Recombinant genetic construct, adeno-associated virus for the treatment of metachromatic leukodystrophy

Abstract

The claimed group of inventions relates to the field of medicine, more precisely to gene therapy for a disease that is incurable as of the date of submission of the application materials - metachromatic leukodystrophy (MLD), which is a severe hereditary neurodegenerative disease that occurs due to deficiency of the lysosomal enzyme arylsulfatase A (ARSA). The essence of the technical solution is a recombinant genetic construct containing CMV / chicken β-actin hybrid enhancer promoter and the codon-optimized ARSA gene sequence represented by SEQ ID NO: 1. Adeno-associated virus of serotype Olig001 for the treatment of metachromatic leukodystrophy, containing a recombinant genetic construct according to claim 1.

Description

[0001] Recombinant genetic construct, adeno-associated virus for the treatment of metachromatic leukodystrophy

[0002] FIELD OF THE INVENTION

[0003] The claimed group of inventions relates to the field of medicine, more precisely to gene therapy for a disease that is incurable as of the date of submission of the application materials - metachromatic leukodystrophy (MLD), which is a severe hereditary neurodegenerative disease that occurs due to deficiency of the lysosomal enzyme arylsulfatase A (ARSA). As a result, ARSA deficiency leads to damage of myelin sheath of the nerve fibers of the central (CNS) and peripheral nervous system (PNS), which covers most of the nerve fibers of the central (CNS) and peripheral nervous system (PNS). Claimed technical solution showsrecombinant genetic construct, containing CMV / chicken 0-actin hybrid enhancer promoter (CMVenCBh) and a codon-optimized ARSA gene sequence, based on which adeno-associated virus serotype OligOOl was obtained (AAVOligOOl- CMVenCBh-coARSA).

[0004] A recombinant genetic construct containing CMV / chicken p-actin hybrid enhancer promoter and a codon-optimized ARSA gene sequence,

[0005] Metachromatic leukodystrophy treatment method helps to restore the deficiency of the ARSA enzyme in the nervous system of a patient with MLD, helping to stop the progression of MLD, resulting in a cure for this disease.

[0006] THE IMPLEMENTATION OF THE INVENTION

[0007] Further, to avoid ambiguous understanding of the text by the applicant, the terms used in the application material and their interpretation are given:

[0008] HSC - hematopoietic stem cells;

[0009] BBB - blood-brain barrier; cDNA - complementary deoxyribonucleic acid;

[0010] BM - bone marrow;

[0011] MLD - metachromatic leukodystrophy; mRNA - messenger ribonucleic acid;

[0012] PNS - peripheral nervous system;

[0013] CSF - cerebrospinal fluid;

[0014] CNS - central nervous system; v-CMV enCBh- ARS A - plasmid vector encoding a codon optimized sequence of the ARSA gene containing a CMVenCBh promoter;

[0015] AAV - Adeno-associated virus;

[0016] AAVOligOOl -CMVenCBh-coARSA - adeno-associated virus serotype OligOOl, containing a unique sequence of the codon-optimized A SA gene, containing the CMVenCBh promoter;

[0017] ARSA - arylsulfase A enzyme;

[0018] ARSA - gene encoding arylsulfatase A;

[0019] CMVenCBh - CMV / chicken [3-actin hybrid enhancer (CBh) promoter;

[0020] IHC - immunohistochemical analysis.

[0021] Codon adaptation index (CAI) - the most widely used method for analyzing codon usage bias. CAI measures the deviation of a protein coding gene sequence relative to a reference set of genes. CAI is used as a quantitative method to predict the expression level of a gene based on its codon sequence.

[0022] GC composition - proportion of guanine (G) and cytosine (C) among all nucleotides in the nucleotide sequence under consideration.

[0023] Hairpin - nucleic acids that are formed when two sequences that are complementary to each other are linked together, bending towards each other and forming an unpaired region at the end - a loop.

[0024] BACKGROUND OF THE INVENTION

[0025] Metachromatic leukodystrophy is a rare hereditary disease from the group of lysosomal storage diseases with an autosomal recessive mechanism of inheritance of metabolic disorders. As of the date of submission of the claimed technical solution, no medicines for the treatment of MLD have been identified in the prior art. Therapy is reduced to the relief of pain and symptoms of the disease. In cases of mild forms of the disease, characterized by moderate manifestations of the clinical picture, there is a possibility of bone marrow transplantation. However, these methods are only being developed and are undergoing clinical trials, as a result of which the possibility of slowing down the progression of the disease, as well as the possibility of completely stopping the development of the pathological process at the level of cells of the central nervous system, will be clarified.

[0026] The brief essence disease is the accumulation of sulfatides contained in myelin, as well as in various cells and tissues of the body, but mainly in the cells of the CNS and PNS. Accumulation arises due to a deficiency of the lysosomal ARSA enzyme. Malfunction or deficiency of the ARSA enzyme occurs due to mutations in the ARSA genes.

[0027] In MLD, sulfatides accumulate in oligodendrocytes, microglia, some CNS neurons, Schwann cells, PNS macrophages, as well as in cells of internal organs, such as the gallbladder, which increases the likelihood of malignant neoplasms of this organ (McFadden K., Ranganathan S., Pathology of the gallbladder in a child with metachromatic leukodystrophy. Pediatr Dev Pathol, 2015. 18(3): p. 228-230]. Clinical manifestations and the degree of neurodegeneration in MLD are varied and depend on the type (kind) of the mutation and the degree of enzyme deficiency. MLD is subdivided into late infantile, juvenile and adult forms [Brown TM, et al., Development of the Impact of Juvenile Metachromatic Leukodystrophy on Physical Activities scale, 2017. 2(1): p. 15.]. Clinical manifestation in the late infantile form of MLD begins before the age of 3 years. This form is considered the most severe and is characterized by severe ARSA deficiency, which leads to rapid neurodegeneration. In addition to the defeat of the central nervous system, peripheral neuropathy is detected. The juvenile form develops at the age of 3-16 years and is characterized by a less pronounced clinical manifestation in comparison with the late infantile form. In late infantile and early juvenile forms, the disease progresses rapidly, and, in the absence of therapy, death occurs within a few years from the onset of the disease. Clinical manifestation in the adult form of MLD usually begins after 16 years of age. The adult form of MLD progresses slowly, often misdiagnosed as early-onset dementia or schizophrenia.

[0028] The claimed technical solution is based on the idea that the delivery of the ARSA enzyme to the CNS of patients with MLD can stop the progression of the disease by restoring the metabolism of sulfatides, the accumulation of which leads to the development of neurodegeneration in patients with MLD. The claimed technical solution ensures the possibility of delivering ARSA using known gene therapy methods. AAV is known to be used to treat several genetic diseases. Moreover, the OligOOl serotype has a preferential tropism for the cell surface of oligodendrocytes, and oligodendrocytes are particularly sensitive to pathological lysosomal accumulation, which leads to demyelination and progressive dysfunction of the nervous system. CMV / chicken p-actin hybrid enhancer (CBh) promoter (CMVenCBh) provides longer and stronger expression of the enzyme. Thus, by using an oligodendrocyte-targeted serotype and a strong promoter to express the transgene, achieving the maximum effect of gene therapy becomes possible. The specified technical result becomes possible due to intrathecal administration of AAVOligOOl- CMVenCBh-coARSA, which prevents MLD development. The claimed technical solution provides the possibility of replenishing the deficiency of the ARSA enzyme in the central and peripheral nervous systems and, as a result, provides the possibility of curing the hereditary disease.

[0029] The claimed technical solution provides the opportunity to solve the technical problem of restoring the enzymatic activity of ARSA in the human body and, as a result, improving the quality of life of patients with MLD with the help of AAVOligOOl-CMVenCBh- coARSA, which corresponds to the conditions of patentability imposed on inventions, namely, absolute novelty and inventive step.

[0030] The resulting AAV, obtained based on a recombinant genetic construct, provides the ability to deliver the missing ARSA enzyme to the CNS and PNS, as a result of which, according to the applicant, it is possible to stop neurodegeneration and improve the quality of life of patients.

[0031] Thus, from the prior art examined by the applicant, it has been revealed that MLD is a severe hereditary neurodegenerative disease caused by a deficiency of the ARSA enzyme, as a result of which there is an accumulation of sulfatides contained in myelin, and as a result of demyelination, severe neurodegeneration and neuroinflammation of the human nervous system develops. The solution to this problem seems relevant due to the absence, as of the date of submission of the claimed technical solution, of effective methods of MLD treatment.

[0032] As of the date of submission of application materials, it was revealed that symptomatic therapy is predominantly used for treatment, while new approaches to the treatment of these diseases are also being actively studied.

[0033] A drug, namely Libmeldy based on hematopoietic stem cells transduced by a lentivirus [https: / / www.libmeldy.eu / ], is known to be expensive - $2.8 million, but when treated with this drug patients who already had symptoms of the disease at the time of therapy, do not have a beneficial effect on human motor activity.

[0034] In addition, promising therapeutic strategies in development that are being actively investigated at the date of submission of application materials have been identified, namely:

[0035] - bone marrow or hematopoietic stem cell transplantation [Hematopoietic Stem Cell Transplantation with Mesenchymal Stromal Cells in Children with Metachromatic Leukodystrophy, Karin Melanie Cabanillas Stanchi et al., 2022],

[0036] - enzyme replacement therapy [Efficacy of enzyme replacement therapy in an aggravated mouse model of metachromatic leukodystrophy declines with age, Frank Matthes, et al., 2012]

[0037] - restoration of functional enzyme expression using gene therapy methods [https : / / www.libmeldy.eu / ] .

[0038] However, these therapies do not show the required level of effectiveness.

[0039] According to the applicant, the lack of effectiveness of known approaches, such as enzyme replacement therapy, is due to the fact that when administered intravenously, drugs do not easily overcome the BBB. Moreover, according to the applicant, the effectiveness of treatment can be increased by direct injection of the recombinant ARSA enzyme into the brain, due to the absence of the need to overcome the BBB. However, such approaches are difficult to apply to humans, since there are quite serious problems:

[0040] - need for serious surgical intervention,

[0041] - poor biodistribution of the therapeutic drug and, as a consequence, the need for multiple injections.

[0042] However, such approaches are being actively studied and are undergoing clinical trials as of the date of submission of application materials.

[0043] Thus, according to the applicant, gene therapy using viral vectors capable of overcoming the BBB may prove to be very effective for the treatment of MLD.

[0044] One of the safest and most effective gene therapy strategies, according to the applicant, is the use of drugs based on recombinant AAVs, which are the most effective (promising) vectors for delivering ARSA to the human nervous system.

[0045] It should be emphasized that AAVs can transsynaptically infect neurons in a wide range from the injection site via anterograde transport [Zingg, B. et al. A AV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors. Neuron 2017, 93, 33-47]. Evidence for this statement is that the introduction of AAV5-ARSA into the brain of mice with a model of MLD allowed for long-term expression of the ARSA gene in the brain [Sevin, C. et al. Intracerebral adeno- associated virus-mediated gene transfer in rapidly progressive forms of metachromatic leukodystrophy] .

[0046] Promising results were also obtained using AAV9 encoding ARSA and a green fluorescent protein reporter gene. Specifically, injection of AAVrh.lO-ARSA into the brains of 8 -month-old mice with MLD resulted in correction of the accumulation of certain types of sulfatides in oligodendrocytes. In a phase Eli clinical trial of this virus (study NCT01801709), animals received 12 injections of AAVrh.lO-ARSA transducing units into the white matter of the brain. However, in experimental animals with early-stage disease, symptoms continued to worsen. A single intravenous administration of AAVPHP.eB- hARSA-HA to mice with a MLD model resulted in stable expression of the ARSA enzyme in the brain and spinal cord and showed complete correction of sulfatide accumulation in the spinal cord of model animals.

[0047] Bone marrow (BM) and hematopoietic stem cell (HSC) transplantation are also often used to treat MLD and other LSDs, since healthy donor cells synthesize the normal enzyme at a physiological level and in some cases, usually in less aggressive forms of the disease, this procedure helps to increase the enzyme activity and alleviate the symptoms of the disease. However, transplantation of BM and HSC without additional genetic modification does not always have a sufficiently high and stable therapeutic effect in the treatment of LSD in general.

[0048] Thus, the above-described methods of treating MLD are not effective enough, which makes the problem of finding new drugs and treatment methods urgent.

[0049] The applicant has analyzed the identified state of the art on scientific and patent information in the field of therapy for MLD and identified several analogues that are currently used to alleviate symptoms and to stop the aggravation of neurodegeneration.

[0050] Several functional (by indication) and structural analogues have been identified from the art search, used to treat lysosomal storage diseases known worldwide, which are virtually incurable as of the date of submission of application materials.

[0051] The invention is known according to patent WO2023133584 “Compositions useful for the treatment of metachromatic leukodystrophy”. The invention is a recombinant replication- deficient adeno-associated virus (rAAV) that can be used to treat a disease associated with a mutation in the arylsulfatase A (ARSA) gene. rAAV contains a vector genome containing inverted terminal repeats (ITRs) and a nucleic acid sequence encoding a functional human arylsulfatase A (hARSA) under the control of regulatory sequences that direct the expression of hARSA in the target cell. Claim: 1. A pharmaceutical composition for the treatment of metachromatic leukodystrophy or a disease associated with a mutation in the arylsulfatase A gene (ARSA), wherein claimed composition comprises a recombinant adeno-associated virus (rAAV) containing the AAVhu68 capsid; and a vector genome) comprising: AAV 5' inverted terminal repeats (ITR), a CB7 promoter containing a CMV IE enhancer and a CB promoter, and a nucleic acid sequence encoding a functional human arylsulfatase A (hARSA) operably linked to a regulatory sequence containing the CB7 promoter that directs expression of hARSA, a polyA signal, and a 3' AAV ITR, wherein the hARSA coding sequence comprises the sequence from nucleotide (nt) 1 to nt 1521 of SEQ ID NO: 1 or a sequence at least 95% to 99.9% identical thereto, which encodes a functional hARSA; and at least one aqueous buffer, at least one carrier, at least one excipient and / or at least one preservative, claimed composition being delivered as a single therapeutic dose by intrathecal administration. 2. The pharmaceutical composition according to claim 1, wherein the regulatory elements further comprise one or more of a Kozak consensus sequence, intron, additional enhancer and / or TATA signal. 3. The pharmaceutical composition according to claim 1 or 2, wherein the sequence encoding hARSA is SEQ ID NO: 1 or SEQ ID NO: 3. 4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the vector genome comprises a 5 '-AAV ITR, an expression cassette having the sequence SEQ ID NO: 28, and 3'- AAV ITR. 5. A pharmaceutical composition according to any one of claims 1-4, wherein the 5'-ITR of AAV has the sequence SEQ ID NO: The 25 and / or 3 -ITR of AAV has the sequence SEQ ID NO: 26. 6. A pharmaceutical composition according to any one of claims 1 to 5, wherein the vector genome comprises nt 1-nt 3883 SEQ ID NO: 5 (SEQ ID NO: 27). 7. A pharmaceutical composition according to any one of claims 1-6, wherein the AAVhu68 capsid is derived from a sequence encoding the amino acid sequence of SEQ ID NO: 7. 8. A pharmaceutical composition according to any one of claims 1 to 5, wherein the composition comprises artificial cerebrospinal fluid comprising a buffered saline solution and one or more of sodium, calcium, magnesium, potassium or mixtures thereof; and a surfactant, wherein the surfactant is optionally present in an amount of from 0.0005% to about 0.001% of the pharmaceutical composition and / or wherein the composition has a pH in the range of from 6.5 to 8.5. 9. A pharmaceutical composition according to any one of claims 1 to 8, wherein the composition is suitable for intracistema magna (ICM) injection or intracerebroventricular administration. 10. A pharmaceutical composition according to any one of claims 1 to 8, wherein a single dose contains from 3x10 10 genome copies (GC) / gram of brain weight to 3.5 x 10 11 GC / gram of brain weight. 11. The pharmaceutical composition of claim 10, wherein the dose is: (a) about 3.3x10 10 genome copies (GC) / gram of brain weight; (b) about 1.1 x10 11 genome copies (GC) / gram of brain weight; or (c) about 3.3x10 11 genome copies (GC) / gram of brain weight. 12. Use of rAAV.hARSA in the manufacture of a drug for the therapeutic treatment of metachromatic leukodystrophy or arylsulfatase A (ARSA) gene mutation-associated disease, said medicament being useful following intrathecal administration of a single dose containing 3x10. From 10 genome copies (GC) / gram brain mass to 3.5 x 10 11 GC / gram brain mass in a patient. 13. Use according to claim 12, wherein the dose is: (a) about 3.3x10 10 genome copies (GC) / gram of brain weight; (b) about 1.1x10 11 genome copies (GC) / gram of brain weight; or (c) about 3.3x10 11 genome copies (GC) / gram of brain weight. 14. The use according to claim 11 or 12, wherein the rAAV comprises an AAVhu68 capsid and a vector genome, wherein said vector genome comprises: AAV 5' inverted terminal repeats (ITR), a CB7 promoter containing a CMV IE enhancer and a CB promoter, and a nucleic acid sequence encoding a functional human arylsulfatase A (hARS A) operably linked to regulatory sequences containing the CB7 promoter that directs expression of hARS A, a polyA signal, and a 3' AAV ITR, wherein the hARS A encoding sequence comprises a nucleotide sequence (nt) from 1 to nt 1521 of SEQ ID NO: 1 or a sequence at least 95-99.9% identical thereto that encodes a functional hARSA. 15. A method of treating a subject suffering from metachromatic leukodystrophy or a disease associated with a mutation in the arylsulfatase A (ARSA) gene, wherein the method comprises administering to the subject a single dose of a recombinant AAV by ICM injection, wherein the recombinant AAV comprises an AAVhu68 capsid and a vector genome packaged therein, wherein said vector genome comprises an AAV ITR, a hARSA encoding sequence comprising SEQ ID NO: 1, or a sequence at least 95% identical thereto that encodes a functional hARSA, and regulatory sequences that direct expression of the functional hARSA in a target cell, wherein a single dose is (i) about 3.3x10 10 genome copies (GC) / gram brain weight; (ii) about 1.1 x10 11 GC / gram brain weight; or (iii) about 3.3 x 10 11 GC / gram brain weight. 16. The method according to claim 15, wherein the rAAV comprises an AAVhu68 capsid; and a vector genome comprising: AAV 5' inverted terminal repeats (ITR), a CB7 promoter containing a CMV IE enhancer and a CB promoter, and a nucleic acid sequence encoding a functional human arylsulfatase A (hARSA) operably linked to regulatory sequences containing the CB7 promoter that drives expression of hARSA, a polyA signal, and a 3' AAV ITR, wherein the hARSA coding sequence comprises the sequence from nucleotide (nt) 1 to nt 1521 of SEQ ID NO: 1 or a sequence at least 95% to 99.9% identical thereto, which encodes a functional hARSA.

[0052] The use of the vector described in the prior art has a drawback - the neurotropic nature of AAVhu68. ARSA must be delivered to oligodendrocytes in the brains of MLD patients, but AAVhu68 does not have these properties, which reduces the drug's effectiveness when used as indicated.

[0053] To solve this problem, it is necessary to develop a more effective method - delivery of the gene of the missing enzyme. AAVOligOOl-CMVenCBh-coARSA can transduce oligodendrocytes, which will stop neurodegeneration and restore the level of the ARSA enzyme.

[0054] An invention under patent RU2692251C2 "Adeno-associated virus-mediated gene transfer to the central nervous system” has been identified from the art search. A brief essence of the prior art is a method for preventing or treating mucopolysaccharidosis type I in humans, which consists in intrathecal administration of a composition based on a recombinant adeno-associated virus (rAAV) 9 and rhlO serotypes containing an open reading frame encoding alpha-L-iduronidase, the essence is a Method for preventing, inhibiting or treating one or more symptoms of mucopolysaccharidosis type I (MPS I) in humans, which includes intrathecal administration to a person in need thereof of a composition containing an effective amount of a vector based on a recombinant adeno- associated virus (rAAV) containing an open reading frame encoding alpha-L-iduronidase for preventing, inhibiting or treating one or more symptoms of MPS I, wherein rAAV is rAAV9 or rAAVrhlO. 2. The method according to claim 1, wherein the person is an adult. 3. The method of claim 1, further comprising administering to a subject an effective amount of a permeability enhancer. 4. The method according to claim 3, wherein the composition contains a permeability enhancer. 5. The method according to claim 3 or 4, wherein the permeability enhancer comprises mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether or EDTA. 6. The method according to claim 1, further comprising administering an immunosuppressant to the subject. 7. The method according to claim 6, wherein the immunosuppressant comprises cyclophosphamide. 8. The method according to claim 6, wherein the immunosuppressant comprises a glucocorticoid, cytostatic agents including an alkylating agent, antimetabolite, cytotoxic antibiotic, antibody or immunophilin-active agent. 9. The method according to claim 7 or 8, wherein the immunosuppressant comprises nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25-) or CD3- antibodies, IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN- , IFN-y, opioid or TNF-a (tumor necrosis factor-alpha) binding agent. 10. The method according to claim 6, wherein the rAAV and the immunosuppressant are administered together, or the immunosuppressant is administered after the rAAV. 11. The method according to claim 6, wherein the immunosuppressant is administered intrathecally. 12. The method according to claim 6, wherein the immunosuppressant is administered intracerebroventricularly. 13. The method according to claim 1, wherein the subject is immunologically tolerant to alpha-L- iduronidase. 14. The method according to any of claims 1-13, wherein alpha-L-iduronidase is damaged or deficient, resulting in a lysosomal storage disease. 15. The method according to any of claims 1-13, wherein the subject is an immunocompetent adult mammal. 16. The method according to any of claims 1-13, wherein the rAAV vector is the rAAV-9 vector. 17. The method according to any of claims 1-13, wherein the rAAV vector is the rAAV rhlO vector.

[0055] Thus, the prior art describes the intrathecal delivery of rAAV9 and the rAAVrhlO vector for the prevention, inhibition or treatment of one or more symptoms of MPS I, showing an absence or deficiency associated with accumulation of a lysosomal enzyme.

[0056] However, the therapeutic effect of vectors containing these capsids has not been studied specifically for the treatment of MLD. To solve this problem, it is necessary to develop and study specific vectors containing the AJiSA gene, as well as methods that allow safe modification of cells of the entire nervous system for long-term expression of a healthy gene. The use of AAVOligOOl-CMVenCBh-coARSA allows achieving a high level of transduction of nervous system cells, due to the ability of these vectors to neurotropism and to effectively transduce CNS cells, thereby ensuring the delivery of the enzyme and increasing its activity.

[0057] From the studied state of the art, an invention was revealed specified in patent RU2671503C2 «Methods and compositions for CNS delivery of arylsulfatase A". The essence consists in intrathecal administration of a composition containing arylsulfatase A (ARSA) protein, polysorbate and phosphate for the treatment of metachromatic leukodystrophy disease. Moreover, the specified phosphate is contained in an amount of not more than 10 mM. The group of inventions ensures successful intrathecal administration of the composition without significant toxicity and immune response. The essence consists in a stable composition for intrathecal administration, containing arylsulfatase A (ASA) protein in a concentration of at least 5 to 50 mg / ml, polysorbate and phosphate, wherein said phosphate is contained in an amount of not more than 10 mM. 2. Stable composition according to claim 1, characterized in that the ASA protein is present in a concentration selected from 10, 30 or 50 mg / ml. 3. Stable composition according to claim 1, characterized in that it additionally contains NaCl. 4. Stable composition according to claim 1, characterized in that the ASA protein contains the amino acid sequence SEQ ID NO: 1. 5. Stable composition according to any of claims 1-4, characterized in that the ASA protein is obtained from a human cell line. 6. Stable composition according to any of claims 1-4, characterized in that the ASA protein is obtained from CHO cells. 7. Stable composition according to claim 3, characterized in that NaCl is present in a concentration of up to 300 mM. 8. Stable composition according to claim 3, characterized in that NaCl is present in a concentration range of approximately 137-154 mM. 9. Stable composition according to claim 8, characterized in that NaCl is present at a concentration of approximately 154 mM. 10. Stable composition according to any of claims 1-4, characterized in that said polysorbate surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and combinations thereof. 11. Stable composition according to claim 10, characterized in that said polysorbate surfactant is polysorbate 20. 12. Sable composition according to item 11, characterized in that polysorbate 20 is present in a concentration of up to 0.2%. 13. Stable composition according to claim 12, characterized in that polysorbate 20 is present in a concentration of approximately 0.005%. 14. Stable composition of claim 1, further comprising a buffering agent selected from the group consisting of acetate, histidine, succinate, citrate, Tris, and combinations thereof. 15. Stable composition according to any of claims 1 -4, characterized in that the stable composition has a pH of approximately 3-8.0. 16. Stable composition according to claim 15, characterized in that the stable composition has a pH of approximately 6.0-6.5. 17. Stable composition according to claim 16, characterized in that said stable composition has a pH of approximately 6.0. 18. Stable composition according to any of claims 1-4, characterized in that the said composition is a liquid composition. 19. Stable composition according to any of claims 1 -4, characterized in that the said composition is made in the form of a lyophilized dry powder. 20. Stable composition according to paragraph 1, characterized in that said composition additionally contains a stabilizing agent. 21. Stable composition according to claim 20, characterized in that said stabilizing agent is selected from the group consisting of sucrose, glucose, mannitol, sorbitol, PEG 4000, histidine, arginine, lysine, phospholipids and combinations thereof. 22. Metachromatic leukodystrophy (MLD) disease treatment method, comprising intrathecal administration to a subject in need thereof of a composition according to any one of claims 1-21. 23. The method according to claim 22, characterized in that intrathecal administration is performed once every two weeks. 24. The method according to claim 22, characterized in that intrathecal administration is performed once a month. 25. The method according to claim 22, characterized in that intrathecal administration is performed once every two months. 26. The method according to claim 22, characterized in that intrathecal administration is performed in combination with intravenous administration. 27. The method according to claim 26, characterized in that the intravenous administration is performed not more than once a month. 28. The method according to claim 26, characterized in that the intravenous administration is performed not more than once every two months. 29. The method according to claim 22, characterized in that intrathecal administration is performed in the absence of intravenous administration. 30. The method according to claim 22, characterized in that intrathecal administration is performed in the absence of concomitant immunosuppressant therapy.

[0058] Thus, the invention describes a method for delivering a composition consisting of the ARSA protein, polysorbate and phosphate into the central nervous system by intrathecal administration without significant toxicity and immune response. The disadvantages of the prior art are that, firstly, when introducing the enzyme into the of cerebrospinal fluid (CSF), there is a risk of complications. - secondly, there is a difficulty in achieving a uniform distribution of the enzyme throughout the nervous system; In addition, previous clinical trials of similar products in patients with other LSDs affecting the CNS did not show the expected effectiveness, neurological disorders continued.

[0059] The closest analogue in technical essence and achieved technical result is the invention under patent RU 2769577 “Preparation for the treatment of metachromatic leukodystrophy and its treatment method”. The essence consists in a gene and gene-cell preparation for the therapy of MLD, consisting of intravenous or intrathecal administration of a preparation containing a recombinant adeno-associated virus of serotype 9, with a unique sequence of the codon-optimized ARSA gene (AAV9-coARSA) or in the transplantation of human mesenchymal stem cells (MSCs), genetically modified AAV9-coARSA (MSCs-ARSA). Claim: A drug for the treatment of metachromatic leukodystrophy, comprising a recombinant adeno-associated virus serotype 9 containing a codon-optimized ARSA gene sequence presented in SEQ ID NO:1 A drug for the treatment of metachromatic leukodystrophy, consisting of mesenchymal stem cells genetically modified with a recombinant adeno-associated virus serotype 9 containing a codon-optimized ARSA gene sequence presented in SEQ ID NO: 1. A method for treating metachromatic leukodystrophy, which consists of a single intravenous administration of the drug according to claim 1 or 2 or a single intrathecal administration of the drug according to claim 1.

[0060] The disadvantage of the prior art is the short-term expression of the transgene, which subsequently leads to the progression of the disease. Also, serotype 9 does not target oligodendrocyte transduction, which reduces the effectiveness of the drug when used as indicated.

[0061] In this case, the use of AAVOligOOl-CMVenCBh-coARSA according to the declared technical solution allows to achieve a high and long-term level of transduction of cells of the human nervous system due to the ability of this vector to oligodentrocyrian tropism, thereby ensuring the delivery of the enzyme to the central nervous system and increasing its activity.

[0062] To solve the technical problems described above, it is necessary to develop a method that allows the correction of cells of the nervous system, namely, modification for long-term expression of a healthy gene. When administered intrathecally AAVOligOOl-CMVenCBh-coARSA oligodendrocytes will be transduced, CMVenCBh promoter will provide strong and long- lasting expression. This will allow long-term expression of ARSA.

[0063] SUMMARY OF THE INVENTION

[0064] Technical result of the claimed technical solution is:

[0065] - development of recombinant genetic construct containing CMV / chicken p-actin hybrid enhancer promoter and a codon-optimized ARSA gene sequence represented by SEQ ID NO:1;

[0066] - development of adeno-associated virus - AAVOligOOl-CMVenCBh-coARSA, providing the possibility of MLD therapy.

[0067] Essence of the claimed technical solution is a recombinant genetic construct containing CMV / chicken p-actin hybrid enhancer promoter and the codon-optimized ARSA gene sequence represented by SEQ ID NO:1. Adeno-associated virus of serotype OligOOl for the treatment of metachromatic leukodystrophy, containing a recombinant genetic construct according to claim 1.

[0068] BRIEF DESCRIPTION OT THE DRAWING

[0069] Claimed technical solution is illustrated in Fig. 1 - Fig. 5.

[0070] In Fig, la, lb, lc, Id, le. If, lg, Ih there are diagrams showing the level of ARSA enzymatic activity in homogenates of pig CNS organs on the 35th day after administration of AAVOligOOl-CMVenCBh-coARSA. Y-axis indicates ARSA enzymatic activity (nM / mg / h). The X-axis shows samples of organ homogenates from the group of intact pigs (designation control), samples of organ homogenates from pigs that were intrathecally injected with recombinant AAVOligOOl-CMVenCBh-coARSA: Fig. la -right frontal lobe, Fig. lb - parietal lobe, Fig. lc - occipital lobe of the cerebral cortex, Fig. Id - cerebellum, Fig. le - brainstem, Fig. If - cervical spinal cord, Fig. lg - thoracic spinal cord, Fig. lh - lumbar spinal cord.

[0071] The X-axis from left to right shows the data for the following samples: Fig. la - sample of the right frontal lobe of pigs (control results, results of experimental pig No.l, results of experimental pig No. 2).

[0072] Fig. lb - sample of parietal lobe of pigs (control results, results of experimental pig No. 1, results of experimental pig No. 2).

[0073] Fig. lc - sample of the occipital lobe of pigs (results of experimental pig No.l, results of experimental pig No.2, results of experimental pig No. 3).

[0074] Fig. Id - sample of cerebellum of pigs (control results, results of experimental pig No. 1, results of experimental pig No. 2, results of experimental pig No. 3).

[0075] Fig. le - sample of trunk of pigs (control results, results of experimental pig No. 1, results of experimental pig No. 2).

[0076] Fig. If - sample of cervical spinal cord of pigs (control results, results of experimental pig No. 1, results of experimental pig No. 2, results of experimental pig No. 3).

[0077] Fig. 1g - sample of thoracic spinal cord of pigs (control results, results of experimental pig No. 1, results of experimental pig No. 2, results of experimental pig No. 3).

[0078] Fig. lh - sample of lumbar spinal cord of pigs (control results, results of experimental pig No. 1, results of experimental pig No. 2, results of experimental pig No. 3).

[0079] Statistical significance is a way to understand how reliable and non-random the results of a study or experiment are. Significance is indicated by asterisks:

[0080] - p < 0.05 - the probability of obtaining such results by chance is less than 5%. The results are considered statistically significant.

[0081] - **: p < 0.01 — the probability of obtaining results by chance is less than 1%. This is a stricter criterion, and the results are considered more reliable.

[0082] - ***: p < 0.001 — the probability of obtaining results by chance is less than 0.1%. This is a very high significance value, indicating that the results are truly important.

[0083] > **** .nQt usuaUyuse(iinscientific practice, but if it was, it could indicate an even more strict criterion.

[0084] In Fig. 2a and 2b present diagrams showing the dynamics of ARSA enzymatic activity before administration, on days 7 and 28. Y-axis indicates ARSA enzymatic activity (nM / mg / h). The X-axis shows samples of cerebrospinal fluid and plasma from pigs No. 1- No. 3, which were injected intrathecally with recombinant AAVOligOOl-CMVenCBh- coARSA: Fig. 2a - in the cerebrospinal fluid, Fig. 2b - in pig plasma. X-axis of Fig. 2a from left to right shows the data of the following samples:

[0085] Cerebrospinal fluid sample from a control pig (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on the 7th and 28th day);

[0086] Cerebrospinal fluid sample from experimental pig No. 1 (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on days 7 and 28);

[0087] Cerebrospinal fluid sample from experimental pig No. 2 (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on day 28);

[0088] Cerebrospinal fluid sample from experimental pig No. 3 (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on days 7 and 28).

[0089] X-axis of Fig. 2b from left to right shows the data of the following samples:

[0090] Plasma sample from the control group pig (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on the 7th and 28th day);

[0091] Plasma sample of experimental pig No. 1 (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on days 7 and 28);

[0092] Plasma sample of experimental pig No. 2 (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on days 7 and 28);

[0093] Plasma sample from experimental pig No. 3 (before administration of recombinant AAVOligOOl-CMVenCBh-coARSA, on days 7 and 28).

[0094] In Fig. 3a, 3b, 3c, 3d are diagrams showing the number of copies of the ARSA gene mRNA in the nervous system organs of pigs on the 35th day after administration of AAVOligOOl-CMVenCBh-coARSA. The X-axis shows the RNA samples. The Y-axis shows the number of copies of ARSA gene mRNA per 1 pg of total RNA. Data obtained by quantitative PCR. Fig. 3a on the X-axis from left to right shows RNA samples of the parietal lobe (for experimental pig No. 2, for experimental pig No. 3), occipital lobe of the brain (for experimental pig No. 2, for experimental pig No. 3), Fig. 3b - cerebellum (for control, for experimental pig No. 2), brainstem (for experimental pig No. 2, for experimental pig No. 3), subcutaneous nerve (for control, for experimental pig No. 2, for experimental pig No. 3), Fig. 3c - cervical spinal cord (for control, for experimental pig No. 2, for experimental pig No. 3), thoracic spinal cord (for control, for experimental pig No. 2, for experimental pig No. 3), lumbar spinal cord (for control, for experimental pig No. 2, for experimental pig No. 3), Fig. 3d - dorsal root ganglia of the cervical spinal cord (for experimental pig No. 2, for experimental pig No. 3), dorsal root ganglia of the thoracic spinal cord (for control, for experimental pig No. 2, for experimental pig No. 3), dorsal root ganglia of the lumbar spinal cord (for control, for experimental pig No. 3).

[0095] Fig. 4a, 4b, 4c, 4d shows diagrams where the Y-axis shows the data of the level analysis in pig blood serum: Fig. 4a - alanine aminotransferase (ALT), U / l; Fig. 4b - aspartate aminotransferase (AST), U / l; Fig. 4c - creatinine- J, mg / dl; Fig. 4d - total bilirubin, mg / dl. The X-axis shows serum samples from experimental pigs with intrathecal administration of recombinant AAVOligOOl-CMVenCBh-coARSA after 0, 7, 28 days. Data obtained using enzyme immunoassay (ELISA). Figures 4a, 4b, 4c, 4d show groups of pigs:

[0096] - Control pig, « -Experimental pig 1 , - Experimental pig 2, - experimental pig 3.

[0097] Fig. 5a and 5b show images of organ sections (obtained by confocal microscopy) showing the analysis of ARSA expression by immunohistochemical analysis of cryostat sections of CNS organs - in the top row of Fig. 5a and 5b of the control pig without administration (control) and in the bottom row of Fig. 5a and 5b samples of organ sections from experimental pigs with intrathecal AAVOligOOl-CMVenCBh-coARSA: fig. 5a -from left to right, section of the cerebellar cortex, section of the occipital lobe cortex, section of the lumbar spinal cord, fig. 5b - from left to right, section of the spinal ganglion of the cervical spinal cord, section of the spinal ganglion of the thoracic spinal cord, section of the spinal ganglion of the lumbar spinal cord.

[0098] DETAILED DESCRIPTION OF THE INVENTION

[0099] Further, the applicant provides a description of the claimed technical solution.

[0100] The identified problem and the claimed technical result are achieved by intrathecal administration of adeno-associated virus with serotype OligOOl, containing recombinant genetic construct with the CMVenCBh promoter, with a unique codon-optimized ARSA gene sequence - AAVOligOOl-CMVenCBh-coARSA (hereinafter - AAVOligOOl-CMVenCBh- coARSA). The sequence represented by SEQ ID NO: 1 is given in the Appendix. The idea of the claimed technical solution is generally based on the fact that due to the ability of the OligOOl serotype to effectively transduce oligodendrocytes involved in the pathogenesis of MLD, CMVenCBh promoter initiates long-term and strong transgene expression. This allows achieving a therapeutic effect. As a result of the development, we obtained AAVOligOOl-CMVenCBh-coARSA, which has a therapeutic effect unknown from the prior art, providing the possibility of treating the previously incurable MLD disease.

[0101] The practical implementation of the claimed invention is carried out in 3 stages in the following sequence, namely:

[0102] Stage 1: Obtaining and analyzing a recombinant genetic construct containing CMV / chicken p-actin hybrid enhancer promoter and the codon-optimized AJiSA gene sequence represented by SEQ ID NO:1.

[0103] Stage 2: Production and analysis of the functionality of adeno-associated virus serotype OligOOl - AAVOligOOl-CMVenCBh-coARSA, containing a recombinant genetic construct, obtained at stage 1.

[0104] Stage 3: Study of efficacy and safety of AAVOligOOl-CMVenCBh-coARSA when administered intrathecally to large laboratory animals.

[0105] The applicant further provides a detailed description of the implementation stages of the claimed technical solution.

[0106] Example 1. Stage 1 - obtaining and analyzing a recombinant genetic construct containing the CMV / chicken 3-actin hybrid enhancer promoter and the codon-optimized AUSA gene sequence, represented by SEQ ID NO:1.

[0107] Codon optimization of the nucleotide sequence of a gene is performed. To optimize the codon composition of the AJiSA gene, well-known algorithms are used with the OptimumGene program (GeneScript, USA). There are many factors that influence the level of gene expression, and the algorithm considers as many of them as possible, creating a single gene that can achieve the highest possible level of expression. At the same time, the native gene uses tandem rare codons, which can reduce the efficiency of translation or even turn off the translation mechanism. Codon bias in the gene increased the codon adaptation index from 0.83 to 0.90, which is considered desirable for the highest level of gene expression. The GC composition change was optimized to increase the half-life of mRNA. Hairpin structures that affect ribosome binding and mRNA stability were disrupted. In addition, negative cis sites were tested and successfully modified during the optimization process.

[0108] The synthesis and cloning of the codon-optimized cDNA of the ARSA gene into the plasmid vector pAAV-MCS (Addgene, USA) is carried out by GenScript (USA). Correct assembly of the recombinant genetic construct pAAV-CMVenCBh-coARSA is confirmed by restriction analysis.

[0109] To confirm the functionality of the genetic recombinant construct containing the CMV / chicken -actin hybrid enhancer promoter and the codon-optimized ARSA gene sequence (CMVenCBh-ARSA), it was transfected (genetically modified) into the immortalized primary human embryonic kidney cell line HEK293T. To do this, use the transfection agent TurboFect (Thermo Fisher Scientific Inc., USA) in accordance with the method recommended by the manufacturer. To assess the transfection efficiency, the plasmid vector pAAV-CMVenCBh-Katushka2S encoding the far-red fluorescent protein was used as a positive control.

[0110] The efficiency of in vitro expression of the recombinant protein using the resulting plasmid construct is confirmed by an activity test and Western blot analysis.

[0111] ARSA enzyme activity is determined in HEK293T lysate 24 hours after transfection. The concentration of total protein in the samples was determined using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, USA). Samples are normalized to total protein concentration. To determine ARSA activity, 50 pl of cell lysate sample was incubated with a nitrocatechol sulfate substrate solution (0.01 M p-Nitrocatechol sulfate dipotassium salt (#N7251, Sigma), 0.5 M sodium acetate, 0.5 mM Na PiO?, 10% sodium chloride, pH = 5) for 1 hour at 37 °C, after which the reaction was stopped by adding 1 N sodium hydroxide. Sulfatase dilutions (#S9626, Sigma) are used as standards. Optical density is measured at a wavelength of 515 run.

[0112] Western blot analysis was performed using primary rabbit polyclonal antibodies to ARSA (Cat. No. PAG619Hu01, Cloud-Clone Corp., USA) dilution 1:500 in blocking buffer.

[0113] According to the results of the applicant's analysis, the expected sizes of approximately 33 kDa were obtained in samples of HEK293T cell lysates transfected with pAAV- CMVenCBh-coARSA. Example 2. Stage 2 - obtaining and analyzing the functionality of the adeno- associated virus of serotype QligOOl - AAVOligOOl-CMVenCBh-coARSA, containing the recombinant genetic construct according to Example 1.

[0114] Based on the recombinant genetic construct pAAV-CMVenCBh-coARSA, AAVOligOOl -CMVenCBh-coARSA was obtained. To obtain AAVOligOOl -CMVenCBh- coARSA AAV Helper free system was used. AAV293 cells were inoculated at 1.5 million, the monolayer was 70-80%. The next day, co-transfection was carried out using the calcium phosphate method with three plasmids (vector plasmid, pAAV-RC and pHelper). AAVOligOOl-CMVenCBh-coARSA was harvested 72 hours after transfection. Cells are harvested with a scraper, cryolyzed, centrifuged at 10,000 x g for 10 minutes to get rid of cell debris. The viral stock is stored at minus 80 °C. AAVOligOOl-CMVenCBh-coARSA was concentrated using the AAV Purification Mega Kit (Cell Biolabs, Inc., USA) according to the manufacturer's recommended protocol.

[0115] Example 3. Stage 3 - studying the efficacy and safety of AAVOligOOl-CMVenCBh- coARSA with intrathecal administration to large laboratory animals.

[0116] Efficacy and safety of the obtained AAVOligOOl -CMVenCBh-coARSA was tested on large laboratory animals.

[0117] The study used 4-month-old pigs (weight 5 kg), which were randomly divided into 2 groups, with three individuals in each group - a control group and an experimental group. Pigs were intrathecally (i.e. by administration of AAVOligOOl -CMVenCBh-coARSA into the spinal cord) administered claimed AAVOligOOl -CMVenCBh-coARSA (n=3) in the amount of 2 x 1013genomic copies / kg, while the control group was not administered anything:

[0118] 1) Control group of intact pigs without administration of AAVOligOO 1 -CMVenCBh-coARSA.

[0119] 2) An experimental group of pigs was administered AAVOligOOl - CMVenCBh-coARSA intrathecally at a dose of 2 x 1013genomic copies / kg.

[0120] The animals were kept in specialized premises of the Kazan State Academy of Veterinary Medicine named after N.E. Bauman (KSAVM) under the supervision of qualified personnel. All experiments were performed in accordance with ethical standards and current legislation.

[0121] The experiment was carried out as follows. Before administration, samples of CSF and whole blood were collected from pigs in test tubes containing the anticoagulant sodium citrate and gel, activator (in all experimental animals), then plasma and serum were isolated from whole blood by centrifugation for 20 minutes at 1900 rpm, CSF, plasma and serum were stored at minus 80 °C.

[0122] Then, in all experimental animals, i.e. 7 and 28 days after administration of AAVOligOOl-CMVenCBh-coARSA whole blood and CSF samples were collected again.

[0123] Next, whole blood plasma and CSF samples were used to determine the enzymatic activity of ARSA.

[0124] Subsequently, on day 35 after the administration of AAVOligOOl-CMVenCBh- coARSA, pigs were euthanized using methods that comply with the principles set out in the European Commission Guidelines for the Euthanasia of Experimental Animals.

[0125] Next, the spinal cord (cervical, thoracic, lumbar regions), posterior root ganglia (cervical, thoracic, lumbar regions), cerebellum, occipital lobe cortex, latent nerve, brainstem, right frontal lobe, and parietal lobe were removed.

[0126] All organs were then homogenized for activity testing and RT-PCR or dissected for IHC analysis.

[0127] Next, based on the obtained experimental data, graphs were constructed, shown in Fig. 1-5, which illustrate the results described in Steps 1-3.

[0128] The graphic materials in Fig.l - Fig.5 experimentally proved the possibility of achieving the stated technical results.

[0129] From the data shown in figure 1, it is evident that the enzymatic activity of ARSA in homogenates of CNS organs increased after administration of AAVOligOOl-CMVenCBh- coARSA. In the homogenate of the occipital lobe of the cerebral cortex, ARSA activity increased by 150% in 1 pig. In cerebellar homogenate, a 318% increase in activity was detected in one pig. In brainstem homogenate, a l l 7% increase in activity was detected in one pig. In the cervical spinal cord, an increase in ARSA activity was detected in pigs 1 and 3 by 193% and 137%, respectively. In the thoracic spinal cord, an increase in ARSA activity was detected in all pigs by 105%, 127% and 116%, respectively. In the lumbar spinal cord, an increase in ARSA activity was detected in pigs 1 and 2 by 176% and 123%, respectively. These results show that after genetic modification with the obtained AAVOligOOl- CMVenCBh-coARSA, nervous system cells begin to express a functionally active enzyme in vivo. From the data shown in figure 2, it is evident that after intrathecal administration of AAVOligOOl-CMVenCBh-coARSA, ARSA activity increased in CSF on day 28. In plasma - on day 7. These results indicate that intrathecal administration of AAVOligOOl- CMVenCBh-coARSA to large laboratory animals results in the transduction of cells that then begin to express and secrete a functionally active enzyme that is detected in the plasma and CSF of the animals.

[0130] From the data shown in figure 3, the presence of overexpression of the codon- optimized AJiSA gene in the CNS organs of pigs that have been intrathecally injected with AAVOligOOl-CMVenCBh-coARSA is evident. In the cerebellum, in the subcutaneous nerve, in the cervical, thoracic, lumbar spinal cord, in the ganglia of the posterior roots of the thoracic, lumbar spinal cord of pig No. 1, 4215, 462, 947, 854716, 93228, 2922, 411 copies of AJ?SA gene mRNA per pg of total RNA were detected, respectively. In the cerebellum, brainstem, saphenous nerve, parietal lobe, occipital lobe, cervical, thoracic, lumbar spinal cord, and in the ganglia of the posterior roots of the cervical and thoracic spinal cord of pig No. 2, 5480, 346, 300, 974, 139, 871, 4965, 1501, 1129, 1039 copies of AKS4 gene mRNA per pg of total RNA were detected, respectively. In the brainstem, saphenous nerve, parietal lobe, occipital lobe, cervical, thoracic, lumbar spinal cord, and dorsal root ganglia of the cervical, thoracic, and lumbar spinal cord of pig No. 3, 88, 973, 5472, 205, 761, 1627, 1700, 1957, 1016, and 579 copies of AJiSA gene mRNA per pg of total RNA were detected, respectively.

[0131] From the data shown in figure 4, it is evident that the biochemical parameters in the blood serum of pigs after intrathecal administration of AAVOligOOl-CMVenCBh-coARSA showed an increase in the levels of AST, creatinine-J and bilirubin on the 7th day in one animal. In the remaining experimental animals, the parameters remain unchanged (AST, creatinine-J, total bilirubin). This proves that intrathecal administration of AAVOligOOl- CMVenCBh-coARSA does not lead to immunological changes in the body of the test animals, which in turn confirms the potential safety of delivery of genetic material encoding the ARSA enzyme.

[0132] From the data shown in figure 5, it is evident that the analysis of ARSA expression in cryostat sections of the nervous system organs of pigs by immunohistochemical analysis confirms the successful genetic modification of the nervous system cells of experimental animals after intrathecal administration of AAVOligOOl-CMVenCBh-coARSA. In the cerebellar cortex, Purkinje cells were found to overexpress ARSA compared to controls. Analysis of the occipital lobe of the brain revealed a significant increase in ARSA expression in neurons of the upper layers of the cerebral cortex. Analysis of cross-sections of the cervical and thoracic spinal cord did not show significant differences in ARSA expression in the gray matter of animals in the experimental and control groups. However, single ARSA-overexpressing neurons were found in the anterior and posterior horns of the lumbar spinal cord. Analysis of dorsal root ganglia at the level of the cervical, thoracic and lumbar spinal cord revealed ARSA-overexpressing neurons. Analysis of spinal nerve roots and the sciatic nerve revealed no differences in ARSA expression between animals in the experimental and control groups.

[0133] From the above, it can be concluded that the applicant has solved the identified technical problems and achieved the claimed technical result, namely:

[0134] - recombinant genetic construct was developed (see Example 1);

[0135] - adeno-associated virus serotype OligOOl containing the specified recombinant genetic construct - AAVOligOOl-CMVenCBh-coARSA, providing the possibility of MLD therapy was developed (see Examples 2, 3, Fig. 1-5).

[0136] The claimed technical solution satisfies the “novelty” patentability condition for inventions, since no sources have been identified from the studied prior art that describe features that match in terms of the function they perform and the form of execution of these features listed in the claims, including the purpose characteristic.

[0137] The claimed technical solution satisfies the patentability condition of “inventive step” for inventions, since the applicant has not identified technical solutions from the studied state of the art, characterized using a method for treating MLD, which involves intrathecal administration of recombinant AAVOligOOl-CMVenCBh-coARSA. In addition, the claimed technical solution, according to the applicant, is not obvious to a specialist, since it ensures the implementation of the task of stopping neurodegeneration and significantly alleviating the symptoms of MLD, which is practically incurable at the date of submission of the application materials.

[0138] The claimed technical solution satisfies the "industrial applicability" patentability requirement for inventions, since it can be used on an industrial scale to create products intended for the treatment of MLD.

Claims

CLAIMS1. A recombinant genetic construct containing CMV / chicken P-actin hybrid enhancer promoter, and the codon-optimized ARSA gene sequence represented by SEQ ID NO:1.

2. Adeno-associated virus of serotype OligOOl for the treatment of metachromatic leukodystrophy, containing a recombinant genetic construct according to claim 1.

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