the cervical level (Figure 6C). This 60% preservation is sufficient to avoid demyelination of the lumbar spinal cord, as shown by Luxor staining and quantitative myelin percentage (Figures 6D and E). AAV-treated SOD1 animals had myelin percentages equal to WT animals and significantly higher than untreated SOD1 animals.

[0141] Another aspect that the inventors wanted to investigate was the muscle phenotype. In fact, muscles are severely affected in ALS, following the loss of MN, primarily due to the loss of innervation and neuromuscular junctions (NMJs).

[0142] First, we showed that significant preservation of muscle structure can be assessed by measuring the mean fiber area (Figure 7A). The tibialis muscle of SOD1-treated animals showed a significant increase in mean fiber area compared to untreated animals (Figure 7B), and the reallocation according to fiber cross-sectional area clearly indicated the preservation of fibers in larger cross-sections (Figure 7B). Similar results were observed for the gastrocnemius muscle (Figures 7D and E) and the quadriceps muscle (Figures 7F and 7G).

[0143] Next, focusing on the NMJ phenotype, compared to untreated SOD1 animals, treated animals showed an increased number of innervated junctions (Figures 8A and B), a significantly greater number of NMJs with normal or thicker endplates, and a decreased number of ectopic and fragmented endplates, indicating improved NMJ structure (Figure 8C).

[0144] This result is particularly important and therapeutically significant because, when we evaluated the state of NMJs in WT and SOD1G93A-untreated animals at 3 weeks, we found that NMJs were already pathological in SOD1 animals compared to WT animals, with an increased number of immature junctions after 3 weeks (Figures 8D and E). This means that even when AAV-CYP46A1 is injected, it is still possible to significantly improve the phenotype of treated animals when NMJs do not form properly.

[0145] To complete the study, the inventors also quantified the expression level of Musk in the tibialis muscle at 15 weeks of age. Musk is a receptor involved in the binding of agrin and nAchR (acetylcholine receptor), which are involved in both synaptic stabilization and NMJ maintenance. Expression levels of both MuSK and nAchR are improved in the tibialis and gastrocnemius muscles (Figure 9).

[0146] CYP46A1 overexpression mitigates behavioral changes in a mouse model of ALS after curative treatment. Based on preventive treatment in animals, we investigated whether upregulation of the cholesterol metabolic pathway could improve motor changes in a post-symptomatic in vivo model of ALS: the SOD1G93A model, through increased levels of CYP46A1. Overexpression of CYP46A1 by intravenous administration of AAVPHP.eB-CYP46A1-HA in 8-week-old mice clearly mitigated motor changes in this mouse model. The mice exhibited improved growth compared to untreated animals (Figure 10A).

[0147] Curative administration of AAVPHP.eB-CYP46A1 significantly reduced motor impairment measures measured by the grasp test (Figure 10B) and mitigated changes measured by the inversion test (Figure 10C). Furthermore, the treatment resulted in increased survival in treated mice, along with an average life expectancy increase of 14 days compared to untreated animals (Figure 10D).

[0148] The mice were euthanized at 15 weeks of age for histological and molecular analysis.

[0149] Biodistribution studies revealed approximately 3–4 vector genome copies (VGCs) at all levels of the spinal cord, and between 10–20 copies in the brain (Figure 11A), as well as very low transduction in peripheral tissues (maximum 0.5 VGCs in the liver and less than 0.2 VGCs in other peripheral organs) (Figure 11B). Target binding was verified by quantitative analysis of 24-hydroxycholesterol in the lumbar spinal cord of animals, revealing a 1.3-fold increase in treated animals compared to wild-type animals (Figure 11C).

[0150] Partial preservation of MN was observed at the lumbar level (approximately 60% compared to WT) (Figures 12A and B), similar to the results of prophylactic treatment (Figure 6C), as assessed by quantitative co-staining with VachT and HA (Figure 12A and B).

[0151] This 60% preservation is sufficient to avoid demyelination of the lumbar spinal cord, as repeated here by Luxor staining and quantification of myelin percentage (Figure 12C). AAV-treated SOD1 animals had a myelin percentage equal to that of WT animals and significantly higher than that of untreated SOD1 animals.

[0152] Muscle structure evaluation by measuring average fiber area (Figure 13) revealed a clear phenotypic improvement in treated animals compared to untreated animals. The quadriceps muscle of SOD1-treated animals showed a significant increase in average fiber area compared to untreated animals (Figures 13E and F). Similar results were observed for the tibialis muscle (Figures 13A and B) and the gastrocnemius muscle (Figures 13C and D).

[0153] Next, focusing on the NMJ phenotype, compared to untreated SOD1 animals, treated animals showed an increased number of innervated junctions (Figures 14A and B), a significant increase in the number of NMJs with normal endplates, and a decrease in the number of fragmented and immature endplates, indicating improved NMJ structure (Figure 14C).

[0154] Finally, the effect of treatment on expression levels of both MuSK and nAchR is milder in preventive treatment (Figure 15), but improves as in the gastrocnemius muscle (Figure 15D).

[0155] In summary, these data strongly support CYP46A1 as a relevant target in ALS. Next, the inventors decided to evaluate whether increasing the AAV-CYP46A1 dose could further improve the beneficial effect, and in a cohort of SOD1G93A animals, 5.10 in curative treatment. 11 The mice were injected with vg. Preliminary behavioral results suggest a strong effect and clear improvement in behavior as measured by the grasping score (Figure 16). The mice were present for 12 weeks and will be evaluated for survival.

[0156] Finally, the inventors argued that since CYP46A1 may be a relevant target for both familial and sporadic cellular ALS, it should be targeted not only in patients with SOD1 mutations but also in patients with TARDP and C9ORF72 mutations. To that end, the inventors decided to test their therapeutic strategy against other models of ALS (the first of which is a C9ORF72 mouse model with 500 repeats of GGGGCC).

[0157] The inventors administered 5.1011vg AAV-CYP46A1-HA as a prophylactic treatment to the C9ORF72 model over a period of 4 weeks. Currently, the mice have been followed up to 16 weeks. No differences were observed in the growth curve (Figure 17A). A clear improvement in motor performance was demonstrated based on the notched bar test (Figure 17B). Mouse follow-up is ongoing, and survival will be evaluated.

[0158] Overall, these data support the idea that CYP46A1 overexpression has therapeutic properties, neurophysiological enhancements, and particularly, improved autophagy (Figure 18) in an in vitro model of ALS. Data on post-symptomatic delivery of the treatment encourage treatment of ALS when symptoms have already developed. [ka] [ka]

Claims

1. A composition for use in the preventive treatment of amyotrophic lateral sclerosis (ALS), comprising a vector, the vector comprising a nucleic acid encoding cholesterol 24-hydroxylase, and administered to a presymptomatic subject.

2. The composition for use according to claim 1, wherein the nucleic acid encoding cholesterol 24-hydroxylase encodes the amino acid sequence of SEQ ID NO:

2.

3. The composition for use according to any one of claims 1 to 2, wherein the nucleic acid encoding cholesterol 24-hydroxylase has the sequence of SEQ ID NO:

1.

4. The composition for use according to any one of claims 1 to 3, wherein the vector is selected from the group consisting of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors.

5. The composition for use according to any one of claims 1 to 4, wherein the vector is an AAV vector.

6. The composition for use according to claim 5, wherein the AAV vector is AAV9, AAV10 vector, or AAVPHP.eB.

7. A composition for use according to any one of claims 1 to 6, which is administered intravenously to the subject or directly to the brain.

8. A composition for use according to claim 7, which is administered to the spinal cord and / or motor cortex.

9. A composition for use according to claim 8, which is administered to motor neurons.

10. A composition for use according to any one of claims 1 to 9, administered by intravascular, intravenous, intranasal, intraventricular, or intrathecal injection.

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