RU2821568C1 - Recombinant genetic construct, adeno-associated virus for therapy of metachromatic leukodystrophy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a recombinant genetic construct for overexpression of arylsulfatase A enzyme containing a promoter of a hybrid CMV enhancer/chicken β-actin and a codon-optimized sequence of the ARSA gene represented by SEQ ID NO: 1. Also disclosed is adeno-associated virus of serotype Olig001 for therapy of metachromatic leukodystrophy, containing recombinant genetic construct according to cl. 1.

EFFECT: invention enables therapy of metachromatic leukodystrophy.

2 cl, 5 dwg, 3 ex

Description

The claimed group of inventions generally relates to the field of medicine, more precisely to gene therapy of a disease that was incurable at the date of submission of application materials - metachromatic leukodystrophy (MLD), which is a severe hereditary neurodegenerative disease resulting from a deficiency of the lysosomal enzyme arylsulfatase A (ARSA). As a result, ARSA deficiency leads to damage to the myelin sheath of nerve fibers of the central (CNS) and peripheral nervous system (PNS), covering most of the nerve fibers of the central (CNS) and peripheral nervous system (PNS). In the claimed technical solution, a recombinant genetic construct was developed containing the promoter of the CMV/chicken β-actin hybrid enhancer (CMVenCBh) and a codon-optimized sequence of the ARSA gene, on the basis of which an adeno-associated virus of serotype Olig001 (AAVOlig001-CMVenCBh-coARSA) was obtained.

Recombinant genetic construct containing the CMV/chicken β-actin hybrid enhancer promoter and a codon-optimized ARSA gene sequence,

A method for treating metachromatic leukodystrophy makes it possible 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.

Below, in order to avoid any ambiguous understanding of the text by the applicant, the terms used in the application material and their interpretation are given:

HSC - hematopoietic stem cells;

BBB - blood-brain barrier;

cDNA - complementary deoxyribonucleic acid;

BM - bone marrow;

MLD - metachromatic leukodystrophy;

mRNA - template ribonucleic acid;

PNS - peripheral nervous system;

CSF - cerebrospinal fluid;

CNS - central nervous system;

pAAV-CMVenCBh-ARSA - plasmid vector encoding a codon optimized sequence of the ARSA gene containing the CMVenCBh promoter;

AAV - adeno-associated virus;

AAVOlig001-CMVenCBh-coARSA is an adeno-associated virus of the Olig001 serotype, containing a unique sequence of the codon-optimized ARSA gene containing the CMVenCBh promoter ;

ARSA - arylsulfase A enzyme;

ARSA - gene encoding arylsulfatase A;

CMVenCBh - CMV/chicken β-actin (CBh) hybrid enhancer promoter;

IHC - immunohistochemical analysis.

The codon adaptation index (CAI) is the most common method for analyzing codon usage bias. CAI measures protein coding deviation, 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.

GC composition is the proportion of guanine (G) and cytosine (C) among all nucleotides of the nucleotide sequence in question.

Hairpin - nucleic acids that are formed when two sequences are complementary to each other and connect to each other, bending towards each other and forming an unpaired section at the end - a loop.

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 drugs for the treatment of MLD were identified from the examined state of the art. Therapy comes down to relieving pain and symptoms of the disease. In the case of mild forms of the disease, characterized by moderate manifestations of the clinical picture, there is the possibility of bone marrow transplantation. However, these methods are only being developed and undergoing clinical trials, as a result of which the possibility of slowing the progression of the disease will be clarified, as well as the possibility of completely stopping the development of the pathological process at the level of cells of the central nervous system.

The brief essence of the 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 central nervous system and PNS. The accumulation occurs due to deficiency of the lysosomal enzyme ARSA. ARSA enzyme dysfunction or deficiency occurs due to mutations in the ARSA genes.

In MLD, sulfatides accumulate in oligodendrocytes, microglia, some neurons of the central nervous system, Schwann cells, macrophages of the PNS, as well as in the 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 (type) of mutation and the degree of enzyme deficiency. MLD is divided into late infantile, juvenile and adult forms [Brown T.M., et al., Development of the Impact of Juvenile Metachromatic Leukodystrophy on Physical Activities scale, 2017. 2(1): p. 15.]. Clinical manifestation in late infantile MLD begins before age 3 years. This form is considered the most severe and is characterized by severe ARSA deficiency, which leads to rapid neurodegeneration. In addition to damage to the central nervous system, peripheral neuropathy is detected. The juvenile form develops at the age of 3-16 years and is characterized by less pronounced clinical manifestations in comparison with the late infantile form. In late infantile and early juvenile forms, rapid progression of the disease is observed and, in the absence of therapy, death occurs within several years from the onset of the disease. Clinical manifestation in the adult form of MLD usually begins after 16 years of age. Adult MPD progresses slowly and is often misdiagnosed as early-onset dementia or schizophrenia.

The claimed technical solution is based on the idea that delivery of the ARSA enzyme to the central nervous system of patients with MLD makes it possible to 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 is to enable the delivery of ARSA using gene therapy methods known as such. AAV is known to be used to treat a number of genetic diseases. In this case, the Olig001 serotype has a predominant tropism for the cell surface of oligodendrocytes, and oligodendrocytes are especially sensitive to pathological lysosomal accumulation, which leads to demyelination and progressive dysfunction of the nervous system. The CMV/chicken β-actin (CBh) hybrid enhancer promoter (CMVenCBh) allows for longer and stronger expression of the enzyme. Thus, for transgene expression using an oligodendrocyte-targeted serotype and a strong promoter, achieving maximum gene therapy effect becomes possible. This technical result becomes possible due to the intrathecal administration of AAVOlig001-CMVenCBh-coARSA, which helps prevent the development of MLD. The claimed technical solution provides the possibility of replenishing the deficiency of the ARSA enzyme in the central nervous system and PNS and, as a consequence, provides the possibility of curing a hereditary disease.

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 using AAVOlig001-CMVenCBh-coARSA, which meets the patentability conditions for inventions, namely world novelty and inventiveness level.

The resulting AAV, obtained on the basis of 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.

Thus, from the level of technology studied by the applicant, it was revealed that MLD is a severe hereditary neurodegenerative disease caused by a deficiency of the ARSA enzyme, as a result of which the accumulation of sulfatides contained in myelin occurs, 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 lack of effective methods for treating MLD as of the date of presentation of the claimed technical solution.

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

A known drug, namely the drug Libmeldy, based on hematopoietic stem cells transduced by a lentivirus [https://www.libmeldy.eu/], is characterized by a high cost - 2.8 million dollars, however, when treated with this drug in patients who there were already symptoms of the disease at the time of therapy, the treatment does not have a beneficial and effective effect on a person’s motor activity.

In addition, promising therapeutic strategies under development have been identified that are actively being studied as of the date of submission of application materials, namely:

- 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],

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

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

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

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 penetrate the BBB well. At the same time, according to the applicant, direct injection of the recombinant ARSA enzyme into the brain can improve the effectiveness of treatment, 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:

- the need for serious surgical intervention,

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

However, such approaches are being actively researched and undergoing clinical trials at the date of submission of application materials.

Thus, according to the applicant, gene therapy using viral vectors capable of crossing the BBB may be very effective for the treatment of MLD.

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

It should be emphasized that AAVs can transsynaptically infect neurons over a wide range from the injection site through anterograde transport [Zingg, B. et al. AAV-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 to achieve prolonged 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].

Promising results were also obtained using AAV9 encoding ARSA and a green fluorescent protein reporter gene. Namely, the introduction of AAVrh.10-ARSA into the brain of eight-month-old mice with a model of MLD led to the correction of the accumulation of certain types of sulfatides in oligodendrocytes. In a phase I/II clinical study of this virus (see study NCT01801709), experimental animals received 12 injections of AAVrh.10-ARSA transducing units into the white matter of the brain. At the same time, in experimental animals with an early stage, the symptoms continued to worsen. A single intravenous injection of AAVPHP.eB-hARSA-HA into mice with MLD resulted in sustained expression of the ARSA enzyme in the brain and spinal cord and showed complete correction of sulfatide accumulation in the spinal cord of the model animals.

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

Thus, the above-described methods of treating MLD are not effective enough, as a result of which the problem of finding new drugs and treatment methods is urgent.

The applicant analyzed the identified state of the art based on scientific and patent information in the field of MLD therapy and identified a number of analogues that are currently used to relieve symptoms and to stop the worsening of neurodegeneration.

From the studied level of technology, a number of functional (as intended) and structural analogs have been identified that are used to treat lysosomal storage diseases known in the world, which were practically incurable at the date of submission of application materials.

The invention is known according to the patent WO2023133584 “Compositions useful for the treatment of metachromatic leukodystrophy”. The entity is a recombinant replication-defective adeno-associated virus (rAAV) that can be used to treat disease associated with mutation of 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. Claims: 1. Pharmaceutical composition for use in the treatment of metachromatic leukodystrophy or a disease associated with a mutation of the arylsulfatase A ( ARSA ) gene, wherein the composition contains a recombinant adeno-associated virus (rAAV) containing the AAVhu68 capsid; and vector genome) containing: AAV 5' inverted terminal repeats (ITRs), 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 a CB7 promoter that directs hARSA expression, 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 the sequence from at least 95% to 99, 9% identical to it, which encodes functional hARSA; and at least one aqueous buffer, at least one carrier, at least one excipient and/or at least one preservative, said 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 sequence, an intron, an additional enhancer and/or a TATA signal. 3. The pharmaceutical composition according to claim 1 or 2, wherein the hARSA coding sequence is SEQ ID NO: 1 or SEQ ID NO: 3. 4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the vector genome contains 5' -AAV ITR, an expression cassette having the sequence SEQ ID NO: 28, and 3'-AAV ITR. 5. Pharmaceutical composition according to any one of paragraphs. 1-4, where the 5'-ITR AAV has the sequence SEQ ID NO: 25 and/or the 3'-ITR AAV has the sequence SEQ ID NO: 26. 6. The pharmaceutical composition according to any one of claims 1-5, where the vector genome contains nt 1-nt 3883 SEQ ID NO: 5 (SEQ ID NO: 27). 7. Pharmaceutical composition according to any one of paragraphs. 1-6, wherein the AAVhu68 capsid is derived from the sequence encoding the amino acid sequence of SEQ ID NO: 7. 8. The pharmaceutical composition according to any one of claims 1-5, wherein the composition contains artificial cerebrospinal fluid containing buffered saline and one or more 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 6.5 to 8.5. 9. Pharmaceutical composition according to any one of claims 1 to 8, wherein the composition is suitable for intracerebroventricular or intracerebroventricular injection. 10. Pharmaceutical composition according to any one of claims 1 to 8, where a single dose contains from 3x10 10 genome copies (GC)/gram of brain weight to 3.5x10 11 GC/gram of brain weight. 11. The pharmaceutical composition according to claim 10, where the dose is: (a) about 3.3×10 10 genome copies (GC)/gram of brain weight; (b) about 1.1×10 11 genome copies (GC)/gram brain mass; or (c) about 3.3 x 10 11 genome copies (GC)/gram of brain mass. 12. Use of rAAV.hARSA in the manufacture of a medicament for the therapeutic treatment of metachromatic leukodystrophy or a disease associated with mutation of the arylsulfatase A gene (ARSA), which medicament is useful after intrathecal administration of a single dose containing 3x10. From 10 genome copies (GC)/gram brain weight to 3.5 x 10 11 GC/gram brain weight per patient. 13. Use according to claim 12, where the dose is: (a) about 3.3×10 10 genome copies (GC)/gram of brain weight; (b) about 1.1 × 10 11 genome copies (GC)/gram brain mass; or (c) about 3.3 x 10 11 genome copies (GC)/gram of brain mass. 14. Use according to claim 11 or 12, wherein rAAV comprises an AAVhu68 capsid and a vector genome, said vector genome comprising: AAV 5' inverted terminal repeats (ITRs), 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 comprising a CB7 promoter that directs hARSA expression, a polyA signal, and a 3'-AAV ITR, wherein the hARSA encoding sequence comprises a nucleotide (nt) sequence 1 to nt 1521 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 of the arylsulfatase A (ARSA) gene, the method comprising administering to the subject a single dose of recombinant AAV by injection of ICM, wherein the recombinant AAV contains AAVhu68. a capsid and a vector genome packaged therein, wherein said vector genome comprises an AAV ITR, an hARSA coding sequence comprising SEQ ID NO: 1, or a sequence at least 95% identical thereto that encodes a functional hARSA, and regulatory sequences that direct the expression of functional hARSA in a target cell, where a single dose is (i) approximately 3.3×10 10 genome copies (GC)/gram brain weight; (ii) about 1.1×10 11 GC/gram brain mass; or (iii) about 3.3 x 10 11 GC/gram brain mass. 16. The method according to claim 15, where rAAV contains the AAVhu68 capsid; and a vector genome containing: AAV 5' inverted terminal repeats (ITRs), 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 promoter CB7, which drives hARSA expression, polyA signal and 3'-AAV ITR, wherein the hARSA coding sequence contains the sequence from nucleotide (nt) 1 to nt 1521 SEQ ID NO: 1 or a sequence of at least 95% to 99.9% is identical to that which encodes functional hARSA.

The use of the vector described in the known technical solution has the disadvantage of being neurotropic AAVhu68. ARSA must be delivered to the brain oligodendrocytes of MLD patients, but AAVhu68 does not have such properties, which reduces the effectiveness of the drug when used as directed.

To solve this problem, it is necessary to develop a more effective method - delivery of the gene for the missing enzyme. AAVOlig001-CMVenCBh-coARSA has the ability to transduce oligodendrocytes, which will stop neurodegeneration and restore the level of the ARSA enzyme.

From the researched level of technology, an invention was identified under patent RU2692251C2 “Adeno-associated virus-mediated gene transfer to the central nervous system.” The brief essence of the known technical solution is a method for preventing or treating mucopolysaccharidosis type I in humans, consisting of intrathecal administration of a composition based on recombinant adeno-associated virus (rAAV) 9 and rh10 serotypes containing an open reading frame encoding alpha-L-iduronidase, the essence is Method preventing, inhibiting or treating one or more symptoms of mucopolysaccharidosis type I (MPS I) in a human, comprising intrathecal administration to a human in need thereof of a composition containing an effective amount of a recombinant adeno-associated virus (rAAV) vector containing an open reading frame encoding alpha-L -iduronidase for the prevention, inhibition or treatment of one or more symptoms of MPS I, wherein the rAAV is rAAV9 or rAAVrh10. 2. The method according to claim 1, in which the person is an adult. 3. The method of claim 1, further comprising administering to the subject an effective amount of a penetration enhancer. 4. The method according to claim 3, wherein the composition contains a permeation enhancer. 5. The method according to claim 3 or 4, wherein the penetration enhancer contains 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 a person. 7. The method according to claim 6, wherein the immunosuppressant contains cyclophosphamide. 8. The method according to claim 6, wherein the immunosuppressant contains a glucocorticoid, cytostatic agents, including an alkylating agent, an antimetabolite, a cytotoxic antibiotic, an antibody or an agent active against immunophilin. 9. The method according to claim 7 or 8, in which the immunosuppressant contains nitrogen mustard, nitrosourea, a platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, aimed at the IL-2 receptor (CD25- ) or CD3 antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioid or TNF-α (tumor necrosis factor-alpha) binding agent. 10. The method of claim 6, wherein the rAAV and the immunosuppressant are administered together or the immunosuppressant is administered after the rAAV is administered. 11. The method according to claim 6, in which the immunosuppressant is administered intrathecally. 12. The method according to claim 6, in which the immunosuppressant is administered intracerebroventricularly. 13. The method of claim 1, wherein the human is immunologically tolerant to alpha-L-iduronidase. 14. Method according to any one of paragraphs. 1-13, in which alpha-L-iduronidase is damaged or deficient in the individual, resulting in lysosomal storage disease. 15. Method according to any one of paragraphs. 1-13, in which the human is an immunocompetent adult mammal. 16. Method according to any one of paragraphs. 1-13, wherein the rAAV vector is an rAAV-9 vector. 17. Method according to any one of paragraphs. 1-13, wherein the rAAV vector is an rAAV rh10 vector.

Thus, the known invention describes intrathecal delivery of rAAV9 and the rAAVrh10 vector for the prevention, inhibition or treatment of one or more symptoms of MPS I having an absence or deficiency of a lysosomal enzyme storage-associated enzyme.

However, the therapeutic effect of vectors containing these capsids specifically for the treatment of MLD has not been studied. To solve this problem, it is necessary to develop and study specific vectors containing exactly the ARSA gene, as well as methods that make it possible to safely modify cells of the entire nervous system for long-term expression of a healthy gene.

The use of AAVOlig001-CMVenCBh-coARSA allows us to achieve a high level of transduction of cells of the nervous system, due to the ability of these vectors to neurotropism and effectively transduce cells of the central nervous system, thereby ensuring delivery of the enzyme and increasing its activity.

From the researched level of technology, an invention was identified under patent RU2671503C2 “Methods and compositions for delivering arylsulfatase A to the central nervous system.” The essence is a method of intrathecal administration of a composition containing protein arylsulfatase A (ARSA), polysorbate and phosphate for the treatment of metachromatic leukodystrophy disease. In this case, the specified phosphate is contained in an amount of no more than 10 mM. The group of inventions ensures successful intrathecal administration of the composition without causing significant toxicity and immune response. The essence is a stable composition for intrathecal administration containing protein arylsulfatase A (ASA) 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. A 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 said stable composition 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 one of claims. 1-4, characterized in that the ASA protein is obtained from a human cell line. 6. Stable composition according to any one of paragraphs. 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. A 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 in a concentration of approximately 154 mM. 10. Stable composition according to any one of paragraphs. 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. A stable composition according to claim 10, characterized in that said polysorbate surfactant is polysorbate 20. 12. A stable composition according to claim 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. The stable composition according to claim 1, additionally containing a buffering agent selected from the group consisting of acetate, histidine, succinate, citrate, Tris and combinations thereof. 15. Stable composition according to any one of paragraphs. 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 one of paragraphs. 1-4, characterized in that said composition is a liquid composition. 19. Stable composition according to any one of paragraphs. 1-4, characterized in that the specified composition is made in the form of a lyophilized dry powder. 20. Stable composition according to claim 1, characterized in that said composition additionally contains a stabilizing agent. 21. A 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. A method for treating the disease metachromatic leukodystrophy (MLD), including the step of intrathecal administration to a subject in need of the composition according to any one of claims. 1-21. 23. The method according to claim 22, characterized in that intrathecal administration is carried out once every two weeks. 24. The method according to claim 22, characterized in that intrathecal administration is carried out once a month. 25. The method according to claim 22, characterized in that intrathecal administration is carried out once every two months. 26. The method according to claim 22, characterized in that intrathecal administration is used in combination with intravenous administration. 27. The method according to claim 26, characterized in that intravenous administration is carried out no more than once a month. 28. The method according to claim 26, characterized in that intravenous administration is carried out no more than once every two months. 29. The method according to claim 22, characterized in that intrathecal administration is used in the absence of intravenous administration. 30. The method according to claim 22, characterized in that intrathecal administration is used in the absence of concomitant immunosuppressive therapy.

Thus, the invention describes a method of delivering a composition consisting of ARSA protein, polysorbate and phosphate to the CNS by intrathecal administration without causing significant toxicity and immune response.

The disadvantages of the known technical solution are that, firstly, when introducing the enzyme into the CSF there is a risk of complications. Secondly, there is difficulty in achieving uniform distribution of the enzyme throughout the nervous system. Also, previous clinical trials of similar drugs in patients with other LND affecting the central nervous system did not show the expected effectiveness, neurological disorders continued, which does not give reason to believe that the drug will be effective when used as directed.

The closest analogue in terms of technical essence and achieved technical result is the invention under patent RU 2769577 “Drug for the treatment of metachromatic leukodystrophy and method of its treatment.” The essence is a gene and gene-cell drug for the treatment of MLD, consisting of intravenous or intrathecal administration of a drug containing recombinant adeno-associated virus serotype 9, with a unique sequence of the codon-optimized ARSA gene (AAV9-coARSA) or transplantation of human mesenchymal stem cells (MSCs) , genetically modified AAV9-coARSA (MSC-ARSA). Claim: A drug for the treatment of metachromatic leukodystrophy, including a recombinant adeno-associated virus serotype 9 containing a codon-optimized sequence of the ARSA gene, represented by SEQ ID NO: 1 A drug for the treatment of metachromatic leukodystrophy, consisting of mesenchymal stem cells genetically modified by a recombinant adeno-associated virus serotype 9, containing a codon-optimized sequence of the ARSA gene presented in SEQ ID NO: 1. A method of treating metachromatic leukodystrophy, consisting 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.

The disadvantage of the known technical solution is the short-term expression of the transgene, which subsequently leads to the progression of the disease. Also, the 9th serotype is not aimed at transducing oligodendrocytes, which reduces the effectiveness of the drug when used as directed.

At the same time, the use of AAVOlig001-CMVenCBh-coARSA according to the stated technical solution makes it possible 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 tropism of oligodentrocyrs, thereby ensuring delivery of the enzyme to the central nervous system and increasing its activity.

To solve the technical problems described above, it is necessary to develop a method that allows the cells of the nervous system to be corrected, namely, modified for long-term expression of a healthy gene.

When administered intrathecally, AAVOlig001-CMVenCBh-coARSA will transduce oligodendrocytes, the CMVenCBh promoter will ensure strong and long-lasting expression. This will allow long-term expression of ARSA to be achieved.

The technical result of the claimed technical solution is:

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

– development of an adeno-associated virus – AAVOlig001-CMVenCBh-coARSA, which provides the possibility of treating MLD.

The essence of the claimed technical solution is a recombinant genetic construct for overexpression of the enzyme arylsulfatase A, containing the promoter of the CMV/chicken β-actin hybrid enhancer and the codon-optimized sequence of the ARSA gene, 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.

The claimed technical solution is illustrated in Fig. 1 – Fig.5 .

In FIG. Figure 1 shows diagrams that show the level of enzymatic activity of ARSA in homogenates of organs of the central nervous system of pigs on the 35th day after administration of AAVOlig001-CMVenCBh-coARSA; the X axis shows samples of homogenates of organs from the group of intact pigs (designation control), samples of homogenates of organs of pigs that were intrathecally injected with the recombinant AAVOlig001-CMVenCBh-coARSA: 1a – right frontal lobe, 1b – parietal lobe, 1c – occipital lobe of the cerebral cortex, 1d – brain stem, 1e – cerebellum, 1f – cervical spinal cord, 1g – thoracic spinal cord, 1h – lumbar spinal cord.

In FIG. Figure 2 shows diagrams showing the dynamics of ARSA enzymatic activity before administration, on days 7, and 28. The X axis shows samples of CSF and plasma from pigs No. 1 – No. 3, which were intrathecally injected with recombinant AAVOlig001-CMVenCBh-coARSA.: 2a – in cerebrospinal fluid (CSF), 2b – in pig plasma

In FIG. Figure 3 shows diagrams showing the number of ARSA gene mRNA copies in the organs of the nervous system of pigs on day 35 after administration of AAVOlig001-CMVenCBh-coARSA. The X axis shows RNA samples. The Y axis shows the number of ARSA gene mRNA copies per 1 μg of total RNA. Data were obtained using quantitative PCR. 3a – RNA samples of the right frontal lobe, parietal lobe, occipital lobe of the brain, 3b – cerebellum, brain stem, saphenous nerve, 3c – cervical spinal cord (SC), thoracic spinal cord (SC), lumbar spinal cord (SC ), 3d – dorsal root ganglia (DRG) of the cervical spinal cord (SC), dorsal root ganglia (DRG) of the thoracic spinal cord (SC), dorsal root ganglia (DRG) of the lumbar spinal cord (SC).

In FIG. Figure 4 shows diagrams showing data from the analysis of the levels of: 4a - alanine aminotransferase (ALT), 4b - aspartate aminotransferase (AST), 4c - creatinine-J, 4d - total bilirubin in pig blood serum. The X axis shows serum samples from the experimental group of pigs with intrathecal administration of recombinant AAVOlig001-CMVenCBh-coARSA. Data were obtained using enzyme-linked immunosorbent assay (ELISA).

In FIG. Figure 5 shows images of organ sections (obtained by confocal microscopy), which show the analysis of ARSA expression by immunohistochemical analysis of cryostat sections of CNS organs - a control group of pigs without administration (control) and samples of organ sections of an experimental group of pigs with intrathecal AAVOlig001-CMVenCBh-coARSA: 5a - section of the cerebellar cortex, section of the occipital lobe cortex, section of the lumbar spinal cord, 5b - 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.

Next, the applicant provides a description of the claimed technical solution.

The identified problem and the claimed technical result are achieved by intrathecal administration of an adeno-associated virus with the Olig001 serotype, containing a recombinant genetic construct with the CMVenCBh promoter, with a unique codon-optimized sequence of the ARSA gene - AAVOlig001-CMVenCBh-coARSA (hereinafter referred to as AAVOlig001-CMVenCBh-coARSA). The sequence represented by SEQ ID NO:1 is shown in the Appendix.

The idea of the claimed technical solution is generally based on the fact that, due to the ability of the Olig001 serotype to effectively transduce oligodendrocytes involved in the pathogenesis of MLD, the CMVenCBh promoter initiates long and strong expression of the transgene. This allows you to achieve a therapeutic effect. As a result of the development, AAVOlig001-CMVenCBh-coARSA was obtained, which has a therapeutic effect unknown from the studied state of the art, providing the possibility of treating the previously incurable disease MLD.

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

Stage 1: Preparation and analysis of a recombinant genetic construct containing the CMV/chicken β-actin hybrid enhancer promoter and the codon-optimized ARSA gene sequence represented by SEQ ID NO:1.

Stage 2: Obtaining and analyzing the functionality of the adeno-associated virus of serotype Olig001 - AAVOlig001-CMVenCBh-coARSA, containing the recombinant genetic construct obtained at stage 1.

Stage 3: Study of the effectiveness and safety of AAVOlig001-CMVenCBh-coARSA when administered intrathecally to large laboratory animals.

Next, the applicant provides a detailed description of the stages of implementation of the claimed technical solution.

Example 1 . Stage 1 - production and analysis of a recombinant genetic construct containing the CMV/chicken β-actin hybrid enhancer promoter and a codon-optimized gene sequence ARSA represented by SEQ ID NO:1 .

Codon optimization of the nucleotide sequence of the gene is performed. To optimize the codon composition of the ARSA gene, well-known algorithms are used using the OptimumGene program (GeneScript, USA). The level of gene expression is influenced by many factors, the algorithm takes into account as many of them as possible, creating a single gene that can achieve the highest possible level of expression. In this case, the native gene uses tandem rare codons, which can reduce translation efficiency or even disable the translation mechanism. Codon shifting 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. Variation of the GC composition was optimized to increase the half-life of the 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.

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

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

The efficiency of in vitro expression of the recombinant protein using the resulting plasmid construct is confirmed by activity testing and Western blot analysis.

ARSA enzyme activity was 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 μl of a cell lysate sample is incubated with a solution of nitrocatechol sulfate substrate (0.01 M p-Nitrocatechol sulfate dipotassium salt (#N7251, Sigma), 0.5 M sodium acetate, 0.5 mM Na ₄ P ₂ O ₇ , 10 % sodium chloride, pH=5) for 1 hour at 37°C, after which the reaction is stopped by adding 1 N sodium hydroxide. Dilutions of sulfatase (#S9626, Sigma) are used as standards. Optical density is measured at a wavelength of 515 nm.

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

Based on the results of the applicant's analysis, the expected sizes of about 33 kDa were obtained in samples of lysates of HEK293T cells transfected with pAAV-CMVenCBh-coARSA.

Example 2 . Carrying out stage 2 - obtaining and analyzing the functionality of the adeno-associated virus of the Olig001 serotype - AAVOlig001-CMVenCBh-coARSA, containing the recombinant genetic construct according to Example 1.

Based on the recombinant genetic construct pAAV-CMVenCBh-coARSA, AAVOlig001-CMVenCBh-coARSA is obtained. To obtain AAVOlig001-CMVenCBh-coARSA, use the AAV Helper free system. AAV293 cells are seeded in an amount of 1.5 million, the monolayer is 70-80%. The next day, co-transfection is carried out using the calcium phosphate method with three plasmids (vector plasmid, pAAV-RC and pHelper). AAVOlig001-CMVenCBh-coARSA was harvested 72 hours after transfection. The cells are collected with a scraper, cryolyzed, and centrifuged at 10,000 × g for 10 minutes to remove cell debris. The viral stock is stored at minus 80°C. AAVOlig001-CMVenCBh-coARSA was concentrated using AAV Purification Mega Kit (Cell Biolabs, Inc., USA) according to the manufacturer's recommended procedure.

Example 3 . Conducting stage 3 - studying the effectiveness and safety of AAVOlig001-CMVenCBh-coARSA when administered intrathecally to large laboratory animals.

The effectiveness and safety of the resulting AAVOlig001-CMVenCBh-coARSA was tested in large laboratory animals.

The work used pigs aged 4 months (weighing 5 kg), which were randomly distributed into 2 groups, each group containing three individuals - a control group and an experimental group. Pigs were injected intrathecally (i.e. by introducing AAVOlig001-CMVenCBh-coARSA into the spinal cord) with the declared AAVOlig001-CMVenCBh-coARSA (n=3) in an amount of 2 x 10 ¹³ genomic copies/kg, while the control group was not injected with anything:

1) Control group of intact pigs without administration of AAVOlig001-CMVenCBh-coARSA.

2) Experimental group of pigs, which were intrathecally administered AAVOlig001-CMVenCBh-coARSA, at a dose of 2 x 10 ¹³ genomic copies/kg.

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

The experiment was carried out as follows.

Before administration, samples of CSF and whole blood were taken from pigs into tubes containing the anticoagulant NaC 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 was stored , plasma and serum at minus 80°C.

Further, in all experimental animals again, i.e. 7 and 28 days after administration of AAVOlig001-CMVenCBh-coARSA, whole blood was collected and CSF.

Next, whole blood plasma samples and CSF were used to determine ARSA enzymatic activity.

Then, on day 35 after administration of AAVOlig001-CMVenCBh-coARSA, pigs are euthanized using methods that comply with the principles set out in the European Commission Guidelines for the Euthanasia of Experimental Animals.

Next, the spinal cord (cervical, thoracic, lumbar regions), dorsal root ganglia (cervical, thoracic, lumbar regions), cerebellum, occipital lobe cortex, latent nerve, brain stem, right frontal lobe, parietal lobe were removed.

Next, all organs were homogenized for activity testing and RT-PCR or dissected for IHC analysis.

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

The graphic materials in Figure 1 - Figure 5 have experimentally proven the possibility of achieving the stated technical results.

From the data shown in Fig. 1, it is clear that the enzymatic activity of ARSA in homogenates of CNS organs increases after the administration of AAVOlig001-CMVenCBh-coARSA. In the homogenate of the occipital lobe of the cerebral cortex, ARSA activity increases by 150% in 1 pig. In the cerebellar homogenate, an increase in activity of 318% was detected in one pig. In the brainstem homogenate, an increase in activity of 117% 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 indicate that after genetic modification with the resulting AAVOlig001-CMVenCBh-coARSA, cells of the nervous system begin to express a functionally active enzyme in vivo .

From the data shown in Fig. 2, it can be seen that after intrathecal administration of AAVOlig001-CMVenCBh-coARSA, ARSA activity increases in the CSF on day 28. In plasma on day 7. These results indicate that intrathecal administration of AAVOlig001-CMVenCBh-coARSA to large laboratory animals leads to the transduction of cells, which then begin to express and secrete a functional enzyme found in the plasma and CSF of the animals.

From the data shown in Fig. 3 shows the presence of overexpression of the codon-optimized ARSA gene in the CNS organs of pigs that were intrathecally administered AAVOlig001-CMVenCBh-coARSA. In the cerebellum, in the saphenous nerve, in the cervical, thoracic, lumbar spinal cord, in the dorsal root ganglia of the thoracic and lumbar spinal cord of pig No. 1, 4215, 462, 947, 854716, 93228, 2922, 411 copies of ARSA gene mRNA per mcg were detected total RNA, respectively. In the cerebellum, brain stem, saphenous nerve, parietal lobe, occipital lobe, in the cervical, thoracic, lumbar spinal cord, in the dorsal root ganglia of the cervical, thoracic spinal cord of pig No. 2, 5480, 346, 300, 974, 139, 871 were detected , 4965, 1501, 1129, 1039 ARSA gene mRNA copies per μg of total RNA, respectively. In the brain stem, saphenous nerve, parietal lobe, occipital lobe, in the cervical, thoracic, lumbar spinal cord, in the dorsal root ganglia of the cervical, thoracic, lumbar spinal cord of pig No. 3, 88, 973, 5472, 205, 761, 1627 were detected , 1700, 1957, 1016, 579 ARSA gene mRNA copies per μg of total RNA, respectively.

From the data shown in Fig. 4, it can be seen that biochemical parameters in the blood serum of pigs after intrathecal administration of AAVOlig001-CMVenCBh-coARSA showed an increase in the levels of AST, creatinine-J and bilirubin on day 7 in one animal. In the remaining experimental animals, the indicators remain unchanged (AST, creatinine-J, total bilirubin). This proves that intrathecal administration of AAVOlig001-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.

From the data shown in Fig. 5, it can be seen that analysis of ARSA expression in cryostat sections of the organs of the nervous system of pigs by immunohistochemical analysis confirms the successful genetic modification of cells of the nervous system of experimental animals after intrathecal administration of AAVOlig001-CMVenCBh-coARSA. ARSA-overexpressing Purkinje neurons were found in the cerebellar cortex compared to controls. Analysis of the occipital lobe of the brain showed 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 from the experimental and control groups. However, single ARSA-overexpressing neurons were found in the anterior and dorsal horns of the lumbar spinal cord. Analysis of spinal ganglia (SMG) at the level of the cervical, thoracic and lumbar spinal cord revealed ARSA-overexpressing neurons. Analysis of the spinal nerve roots and sciatic nerve did not reveal differences in ARSA expression in animals of the experimental and control groups.

From the above, we can conclude that the applicant has solved the identified technical problems and achieved the stated technical result, namely:

- a recombinant genetic construct has been developed (see Example 1);

- an adeno-associated virus of serotype Olig001 has been developed containing the specified recombinant genetic construct - AAVOlig001-CMVenCBh-coARSA, which provides the possibility of treating MLD (see Examples 2, 3, Fig. 1-5).

The claimed technical solution satisfies the patentability condition of “novelty” imposed on inventions, since from the researched level of technology no sources have been identified that describe features that coincide in the function they perform and the form of implementation of these features listed in the claims, including the characteristics of the purpose.

The claimed technical solution satisfies the patentability condition of “inventive step” imposed on inventions, since from the researched level of technology the applicant has not identified technical solutions characterized by the use of a method of treating MLD, implying intrathecal administration of recombinant AAVOlig001-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 was practically incurable at the date of submission of the application materials.

The claimed technical solution satisfies the patentability requirement of “industrial applicability” for inventions, since it can be used on an industrial scale to create drugs intended for the treatment of MLD.

--->

Sequence Listing Information:

DTD Version: V1_3

File Name: pAAV-ARSA1.xml

Software Name: WIPO Sequence

Software Version: 2.3.0

Production Date: 2023-10-24

General Information:

Current application / IP Office: RU

Current application / Application number: 2023126473/20(058552)

Current application / Filing date: 2023-10-16

Current application/Applicant file reference:-

Earliest priority application / IP Office: RU

Earliest priority application/Application number:

2023126473/20(058552)

Earliest priority application / Filing date: 2023-10-16

Applicant name: Federal State Autonomous

educational institution of higher education "Kazansky

(Volga Region) Federal University" (FGAU HE KFU), RU

Applicant name / Language: ru

Applicant name / Name Latin: Federalnoye gosudarstvennoye

avtonomnoye obrazovatelnoye uchrezhdeniye vysshego obrazovaniya

Kazanskiy (Privolzhskiy) federalnyy universitet (FGAOU VO KFU), RU

Inventor name: Mullagulova Aisylu Ildarovna

Inventor name / Language: en

Inventor name / Name Latin: Mullagulova Aisylu Ildarovna

Invention title: Recombinant genetic construct,

adeno-associated virus for the treatment of metachromatic

leukodystrophy (ru)

Sequence Total Quantity: 1

Sequences:

Sequence Number (ID): 1

Length: 6282

Molecule Type: DNA

Features Location/Qualifiers:

- source, 1..6282

> mol_type, other DNA

>organism, synthetic construct

Residues:

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg

ggcgacctttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa

ctccacatcact 120

aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg

gggtcattag 180

ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc

ccgcctggct 240

gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg

actttcatt 300

gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat

caagtgtatc 360

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc

tggcatttgc 420

ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat

tagtcatcgc 480

tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct

cccccccctc 540

cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga

tggggggcggg 600

gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg

gcggggcgag 660

gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc

cttttatggc 720

gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgc

gtttagtgaa 780

ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa

gacaccggga 840

ccgatccagc ctccgcggat tcgaatcccg gccgggaacg gtgcattgga

acgcggattc 900

cccgtgccaa gagtgacgta agtaccgcct atagagtcta taggcccaca

aaaaatgctt 960

tcttctttta atatactttt ttgtttatct tatttctaat actttcccta

atctctttct 1020

ttcagggcaa taatgataca atgtatcatg cctctttgca ccattctaaa

gaataacagt 1080

gataatttct gggttaaggc aatagcaata tttctgcata taaatatttc

tgcatataaa 1140

ttgtaactga tgtaagaggt ttcatattgc taatagcagc tacaatccag

ctaccattct 1200

gcttttattt tatggttggg ataaggctgg attattctga gtccaagcta

ggcccttttg 1260

ctaatcatgt tcatacctct tatcttcctc ccacagctcc tgggcaacgt

gctggtctgt 1320

gtgctggccc atcactttgg caaagaattg ggattcgaac atcgattgaa

ttcacaagtt 1380

tgtacaaaaa agcaggctga attcaccatg tccatgggcg ccccccggtc

tctgctgctg 1440

gcactggcag caggactggc agtggccaga ccccctaaca tcgtgctgat

cttcgcagac 1500

gatctgggat acggcgacct gggctgctat ggccacccaa gctccaccac

acccaatctg 1560

gaccagctgg cagcaggagg actgaggttc accgattttt acgtgccagt

gagcctgtgt 1620

accccatcca gggcagcact gctgacaggc aggctgccag tgcgcatggg

catgtatcct 1680

ggcgtgctgg tgccatctag caggggagga ctgccactgg aggaggtgac

cgtggcagag 1740

gtgctggcag ccagaggcta cctgacagga atggccggca agtggcacct

ggggagtggga 1800

cctgagggcg ccttcctgcc accccaccag ggcttccacc ggtttctggg

catcccttat 1860

tcccacgacc agggcccatg ccagaacctg acctgttttc ctccagcaac

accatgcgac 1920

ggagatgtg atcagggact ggtgcctatc ccactgctgg ccaatctgtc

tgtggaggca 1980

cagccacctt ggctgcctgg actggaggca aggtacatgg cattcgcaca

cgacctgatg 2040

gcagatgcac agcggcagga tagacctttc tttctgtact atgcctccca

ccaccaccac 2100

tatccacagt tcagcggcca gtcctttgca gagaggagcg gaaggggacc

attcggcgac 2160

tccctgatgg agctggatgc cgccgtgggc accctgatga cagcaatcgg

cgacctggga 2220

ctgctggagg agaccctggt catcttcacc gccgataacg gccccgagac

aatgcggatg 2280

tctagaggag gatgcagcgg actgctgaga tgtggcaagg gaaccacata

cgagggaggc 2340

gtgcgcgagc ctgcactggc attttggcca ggacacatcg cacctggagt

gaccaccagag 2400

ctggcatcct ctctggacct gctgccaaca ctggcagcac tggcaggagc

accactgcct 2460

aatgtgaccc tggacggctt cgatctgtct ccactgctgc tgggcaccgg

caagtccccc 2520

agacagtctc tgttctttta ccccagctat cctgatgagg tgcggggcgt

gtttgccgtg 2580

agaaccggca agtacaaggc ccacttcttt acacagggct ctgcccacag

cgacaccaca 2640

gccgatcctg catgccacgc aagctcctct ctgaccgcac acgagccacc

actgctgtac 2700

gacctgtcca aggaccccgg cgagaactat aatctgctgg gaggagtggc

aggagcaacc 2760

cctgaggtgc tgcaggccct gaagcagctg cagctgctga aggcacagct

ggacgcagca 2820

gtgacattcg gaccaagcca ggtggcaagg ggagaggacc ccgcactgca

gatctgctgt 2880

caccctggat gcaccccaag gccagcatgc tgtcactgtc ctgatccaca

cgcctgagga 2940

tccacccagc tttcttgtac aaagtggtgg atcctctaga gtcgacctgc

agaagcttgc 3000

ctcgagcagc gctgctcgag agatctacgg gtggcatccc tgtgacccct

ccccagtgcc 3060

tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa

taaaattaag 3120

ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga

ggggggtggt 3180

atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct

attgggaacc 3240

aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct

gggttcaagc 3300

gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga

ccaggctcag 3360

ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc

tggtctccaa 3420

ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat

tacaggcgtg 3480

aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg

gaccgagcgg 3540

ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg

ctcgctcact 3600

gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc

ctcagtgagc 3660

gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct

tacgcatctg 3720

tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt

agcggcgcat 3780

taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc

agcgccctag 3840

cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc

tttccccgtc 3900

aagctctaaa tcggggggctc cctttagggt tccgatttag tgctttacgg

cacctcgacc 3960

ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga

tagacggttt 4020

ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc

caaactggaa 4080

caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg

ccgatttcgg 4140

cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt

aacaaaatat 4200

taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc

gcatagttaa 4260

gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt

ctgctcccgg 4320

catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag

aggttttcac 4380

cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt

ttataggtta 4440

atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga

aatgtgcgcg 4500

gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc

atgagacaat 4560

aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt

caacatttcc 4620

gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct

4680

cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt

tacatcgaac 4740

tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt

tttccaatga 4800

tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac

gccgggcaag 4860

agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac

tcaccagtca 4920

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct

gccataacca 4980

tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg

aaggagctaa 5040

ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg

gaaccggagc 5100

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca

atggcaacaa 5160

cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa

caattaatag 5220

actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt

ccggctggct 5280

ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc

attgcagcac 5340

tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg

agtcaggcaa 5400

ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt

aagcattggt 5460

aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt

catttttaat 5520

ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc

ccttaacgtg 5580

agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct

tcttgagatc 5640

ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta

ccagcggtgg 5700

tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc

ttcagcagag 5760

cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac

ttcaagaact 5820

ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct

gctgccagtg 5880

gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat

aaggcgcagc 5940

ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg

acctacaccg 6000

aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa

gggagaaagg 6060

cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg

gagcttccag 6120

ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga

cttgagcgtc 6180

gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc

aacgcggcct 6240

ttttacggtt cctggccttt tgctggcctt ttgctcacat gt

6282

END

<---

Claims (2)

1. A recombinant genetic construct for overexpression of the arylsulfatase A enzyme, containing a CMV/chicken β-actin hybrid enhancer promoter and a codon-optimized ARSA gene sequence represented by SEQ ID NO: 1. 2. Adeno-associated virus of serotype Olig001 for the treatment of metachromatic leukodystrophy, containing a recombinant genetic construct according to claim 1.