FIELD OF THE INVENTION

The invention relates to the field of medicine in general, more specifically to the technology for the development of a gene and gene-cell products and methods of treatment of an incurable hereditary disease of metachromatic leukodystrophy (MLD) as of the date of submission of the application materials, which is a severe hereditary neurodegenerative disease resulting from a deficiency of the lysosomal enzyme arylsulfatase A (ARSA) or sphingolipid activator protein B (SAP-B or saposin B) in humans . As a result, ARSA deficiency leads to damage to the myelin sheath covering most of the nerve fibers of the central (CNS) and peripheral nervous system (PNS).

BACKGROUND OF THE INVENTION

As a result of the implementation of the claimed technical solution, it becomes possible to solve problems that are unrealizable at the date of filing the application, such as the treatment of patients with MLD, more specifically, the claimed technical solution ensures the delivery of ARSA to the CNS and PNS by minimally invasive procedures.

The solution of this problem in the world seems to be relevant due to the lack of effective and safe methods of therapy for MLD as of the date of submission of this application. At the date of submission of application materials, only symptomatic therapy is offered as treatment, however, new approaches to the treatment of these diseases are being actively explored. Promising therapeutic strategies are under development and are currently being actively investigated, namely bone marrow or hematopoietic stem cell transplantation, enzyme replacement therapy, and recovery of functional enzyme expression using gene therapy methods. However, these therapies do not show the required level of effectiveness. The weak effectiveness of such approaches as enzyme replacement therapy is due to the fact that, when administered intravenously, the substances do not overcome the BBB poorly. From this point of view, the direct intracerebral injection of the recombinant ARSA enzyme has an advantage, however, such approaches are difficult to apply to humans, since there are such disadvantages as the need for major surgery, poor biodistribution of the therapeutic product and, therefore, the need for multiple injections. Nevertheless, such approaches are being actively investigated and are undergoing clinical trials (NCT01510028, S. Stroobants, Intracerebroventricular enzyme infusion corrects central nervous system pathology and dysfunction in a mouse model of metachromatic leukodystrophy, 2011).

Gene therapy using viral vectors capable of crossing the BBB may be effective. There are in vivo studies using adeno-associated viral vector serotype 5 (AAV5). Intracerebral delivery of AAV 5 to the brain of mice with the MLD model made it possible to achieve prolonged expression of ARSA in the brain (3-15 months) and prevent neuropathological and neuromotor disorders [Sevin C., et al., Intracerebral adeno-associated virus-mediated gene transfer in rapidly progressive forms of metachromatic leukodystrophy, 2006. 15(1): p. 53-64.].

Good results have also been obtained using AAV9. Injection of AAV9, encoding ARSA and the green fluorescent protein reporter gene, into the jugular vein of neonatal MLD mice resulted in long-term expression of the enzyme, and sulfatide accumulation in the brain and spinal cord was significantly reduced and did not differ from that in wild-type mice. AAVrh.10 was found to spread more efficiently from intracerebral injection sites than AAVl, AAV2, AAV5, AAV7 or AAV8 do. The introduction of AAVrh.lO-ARSA into the brain of MLD mice resulted in a decrease in the accumulation of certain types of sulfatides in oligodendrocytes. Clinical trials of AAVrh.lO-ARSA have been carried out. AAVrh.lO- ARSA was injected into the cerebral white matter. ARSA activity in the cerebrospinal fluid (CSF) increased significantly after injection, reaching 20-70% of control values at the last assessment. However, after discontinuation of therapy, the condition of the patients continued to worsen (NCT01801709).

Bone marrow (BM) and hematopoietic stem cell (HSC) transplantation is often used for the treatment of MLD and other LSD, since the cells of a healthy donor synthesize the normal enzyme at the physiological level and in some cases, usually in less aggressive forms of the disease, this procedure helps to increase activity enzyme and alleviate the symptoms of the disease. However, BM and HSC transplantation without additional genetic modification does not always have a sufficient therapeutic effect in the treatment of LSD (S. Groeschel, Long-term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Patients With Juvenile Metachromatic Leukodystrophy Compared With Nontransplanted Control Patients, 2016).

Thus, the above methods of treating MLD are not effective enough and the search for new drugs and methods of treatment is still relevant.

The applicant has analyzed the identified state of the art on scientific and patent information in the field of therapy for MLD, and identified a number of analogues that are currently used to alleviate symptoms and to stop the aggravation of neurodegeneration in patients with MLD.

From the studied state of the art, an invention was revealed specified in patent RU2727015C2 "AAV vectors targeting the central nervous system". The essence of the known technical solution is a nucleic acid encoding an AAV capsid protein, where the nucleic acid contains an AAV capsid encoding sequence that encodes a capsid protein containing: a polypeptide having the sequence of SEQ ID NO: 53, with up to 3 amino acids of the specified polypeptide replaced, and substitutions take place in the VP3 portion of the AAV capsid protein or a polypeptide having the sequence of SEQ ID NO: 56, wherein up to 1 amino acid of the said polypeptide is changed and the substitution takes place in the VP3 portion of the AAV capsid protein. Nucleic acid is at least 99% identical to SEQ ID NO: 13. Nucleic acid wherein the VP 1 /VP2 portion of the sequence is 100% identical to the VP 1 /VP2 portion of SEQ ID NO: 13. Nucleic acid is at least 99% identical to SEQ ID NO: 10. Nucleic acid, wherein the VP1/VP2 portion of the sequence, is 100% identical to the VP1/VP2 portion of SEQ ID NO: 10. The nucleic acid is a plasmid, phage, viral vector, bacterial artificial chromosome or yeast artificial chromosome, AAV vector containing a coding sequence. Additionally contains a sequence encoding rep AAV. An in vitro cell for producing an AAV virus particle containing a nucleic acid stably inserted into the genome. The viral particle for delivering the nucleic acid to a cell is an AAV particle, an adenoviral particle, a herpes virus or a baculovirus particle. An AAV capsid protein comprising a polypeptide having the sequence of SEQ ID NO: 53, wherein up to 3 amino acids of said polypeptide are substituted and the substitutions take place in the VP3 portion of the AAV capsid protein. A polypeptide having the sequence of SEQ ID NO: 56, wherein up to 1 amino acid of the said polypeptide has been changed and the change takes place in the VP3 portion of the AAV capsid protein. An AAV capsid protein containing an amino acid sequence identical to SEQ ID NO: 53 and SEQ ID NO: 56. An AAV particle containing: the AAV vector genome and an AAV capsid containing the AAV capsid proteins, where the AAV capsid encapsulates the AAV vector genome. The AAV particle contains a heterologous nucleic acid and encodes an antisense RNA, miRNA or RNAi, therapeutic polypeptide, growth or differentiation factor, insulin-like growth factor- 1, glial neurotrophic factor, neutrophin-3, neutrophin-4, artemin, neurterin, persephin, brain-derived neurotrophic factor (BDNF), nerve growth factor, ciliary neurotrophic factor, transforming growth factor alpha, platelet growth factor, leukemia inhibitory factor, prolactin, monocarboxylate transporter 1 or nuclear factor 1A, reporter protein, is also operably linked to a constitutive promoter, operably linked to a specific for CNS cells or a CNS cell-preferred promoter. The promoter is a neuron-specific enolase, synapsin, MeCP2, gliofibrillary acidic protein, SlOOp, wdrl6, Foxj 1, LRP2, myelin basic protein, cyclic nucleotide phosphodiesterase, proteolipid protein, Gtx, or Sox 10. A method for producing a recombinant AAV particle containing an AAV capsid, comprising: providing a cell in vitro with a nucleic acid, an AAV rep coding sequence, an AAV vector genome containing a heterologous nucleic acid, and helper functions for producing a productive AAV infection; and providing assembly of a recombinant AAV particle comprising an AAV capsid and a capsid-encapsulated AAV vector genome. A pharmaceutical composition for delivering a nucleic acid of interest to a mammalian patient contains the nucleic acid. A method for delivering a nucleic acid of interest to a CNS cell, comprising bringing the cell into contact with an AAV particle, administering an effective amount of AAV particles, or a pharmaceutical formulation. The AAV particle is delivered directly to the CNS via intrathecal, intracerebral, intraventricular, intranasal, intra-auricular, intraocular, or periocular way, or any combination thereof. A method for delivering a nucleic acid of interest to a region of the CNS adjacent to an area of impaired BBB barrier in a mammalian patient, comprising intravenously administering an effective amount of AAV particles or a pharmaceutical composition.

Thus, more briefly, the essence of the known technical solution are chimeric AAV capsid sequences capable of large-scale gene transfer to the CNS with minimal tropism for peripheral organs. Chimeric capsids can be used to generate AAV vectors for research or therapeutic use when gene transfer to oligodendrocytes is desired without extensive biodistribution of the vector in neurons or peripheral organs.

The disadvantage of the known technical solution is that the therapeutic effect of vectors containing chimeric capsids specifically for the treatment of MLD has not been studied yet. It is indicated that capsids have a tropism for oligodendrocytes, and in the case of MLD, transduction of all cells of the nervous system and the CNS and PNS is necessary, which does not lead to therapeutic improvements. Thus, the known technical solution does not provide the possibility of a highly effective cure for MLD, that is, the treatment of MLD requires the development of a drug product and the study of specific vectors containing the ARSA gene, as well as the development of methods that allow to safely modify cells of the entire nervous system for long-term expression of a healthy gene, leading to a cure for the previously incurable MLD disease.

The use of AAV9-coARSA in the claimed technical solution makes it possible to achieve a high level of transduction of cells of the nervous system, due to the ability of this vector to cross the BBB, thereby ensuring the delivery of the enzyme and increasing its activity. AAV9 is also able to cross the BBB, and intravenous administration of the drug allows transduction of both CNS and PNS cells. Due to the ability of MSCs to migrate to the BBB, the introduction of MSC-ARSA ensures an even distribution of the enzyme throughout the nervous system and a complete cure for a previously incurable disease.

From the studied state of the art, an invention was revealed specified in patent CN108707627A "Lentiviral vector, method for its production and use in MLD". The essence of the known technical solution is the MLD lentiviral vector, characterized in that the vector is modified at the 5'-end of the pTYF lentiviral vector, and the specific modification is as follows: the 5 '-end of the splicing donor site is removed or constructed, and the constructed donor site is not a potential site for homologous recombination between the packaging vector and the reference lentivirus, it still functions as a virus packaging signal. The lentiviral vector additionally contains the ARSA gene. The nucleotide sequence of the ARSA gene is shown in SEQ ID NO. 1 or has at least 80% homology, preferably at least 85% homology with it, in addition, a nucleotide sequence with at least 95% homology is preferred. The sequence further comprises a promoter sequence which is EFla and/or CMV, preferably EFla, the EFla nucleotide sequence is as shown in SEQ ID NO 2 or has a nucleotide sequence with at least 90%-95% homology. Packaging helper plasmid pNHP and pHEF-VSV-G co transfected with a mammalian cell. Preferably the mammalian cells are HEK293T and/or TE671 cells. Lentivirus production method: point mutation of the donor splicing site at the 5'-end of the pTYF lentiviral vector, whole gene synthesis promoter and ARSA gene sequence inserted into the lentiviral vector. The constructed lentiviral vector and the packaging helper plasmid were co-transfected into cells to obtain recombinant lentivirus. The insertion site is located between the BamHI and Spel cleavage sites. The packaging helper plasmid is pNHP and pHEF-VSV-G. The culture time after co-transfection of mammalian cells is 24-72 hours. The recombinant cell is a recombinant stem cell, preferably a peripheral blood stem cell and/or a mesenchymal stem cell. Pharmaceutical composition contains lentiviral vector and recombinant lentivirus. The product contains a lentiviral vector, recombinant lentivirus, recombinant cell or pharmaceutical. Application: as drug products and/or agents for the treatment of MLD. The composition further includes a pharmaceutically acceptable excipient.

Thus, more briefly, the essence of the known technical solution is the use of drug products and/or agents for the treatment of MLD, namely, stem cells transduced with lentivirus capable of stably expressing a large amount of the ARSA gene.

The disadvantages of using the vector described in the known technical solution are, for example, the significant limitations of lentiviral vectors that are able to integrate into the genome of the cell, which raises concerns about safety, since there is a risk of malignant neoplasm of cells.

To solve this problem, it is necessary to develop a safer method of treatment of MLD. The A A Vs in the claimed technical solution are the safest gene therapy vectors known to date. They do not integrate into the cell genome and do not cause insertional mutagenesis, which causes a malignant neoplasm of cells. Also, AAVs do not cause any human disease or immune response. At the same time, AAV9 show a high level of transduction of both nerve cells and MSCs.

From the studied state of the art, an invention was revealed specified in patent of the Russian Federation RU 201891842 "Methods and compositions for CNS delivery of arylsulfatase A". The essence of the known technical solution is a method of treatment of MLD, which includes the step of intrathecal administration to a subject in need of treatment of a recombinant ARSA enzyme at a therapeutically effective dose and with an interval between injections during the treatment period sufficient to improve, stabilize or reduce the rate of deterioration of one or more motor functions compared to baseline. Administration of the recombinant ARSA enzyme further improves, stabilizes, or reduces the rate of deterioration of one or more cognitive, adaptive, and/or executive functions. One or more motor functions include a gross motor function. Gross motor function is assessed using the Gross Motor Functioning Measure (GMFM). The GMFM is GMFM-88. Baseline GMFM- 88 score is higher than 40% or less than 40%. Administration of the recombinant ARSA enzyme results in a decrease in GMFM-88 score of less than 10%, 20%, 30%, 40%, or 50% and stabilization. A method of treatment of MLD, which includes the step of intrathecal administration to a subject in need of treatment of recombinant ARSA at a therapeutically effective dose and with a certain interval between injections during the treatment period sufficient to reduce the levels of a biomarker that accumulates in MLD in physiological fluid, selected from the group consisting of CSF, urine, blood and serum compared to the baseline level of the biomarker. The biomarker is selected from the group consisting of sulfatide, lysosulfatide, and combinations thereof. The baseline of sulphatides in the CSF exceeds approximately 0.1 -0.3 pg/ml. Administration of the recombinant ARSA enzyme results in a reduction in CSF sulphatide levels of greater than about 0.1 -0.2 pg/mL. A method of treatment of MLD syndrome, comprising the step of intrathecal administration to a subject in need of treatment of a recombinant ARSA enzyme at a therapeutically effective dose and with some interval between administrations during the treatment period sufficient to increase the levels of the biomarker reduced in MLD in the cerebral tissue compared with baseline biomarker. The biomarker is a metabolite where the metabolite is N-acetylaspartate. N- acetylaspartate levels are assessed by proton magnetic resonance spectroscopy (MRS). A method for treating MLD syndrome, comprising the step of intrathecal administration to a subject in need of treatment of a recombinant ARSA enzyme at a therapeutically effective dose and with some interval between administrations during the treatment period sufficient to stabilize or reduce involvement in brain damage compared to baseline. Brain damage is assessed by the MLD severity score, determined using MRI. Administration of the recombinant ARSA enzyme resulted in a reduction in the MLD MR imaging severity score in a patient compared to baseline. A therapeutically effective dose is 10 mg to 200 mg or more. The interval between injections is one to two weeks or even one month. The subject for the product administration is a mammal or human 12 months of age or older, in diagnosing, showing symptoms of MLD, or at risk of developing MLD. ARSA is administered into the spinal canal in the lumbar region by lumbar puncture through intermittent or continuous access to an implanted intrathecal drug delivery system. The treatment period is at least 6 to 24 months. The patient does not experience serious adverse effects associated with the administration of recombinant ARSA. The recombinant ARSA enzyme, when intrathecally administered to a subject at risk of developing or suffering from MLD, at a therapeutically effective dose and at some interval between administrations during the treatment period, is sufficient to improve, stabilize or reduce the rate of deterioration of one or more motor functions and brain damage compared to baseline, also to reduce CSF sulfatide level compared to baseline. The recombinant ARSA enzyme contains an amino acid sequence at least 85%-98% identical at the amino acid level with the sequence of SEQ ID NO: 1. The method or recombinant ARSA enzyme according to any one of the preceding claims, wherein the recombinant ARSA enzyme comprises of the amino acid sequence of SEQ ID NO: 1. The method or recombinant ARSA enzyme according to any one of the preceding claims, wherein the recombinant ARSA enzyme contains an amino acid sequence containing no more than four mismatches from SEQ ID NO: 1. The method or recombinant ARSA enzyme according to any one of the preceding claims, wherein the recombinant ARSA enzyme contains an amino acid sequence containing no more than three mismatches from SEQ ID NO: 1. The method or recombinant ARSA enzyme according to any one of the preceding claims, wherein the recombinant ARSA enzyme contains an amino acid sequence containing no more than two mismatches with SEQ ID NO: 1. The method or recombinant ARSA enzyme according to any one of the preceding claims, wherein the recombinant ARSA enzyme comprises an amino acid sequence containing no more than one mismatch with SEQ ID NO: 1.

Thus, more briefly, the essence of the known technical solution is a method for enzyme replacement therapy for MLD and a pharmaceutical composition for direct delivery of recombinant ARSA to the CNS (intrathecal injection of the enzyme into the CSF).

The disadvantages of the known technical solution is:

- firstly, when the enzyme is introduced into the CSF, there is a risk of complications;

- secondly, there is a difficulty in achieving a uniform distribution of the enzyme throughout the nervous system;

- thirdly, one injection of such a product (recombinant enzyme) is not enough, it is necessary to make injections throughout the patient's life, and this increases the likelihood of risks, in addition, constant costs are required for the procurement of a recombinant enzyme. 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.

To solve this problem, it is necessary to develop a product and a method that can correct the cells of the nervous system, namely, reprogram or modify them for long-term expression of a healthy gene.

With intrathecal and/or intravenous administration of AAV9-coARSA in the claimed technical solution, neurons will be transduced in a wide range from the injection site through anterograde neuronal transport. This ability of AAV9 allows you to reach more affected areas of the brain and achieve a therapeutic effect in a minimally invasive way. Due to the ability of AAV9 to cross the BBB, intravenous administration of the gene product also allows the transduction of cells of the nervous system. MSCs are also able to migrate to the BBB, especially during inflammation. The introduction of genetically modified MSCs with ARSA overexpression ensures uniform distribution of the enzyme throughout the nervous system.

From the studied level of technology, an invention was revealed specified in patent of the Russian Federation RU 2665381 "Intraventricular delivery of enzymes in lysosomal storage diseases". The essence of the known technical solution is a method for the prevention or treatment of lysosomal storage disease, which is caused by an enzyme deficiency in a patient, including intraventricular administration of the enzyme into the patient's brain, while the administration of a single dose of the enzyme takes more than three to eight hours. Said prevention or treatment includes administering the enzyme to the lateral ventricles and/or the fourth ventricle of the brain. The amount of enzyme administered to a patient is sufficient to reduce the enzyme levels in the patient's liver, lungs, spleen, or kidneys. Prevention or treatment includes monitoring the patient's sphingomyelin levels and administering additional enzyme in response to recorded levels of the enzyme. The enzyme is administered via an indwelling catheter and includes multiple infusions. Lysosomal storage diseases are: mucopolysaccharidosis I caused by alpha-L-iduronidase deficiency, mucopolysaccharidosis II caused by iduronate-2-sulfatase deficiency, Gaucher disease caused by glucocerebrosidase deficiency, Pompe disease caused by alpha-glucosidase deficiency, Classic late infantile Batten disease (CLN2) caused by tripeptidyl peptidase deficiency, as well as diseases listed in Table 1. Thus, more briefly, the essence of the known technical solution is the intraventricular delivery of lysosomal hydrolytic enzymes to the brain of patients with lysosomal storage diseases.

The disadvantage of the known technical solution is that when the enzyme is introduced into the brain, there is a high invasiveness of this procedure, as well as cytotoxicity of the enzyme at high concentrations and a limited rate of parenchymal diffusion in the brain.

The closest to the claimed technical solution in terms of technical essence and the achieved technical result aimed at the treatment of MLD, selected as a prototype, is the invention specified in patent WO2020227166A1 "Compositions useful in treatment of metachromatic leukodystrophy". The essence of the prototype is adeno-associated virus (AAV) used for the treatment of MLD. AAV includes: an AAVhu68 capsid and a vector genome packaged in an AAV capsid, where the vector genome contains inverted terminal repeats and a nucleic acid sequence encoding a functional human ARSA under the control of regulatory sequences that regulate ARSA expression. The coding sequence for ARSA includes the sequence of nucleotides from 55 to 1521 of SEQ ID NO: 1 or a sequence at least 95-99.9% identical to it, encoding a functional ARSA. The functional ARSA protein contains a signal peptide and amino acid sequence (aa) 19 aa 507 from SEQ ID NO: 2. The signal peptide has an amino acid sequence of 1 to 18 aa from SEQ ID NO: 2 or an amino acid sequence of 1 to 20 aa from SEQ ID NO: 4. The regulatory sequences direct the expression of ARSA in cells of the nervous system and contain a ubiquitous promoter, including the chicken b-actin promoter. The regulatory elements comprise one or more of a Kozak sequence, a polyadenylation sequence, an intron, an enhancer, and a TATA signal. The ARSA coding sequence is at least 95-99.9% identical to SEQ ID NO: 1 and encodes a functional ARSA. The ARSA coding sequence is SEQ ID NO: 1 or SEQ ID NO: 3. The vector genome has a sequence from 1 nucleotide to 3883 nucleotides of SEQ ID NO: 5. The AAVhu68 capsid is produced from the sequence encoding the predicted amino acid sequence of SEQ ID NO: 7.

The aqueous buffer of the pharmaceutical composition consists of: artificial cerebrospinal fluid containing buffered saline, a mixture of sodium, calcium, magnesium, potassium and a surfactant. The surfactant is present in an amount of 0.0005% to about 0.001% of the pharmaceutical composition. The solution has a pH in the range of 7.5 to 7.8. The composition buffer is suitable for intrasystemic large injection, intravenous delivery, intrathecal administration, or intracerebroventricular administration.

A vector containing an expression cassette, wherein the expression cassette contains a nucleic acid sequence encoding a functional ARSA under the control of regulatory sequences that control the expression of ARSA. The functional ARSA protein includes a signal peptide and amino acid sequence 19 aa 507 of SEQ ID NO: 2. The signal peptide has an amino acid sequence of 1 to 18 aa from SEQ ID NO: 2 or an amino acid sequence of 1 to 20 aa from SEQ ID NO: 4. The coding sequence for ARSA has a sequence of nucleotides from 55 to 1521 of SEQ ID NO: 1 or a sequence at least 95-99.9% identical to it, which encodes a functional ARSA. The ARSA coding sequence is SEQ ID NO: 1 or SEQ ID NO: 3. The vector is a viral vector selected from recombinant adeno-associated virus, recombinant parvovirus, recombinant lentivirus, recombinant retrovirus, or recombinant adenovirus. Or a non- viral vector selected from naked DNA, naked RNA, inorganic particle, lipid particle, polymer-based vector, or chitosan-based formulation.

A method of treatment of MLD or a disease associated with an ARSA gene mutation, comprising administering an effective amount of AAV. A method in which the vector is administered by suboccipital injection under computed tomography guidance into the cistema magna. This includes delivery of the AAV, pharmaceutical composition, or vector in a single dose. AAV is administered at a dose between 3.00 x 10 ¹⁰ genome copies (GC) per gram (GC/g) of brain weight and 1.00 x 10 ¹² GC/g of brain weight. Following administration, the subject's symptoms of the disease are improved and/or the progression of the disease is delayed. It is suitable for patients who are younger than 7 years of age for the need to alleviate the symptoms of MLD or an ARSA gene mutation disease and/or to delay the progression of MLD or an ARSA gene mutation disease.

The AAV production system includes a cell culture containing: a nucleic acid sequence encoding the AAVhu68 capsid protein, a vector genome, a sufficient number of reproductive functions and auxiliary functions of AAV to ensure the packaging of the vector genome into the AAVhu68 capsid. The vector genome has a sequence of nucleotides 1 to nucleotides 3883 of SEQ ID NO: 5. The cell culture is a 293 human embryonic kidney cell culture. REP AAV is derived from AAV2. The AAV REP coding sequence and the CAP genes are on the same nucleic acid molecule, with a spacer optionally present between REP and CAP. The spacer is the polynucleotide sequence of SEQ ID NO: 24. Thus, more briefly, the essence of the prototype is the intracerebral administration of a recombinant AAV having an AAVhu68 capsid and a vector genome containing a nucleic acid sequence encoding a functional ARSA.

The disadvantages of the prototype is that it describes the intracerebral delivery of AAVhu68 encoding ARSA. Intracerebral delivery (injection directly into the brain) is a highly invasive technique that can lead to serious complications. In addition, the applicant identified clinical studies (NCT01801709), which show that this method of delivery does not help stop the progression of lysosomal storage diseases associated with disruption of the nervous system, due to this prototype as a whole does not provide the possibility of a complete, reliable and accessible to a wide range of cure patients.

To prevent such shortcomings, it is necessary to develop a drug product and a less invasive method that can safely and at the same time effectively deliver the missing enzyme to the CNS and PNS for effective treatment of patients. Using the methods of gene and genecell therapy described in the claimed technical solution allows to achieve the best transduction of neurons from the injection site, allowing to achieve a greater number of transduced brain regions with intrathecal and/or intravenous administration of AAV9- coARSA. Due to the ability of AAV9 to cross the BBB, intravenous administration of AAV9-coARSA will also allow transduction of nervous system cells. MSC-ARSA transplantation ensures uniform distribution throughout the nervous system due to the ability of MSCs to migrate to the BBB.

SUMMARY OF THE INVENTION

The claimed technical solution has developed a drug product for gene and gene-cell therapy and a method for treating MLD, which consists in 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 in transplantation of mesenchymal human stem cells (MSCs) genetically modified with AAV9-coARSA (MSC-ARSA). The claimed drug product and methods for delivering the ARSA enzyme make it possible to restore the deficiency of the ARSA enzyme in the nervous system of a terminally ill person, due to the fact that the claimed technical solution makes it possible to prevent the cause of the disease. Metachromatic leukodystrophy is a rare hereditary disease from the group of lysosomal storage diseases with an autosomal recessive mechanism of inheritance of metabolic disorders. At the date of submission of the claimed technical solution, no drug for the treatment of MLD from the studied prior art has been identified. Therapy is reduced to the relief of 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 (including stem cells ). 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. However, the data obtained during the study of the peripheral nervous system are not so dramatic, and the long-term outcomes of the therapy performed are ambiguous to date. At the same time, other treatment options for metachromatic leukodystrophy are being developed. These include gene therapy, enzyme replacement therapy, substrate-lowering therapy, and self-enzyme enhancement.

Thus, the brief essence of this 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. Accumulation occurs due to deficiency of the lysosomal enzyme ARSA or the SapB activator protein. Malfunction or deficiency of the ARSA enzyme occurs due to mutations in the ARSA and PSAP genes.

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.

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 technical solution consists in the delivery of ARSA by the methods of gene (intrathecal or intravenous injection of AAV9-coARSA) and gene-cell (intravenous injection of MSC-ARSA) therapy. AAV9 is known to be able to cross the BBB, deliver the enzyme gene, and efficiently transduce cells of the nervous system. AAV9 is able to transport anterogradely along a neuron and transsynaptically transduce neurons in a wide range from the injection site, and MSCs are able to migrate to the area of neuroinflammation and neurodegeneration, therefore, after intrathecal and/or intravenous administration of AAV9-coARSA and/or intravenous transplantation of MSC-ARSA, the enzyme concentration is brought to normal physiological levels and neurodegeneration is prevented or slowed down. Thus, it becomes possible to restore the enzymatic activity of ARSA and improve the quality of life of patients. The specified technical result becomes possible due to the use of an approach that is not obvious to specialists, used by the applicant. The introduction of AAV9-coARSA and MSC-ARSA can prevent the development of MLD. The methods provide replenishment of the deficiency of the ARSA enzyme in the CNS and PNS.

The claimed technical solution provides an opportunity to solve a technically insoluble obstacle that exists in the world at the date of submission of the claimed technical solution, namely, to ensure that the BBB is overcome to deliver the missing enzyme to the CNS and PNS, and represents the possibility of restoring the enzymatic activity of ARSA in the patient's body, thereby providing an improvement the quality of life of patients suffering from MLD, which makes it possible to draw a logical conclusion about the compliance of the claimed technical solution not only with the "world novelty" criterion, but also with the "inventive step" criterion in accordance with the claimed technical solution, namely, the developed gene and gene-cell drug product and a method of treating MLD, which is intrathecal or intravenous administration of recombinant AAV9 containing the sequence of the codon-optimized ARSA gene, or intravenous administration of MSCs genetically modified with AAV9-coARSA.

The obtained gene and gene-cell drug products provide the possibility of delivering the missing ARSA enzyme to the CNS and PNS, as a result, according to the applicant, it is possible to completely stop neurodegeneration and cure sick patients suffering from MLD.

The purpose and technical result of the claimed technical solution is:

1. Development of a gene and gene-cell drug product for the treatment of MLD.

2. Development of methods for gene and gene cell therapy of MLD using recombinant AAV9-coARSA and MSCs genetically modified with recombinant AAV9- coARSA.

3. Reduction of invasiveness in the treatment of MLD.

4. Improving efficiency in the treatment of MLD.

5. Achieving uniform distribution of the therapeutic enzyme.

The claimed technical result in the form of a uniform distribution of the therapeutic enzyme to the sites of neurodegeneration and neuroinflammation is achieved due to the natural properties of AAV9-coARSA and MSCs transduced by AAV9-coARSA. It is known that MSCs are able to overcome the BBB and migrate to the nervous system during neuroinflammation, which is typical for MLD. AAV9 are also able to cross the BBB and be transported anterogradely along the neuron and transduce neurons in a wide range from the injection site to the CNS and PNS due to the presence of high transducing properties of the AAV 9 serotype.

The essence of the claimed technical solution is a product for gene therapy, including a recombinant adeno-associated virus serotype 9 containing a codon-optimized ARSA gene sequence, represented by SEQ ID NO:l. A product for gene-cell therapy, 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:l. A method for the treatment of metachromatic leukodystrophy using the product according to claim 1, consisting in a single intravenous injection of a recombinant adeno-associated virus serotype 9 containing a codon-optimized ARSA gene sequence presented in SEQ ID NO:l. A method for the treatment of metachromatic leukodystrophy using the product according to claim 1, consisting in a single intrathecal injection of a recombinant adeno-associated virus serotype 9 containing a codon-optimized ARSA gene sequence presented in SEQ ID NO:l. A method for the treatment of metachromatic leukodystrophy using the product according to claim 2, consisting in a single intravenous administration 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 consisting in a combination of methods according to claim 3 and claim 4. A method consisting in a combination of methods according to claim 3 and claim 5. A method consisting in a combination of methods according to claim 4 and claim 5.

Thus, in the claimed technical solution, gene and gene-cell preparations and methods for the treatment of MLD have been developed, consisting in the following: intravenous administration of recombinant AAV9-coARSA; conduct intrathecal administration of recombinant AAV9-coARSA; carry out intravenous administration of MSCs, previously genetically modified with recombinant AAV9-coARSA.

Recombinant AAV9-coARSA expresses the ARSA enzyme gene, is able to cross the BBB and evenly distribute the expression product throughout the CNS and PNS. MSC- ARSA overexpress and secrete the ARSA enzyme and ensure its delivery and uniform distribution throughout the CNS in patients with MLD.

BRIEF DESCRIPTION OT THE DRAWING

The claimed technical solution is illustrated in Figure 1 - Figure 6.

Figure 1 shows the dynamics of ARSA enzymatic activity in porcine plasma: before administration, on days 7, 14, 21, 28, 35. Y-axis indicates ARSA enzymatic activity (nM/mg/h). The x-axis indicates plasma samples of pigs N° 1 - N° 9, which were intravenously injected with genetically modified MSCs (designation MSCs + ARSA), plasma samples of pigs, which were intravenously injected with AAV9-coARSA (designation IY AAV9-coARSA), plasma samples of pigs, which were intrathecally introduced AAV9-coARSA (IT designation AAV9-coARSA).

The X-axis from left to right shows the data of the following plasma samples: Pig plasma sample N° 4 - designation MSCs+ARSA (first group of outcomes measured before administration, on days 7, 14, 21, 28, 35).

Pig plasma sample Ns 5 - designation MSCs+ARSA (second group of outcomes measured before administration, on days 7, 14, 21, 28, 35).

Pig plasma sample ½ 6 · designation MSCs+ARSA (third group of outcomes measured before administration, on days 7, 14, 21, 28, 35).

Pig plasma sample N° 7 - designation IV AAV9-coARSA (fourth group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample Ns 8 - designation IV AAV9-coARSA (fifth group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample N° 9 - designation IV AAV9-coARSA (sixth group of results, measured before administration, on days 7, 14, 21, 28, 35).

Pig plasma sample N° 1 - designation IT AAV9-coARSA (seventh group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample ]f» 2 - designation IT AAV9-coARSA (eighth group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample N° 3 - designation IT AAV9-coARSA (ninth group of results, measured before administration, on days 7, 14, 21, 28, 35).

Figure 2 shows the dynamics of the enzymatic activity of ARS A in the spinal cord (CSF) of pigs: before administration, on days 7, 14, 21, 28, 35. Y-axis indicates ARSA enzymatic activity (nM/mg/h). The x-axis indicates CSF samples of pigs N° 1 - N° 9, which were intravenously injected with genetically modified MSCs (designation MSCs + ARSA), CSF samples of pigs, which were intravenously injected with AAV9-coARSA (designation IV AAV9-coARSA), CSF samples of pigs, which were intrathecally introduced AAV9- coARSA (IT designation AAV9-coARSA).

The X-axis from left to right shows the data of the following plasma samples:

Pig plasma sample N° 4 - designation MSCs+ARSA (first group of outcomes measured before administration, on days 7, 14, 21, 28, 35).

Pig plasma sample N° 5 - designation MSCs+ARSA (second group of outcomes measured before administration, on days 7, 14, 21, 28, 35).

Pig plasma sample N° 6 - designation MSCs+ARSA (third group of outcomes measured before administration, on days 7, 14, 21, 28, 35). Pig plasma sample ½ 7 · designation IV AAV9-coARSA (fourth group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample Na 8 - designation IV AAV9-coARSA (fifth group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample Na 9 - designation IV AAV9-coARSA (sixth group of results, measured before administration, on days 7, 14, 21, 28, 35).

Pig plasma sample Ns 1 - designation IT AAV9-coARSA (seventh group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample Ns 2 - designation IT AAV9-coARSA (eighth group of results, measured before administration, at 7, 14, 21, 28, 35 days).

Pig plasma sample Ns 3 - designation IT AAV9-coARSA (ninth group of results, measured before administration, on days 7, 14, 21, 28, 35).

Figure 3a. 3b. 3c. 3d. 3e shows the level of ARSA enzymatic activity in homogenates of the CNS organs of pigs Na 1 - Ns 9 on the 35th day after product administration: the occipital lobe of the cerebral cortex (Figure 3a), cerebellum (Figure 3b), cervical spinal cord (Figure 3c), thoracic spinal cord (Figure 3d), lumbar spinal cord (Figure 3e). Y-axis indicates ARSA enzymatic activity (nM/mg/h). The X-axis shows samples of organ homogenates from a group of intact pigs (designation control), samples of organ homogenates from pigs that were intravenously injected with genetically modified MSCs (designation MSC-ARSA), samples of organ homogenates from pigs that were intravenously injected with AAV9-coARSA (designation IV AAV9-coARSA ), samples of organ homogenates from pigs injected intrathecally with AAV9-coARSA (IT designation AAV 9-co ARS A).

The X-axis from left to right shows the data of the following plasma samples:

Control.

Pig plasma sample Na 1 - IT designation AAV9-coARSA.

Pig plasma sample Na 2 - IT designation AAV9-coARSA.

Pig plasma sample Na 3 - IT designation AAV9-coARSA.

Pig plasma sample Na 4 - designation MSC+ARSA.

Pig plasma sample Na 5 - designation MSC+ARSA.

Pig plasma sample Na 6 - designation MSC+ARSA.

Pig plasma sample Na 7 - designation IV AAV9-coARSA. Pig plasma sample N° 8 - designation IV AAV9-coARSA.

Pig plasma sample ½ 9 - designation IV AAV9-coARSA.

Figure 4 shows an analysis of ARSA expression by immunohistochemical analysis of cryostat sections of CNS organs (ARSA - light (yellow), DAPI - dark (blue)). Vertically represented: A - cerebellar cortex, B - cerebral cortex, C - lumbar spinal cord, D - spinal ganglia. Horizontally presented: samples of sections of organs of the control group of pigs without AAV9-coARSA injection (1), samples of sections of organs of the experimental group of pigs with intravenous injection of AAV9-coARSA (2), samples of sections of organs of the experimental group of pigs with intrathecal injection of AAV9-coARSA (3) and samples of sections of organs of the experimental group of pigs with intravenous injection of MSCs genetically modified with AAV9-coARSA (4).

Figure 5a and Figure 5b shows the number of copies of the mRNA of the ARSA gene in the organs of the nervous system of pigs on days 1, 2, 3, 35 days after product administration. Fig. 5a - outcomes of intrathecal administration of AAV9-coARSA. Fig. 5b - outcomes of intravenous administration of AAV9-coARSA to pig No. 9. Along the x-axis in Figure 5a - from left to right, RNA samples of the cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord are indicated, in Figure 5b - from left to right, the spinal cord of the thoracic region and the ganglia of the posterior roots of the thoracic region are indicated. The Y-axis indicates the number of copies of the ARSA gene mRNA per 1 pg of total RNA. Data obtained by quantitative PCR.

Figure 6a. 6b, 6c, 6d shows alanine aminotransferase (ALT symbol) assay data (Fig. 6a), aspartate aminotransferase (AST symbol) (Fig. 6b), creatinine-J (Fig. 6c) and total bilirubin (Fig. 6d) in the blood serum of pigs, respectively, before administration, on the 7th day, on the 35th day. Data obtained using enzyme immunoassay (ELISA). From left to right, the X-axis shows serum samples from the experimental group of pigs with intravenous administration of genetically modified MSCs using AAV9-coARSA (designation MSC- ARSA), the experimental group of pigs with intravenous administration of AAV9-coARSA (designation IV AAV9-coARSA), the experimental group of pigs with intrathecal administration of AAV9-coARSA (IT designation AAV9-coARSA).

The X-axis from left to right shows the data of the following serum samples:

Pig serum samples - designation MSC+ARSA (first group of results, measured before injection, at 7, at 35 days). Serum samples of pigs - designation IV AAV9-coARSA (second group of outcomes, measured before administration, 7, 35 days).

Pig serum samples - designation IT AAV9-coARSA (third group of outcomes, measured before administration, at 7, at 35 days).

THE IMPLEMENTATION OF THE INVENTION

Further, in order to avoid 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 - matrix ribonucleic acid;

MSC - mesenchymal stem cells;

MSC-ARSA - mesenchymal stem cells modified with AAV9-coARSA;

PNS - peripheral nervous system;

CSF - cerebrospinal fluid;

CNS - central nervous system;

AAV9-coARSA — serotype 9 adeno-associated virus containing a unique codon- optimized ARSA gene sequence;

ARSA - arylsulfase A enzyme;

ARSA is the gene encoding arylsulfatase A.

DETAILED DESCRIPTION OF THE INVENTION

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

The set goal and the claimed technical outcome are achieved by intravenous and/or intrathecal administration of AAV9-coARSA, and/or intravenous administration of modified MSC-ARSA. The idea of the claimed technical solution is based on the fact that, due to the ability of MSCs to migrate to the BBB for neuroinflammation, the introduction of genetically modified MSCs with ARSA overexpression ensures uniform distribution of the enzyme throughout the nervous system. With intrathecal or intravenous administration of AAV9- coARSA, neurons are transduced in a wide range from the injection site through anterograde neuronal transport. This ability of AAV9 allows you to reach more transduced brain regions and achieve a therapeutic effect in a minimally invasive way. AAV9 is also able to cross the BBB by intravenous administration of the product. As a result of the development, a gene and gene-cell preparation was obtained, which has a therapeutic effect unknown from the studied state of the art, providing the possibility of treating a previously incurable disease of MLD.

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

Stage 1: Obtaining and analyzing the functionality of the p AAV- ARSA vector plasmid containing a unique codon-optimized ARSA gene sequence;

Stage 2: Obtaining and analysis of the functionality of the product for gene therapy, namely the recombinant adeno-associated virus AAV9-coARSA containing a unique codon- optimized ARSA gene sequence;

Stage 3: Testing the efficacy and safety of intravenous, intrathecal administration of AAV9-coARSA and intravenous administration of MSC-ARSA to large laboratory animals.

Further, the applicant gives examples of each of the stages of the implementation of the claimed technical solution.

Example 1. Carrying out the 1st stage - obtaining and analyzing the functionality of the pAAV-coARSA vector plasmid containing a unique codon-optimized ARSA gene sequence

Produced codon optimization of the nucleotide sequence of the gene. To optimize the codon composition of the ARSA gene, well-known algorithms were used, for example, OptimumGene (GeneScript, USA), which takes into account various factors affecting gene expression levels, such as codon shift, GC composition, content of CpG dinucleotides, mRNA secondary structure, tandem repeats, restriction sites that may interfere with cloning, premature polyadenylation sites, additional minor ribosome binding sites. 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). The correct assembly of the pAAV-coARSA genetic construct was confirmed by restriction analysis.

To confirm the functionality of the p AAV- ARSA genetic construct, it is transfected (genetically modified) with an immortalized HEK293T primary human embryonic kidney cell line. 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 efficiency of transfection, the pAAV-Katushka2S plasmid vector encoding the far-red fluorescent protein is used as a positive control.

The in vitro expression efficiency of the recombinant protein with the resulting plasmid construct was confirmed by an activity test and a Western blot.

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 the activity of ARSA, a 50 mΐ sample of cell lysate is incubated with a solution of the substrate nitrocatechol sulfate (0.01 M p- Nitrocatechol sulfate dipotassium salt (#N7251, Sigma), 0.5 M sodium acetate, 0.5 mM Na4P207, 10% sodium chloride, pH = 5) for 1 hour at 37 ° C, after which the reaction is stopped by adding 1 N sodium hydroxide. Sulfatase dilutions (#S9626, Sigma) are used as standards. Optical density is measured at a wavelength of 515 nm.

Western blot analysis was performed using primary rabbit anti-ARSA polyclonal antibody (Cat. No. PAG619Hu01, Cloud-Clone Corp., USA) diluted 1:500 in blocking buffer. Applicant's analysis of lysate samples of HEK293T cells transfected with pAAV- ARSA yielded expected sizes of about 33 kDa.

Example 2. Carrying out the 2nd stage - obtaining and analyzing the functionality of the product for gene therapy, namely the recombinant adeno-associated virus AAV9- coARSA containing a unique codon-optimized ARSA sene sequence.

Based on the plasmid, a preparation based on the recombinant AAV9-coARSA virus is obtained. AAV Helper free system is used to obtain recombinant AAV9 viruses. Cells AAV293 sowed in the amount of 1.5 million, the monolayer should be 70-80%. The next day, co-transfection was carried out using the calcium phosphate method with three plasmids (vector plasmid, pAAV-RC and pHelper). Virus is 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 -80 °C. The virus is concentrated using the AAV Purification Mega Kit (Cell Biolabs, Inc., USA) according to the method recommended by the manufacturer.

The resulting recombinant virus is transduced with MSCs to obtain a gene-cell preparation. The efficiency of genetic modification with the obtained viral vectors is checked by Western blot analysis and enzyme activity test.

Example 3. Carrying out the 3rd stage - checking the effectiveness and safety of the product and the method using intravenous, intrathecal administration of AAV9-coARSA and intravenous administration of MSC-ARS A to large laboratory animals.

The efficacy and safety of the obtained gene and gene-cell preparations were tested on large laboratory animals.

In the experimental work, pigs were used at the age of 4 months (weighing 5 kg), which were randomly distributed into 4 groups:

1) Control group of intact pigs without product administration;

2) Experimental group of pigs (pigs N° 1-3) injected intrathecally with recombinant AAV9-coARSA encoding the ARSA enzyme at a dose of 1 x 10 ¹² genome copies/kg;

3) An experimental group of pigs (pigs N° 7-9) injected intravenously with recombinant AAV9-coARSA encoding the ARSA enzyme at a dose of 3.77 x 10 ¹³ genome copies/kg;

4) Experimental group of pigs (pigs Na 4-6), which are intravenously injected with MSCs genetically modified with AAV-coARSA in the amount of 2.7 million cells/kg.

Each group has 3 individuals. 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 performed in accordance with ethical standards and current legislation.

Prior to the administration of the study product, samples of CSF and whole blood were taken from pigs in tubes containing sodium citrate anticoagulant and gel, activator; Plasma and serum from whole blood were isolated by centrifugation for 20 minutes at 1900 rpm, CSF, plasma and serum were stored at -80°C. Whole blood and CSF were also collected 7, 14, and 21 days after cell injection. Plasma and CSF samples were used to determine the enzymatic activity of ARSA.

On the 35 th day after the administration of recombinant AAV9-coARSA and MSC- ARSA, pigs were euthanized, using methods that comply with the principles set forth in the European Commission Guidelines for the Euthanasia of Experimental Animals, the spinal cord (cervical, thoracic, lumbar), dorsal root ganglia were removed (cervical, thoracic, lumbar), cerebellum, occipital cortex, hidden nerve, heart, liver, kidneys, spleen, lungs. All organs were homogenized for activity testing and RT-PCR or dissected for IHC analysis.

The results described in Steps 1-3 are shown in FIG. 1 - FIG.6.

Graphic materials illustrating examples of specific implementation of the claimed technical solution in FIG.l - FIG.6, experimentally proved the possibility of achieving the claimed technical outcomes.

Thus, from the data shown in figure 1 , it can be seen that the enzymatic activity of ARSA in the blood plasma of pigs increases on days 7-21.

After intravenous administration of AAV9-coARSA, ARSA activity increased to 124%; after intrathecal administration of AAV9-coARSA, ARSA activity increased to 26%; after intravenous administration of MSC-ARSA, no statistically significant increase in ARSA activity was detected. These results indicate that intrathecal and intravenous administration of AAV9-coARSA to large laboratory animals results in transduction of cells that then begin to express and secrete a functionally active enzyme found in animal plasma.

From the data shown in figure 2, it can be seen that the enzymatic activity of ARSA in porcine CSF increases from 17 to 74% on days 7-21 after intrathecal administration of AAV9-coARSA, compared with pre-administration CSF samples.

After intravenous administration of AAV9-coARSA or MSC-ARSA, no increase in ARSA enzymatic activity in the CSF was observed. These results also confirm that AAV9-COARSA encodes an active ARSA enzyme and also suggest that intrathecal administration of the virus allows more efficient transduction of CNS cells.

From the data shown in FIG. 3, it can be seen that the enzymatic activity of ARSA in homogenates of CNS organs increases after administration of the products. In the homogenate of the occipital lobe of the cerebral cortex, ARSA activity is increased in all pigs except N°1 and N°2. Pigs N° 3, 4, 5, 6, 7, 8, 9 showed an increase of 26%, 30%, 136%, 145%, 54%, 19%, 80%, respectively. In the homogenate of the cerebellum, an increase in ARSA activity was detected in all pigs. Pigs N°l, 2, 3, 4, 5, 6, 7, 8, 9 showed an increase of 23%, 26%, 59%, 93%, 73%, 141%, 31%, 66%, 65%, respectively.

In the homogenate of the cervical spinal cord, an increase in ARSA activity was detected in all pigs, except for Ns 1. Pigs N° 2, 3, 4, 5, 6, 7, 8, 9 showed an increase of 85%, 22%, 74%, 200%, 132%, 147%, 129%, 190%, respectively.

ARSA enzymatic activity is increased in porcine thoracic spinal cord homogenate. Pigs J o 1, 2, 3, 4, 5, 6, 7, 8, 9 showed increases of 99%, 109%, 400%, 273%, 280%, 300%, 194%, 320% and 331%, respectively.

In the homogenate of the lumbar spinal cord, an increase in ARSA activity was detected in all pigs except for Ns 1. Pigs Ms 2, 3, 4, 5, 6, 7, 8, 9 showed an increase of 144%, 41%, 182%, 283%, 233%, 139%, 220%, 415%, respectively. These results indicate that, after genetic modification with the resulting AAV9-coARSA, neuronal cells begin to express the functional enzyme in vivo. Moreover, the increase is observed in all groups of animals.

From the data shown in FIG. 4, it can be seen that the 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. Analysis of ARSA expression in the cerebellar cortex and in the cortex of the occipital lobe of the brain after intrathecal administration showed a different number of Purkinje neurons within the group at S = 54.8 mm2: in 1 pig, single Purkinje neurons overexpressing ARSA were found; in 2 and 3 pigs - at least 100 and 20 overexpressing Purkinje neurons, respectively. 7 and 9 pigs had 5-10 overexpressing Purkinje ARSA neurons, 8 pigs had at least 15 overexpressing Purkinje neurons, respectively.

At the same time, in the control pig (without the introduction of AAV9-coARSA), ARSA expression was lower and most often localized only on the periphery of the cytoplasm of the cell body. Overexpression of ARSA in the control pig, similar to that found in the experimental group, was found only in 1 Purkinje cell per S = 328.8 mm2. Analysis of ARSA expression in the cerebellar cortex after intravenous administration of AAV9-coARSA showed a different number of Purkinje neurons within the group at S = 54.8 mm2: 7 and 9 pigs had 5-10 overexpressing Purkinje ARSA neurons, 8 pigs had at least 15 overexpressing Purkinje neurons, respectively. The discovered Purkinje neurons overexpressing ARSA were most often localized singly within the boundaries of one gyrus. After intravenous administration of MSC-ARSA, ARSA overexpression in the cerebellum, Purkinje neurons, in particular, was not detected; the intensity of ARSA luminescence and its distribution in the cell corresponded to the control group of animals. Analysis of ARSA expression in the occipital cortex of the brain showed a greater expression of ARSA in the subarachnoid space in pigs with intravenous administration of AAV9-coARSA compared with the control group of intact animals and the group with intravenous administration of MSC-ARSA. However, statistical processing of the average ARSA luminescence intensity in this area did not reveal any significant difference between the groups. Both in pigs of the 2nd and 3rd experimental groups, and in the control group, ARSA+ cells were found on cross sections of the cerebral cortex, the specific luminescence in which was localized on the periphery of the cytoplasm of the cell body and partially in the processes. Analysis of ARSA expression in the cervical, thoracic and lumbar regions of the spinal brain did not show significant differences in the expression of ARSA in the white and gray matter of the group of animals with intrathecal administration and control animals. In the white matter, ARSA expression was markedly higher than in the gray matter, and was localized mainly in the processes and bodies of glial cells. The overall pattern of ARSA expression in the lumbar spinal cord was similar to that described above. However, single motor neurons overexpressing ARSA were found in the ventral horns of the lumbar spinal cord of the experimental pigs, which was not observed in the animals of the control group. Analysis of ARSA expression in the spinal ganglia (cervical region) revealed ARSA-overexpressing neurons after intravenous administration of AAV9- coARSA. The number of neurons overexpressing ARSA varied greatly within the group, but no significant difference in this indicator between the spinal ganglia was found. The expression of ARSA in the spinal ganglia of the cervical region after intravenous administration of MSC-ARSA corresponded to that in the control.

Analysis of ARSA expression in the spinal ganglia (thoracic region) revealed neurons overexpressing ARSA after intravenous administration of AAV9-coARSA. The number of neurons overexpressing ARSA varied greatly within the group, however, there was no significant difference in this indicator between the spinal ganglia. The expression of ARSA in the spinal ganglia of the thoracic region after intravenous administration of MSC- AAV9-COARSA corresponded to that in the control. An analysis of ARSA expression in the spinal ganglia (lumbar region) revealed ARSA overexpressing neurons after intravenous administration of AAV9-coARSA. The number of neurons overexpressing ARSA varied greatly within the group, however, there was no significant difference in this indicator between the spinal ganglia. The expression of ARSA in the spinal ganglia of the lumbar spine after intravenous administration of MSC-AAV9-coARSA corresponded to that in the control.

From the data shown in figure 5, overexpression of the codon-optimized ARSA gene is seen in the CNS organs of pigs injected intrathecally with AAV9-coARSA. In the cerebellum, cervical, thoracic, lumbar spinal cord of pig No. 1, 28,951, 8,408, 7,818, 30,439 copies of the ARSA gene mRNA per pg of total RNA, respectively, were detected. In the cerebellum, cervical, thoracic, lumbar spinal cord of pig No. 2, 243,934, 17,031, 1,804, 6,889 copies of the ARSA gene mRNA per pg of total RNA, respectively, were detected.

In the cerebellum, cervical, thoracic, lumbar spinal cord of pig No. 3, 2,436, 8,104, 2,027, 8,104 copies of the ARSA gene mRNA per pg of total RNA, respectively, were detected. The number of copies of the mRNA of the ARSA gene in the organs of the CNS and PNS of pig No. 9 on day 35 after intravenous administration of AAV9-coARSA in the thoracic spinal cord was detected 1,104 copies of the mRNA of the ARSA gene per pg of total RNA. In the ganglia of the dorsal roots of the thoracic region, 666 copies of mRNA of the ARSA gene per pg of total RNA were detected. In the group with the introduction of MSC-ARSA, mRNA of the ARSA gene was not found in samples of the organs of the nervous system.

From the data shown in figure 6, it can be seen that the biochemical parameters in the blood serum of pigs after intrathecal administration of AAV9-coARSA showed the values of ALT, creatinine-J and bilirubin remain unchanged after the administration of the products. After intravenous administration of AAV9-coARSA and MSC-ARSA, ALT and creatinine-J values remain unchanged. On the 35th day after the administration of the product, the level of AST was statistically significantly lower in the group of animals that were intravenously injected with AAV9-coARSA. Also, the level of bilirubin is significantly reduced on days 7 and 35 after intravenous administration of AAV9-coARSA. After intravenous administration of genetically modified MSCs, on the 35th day the level of total bilirubin is significantly lower compared to the samples before the administration of the product. Thus, from Examples 1 to 3 and Fig 1 - Fig.6 it can be concluded that the applicant has developed the claimed methods using the claimed preparations:

- treatment of metachromatic leukodystrophy using the claimed product for gene therapy, namely, a single intravenous injection of a product that includes a recombinant adeno-associated virus serotype 9 containing a codon-optimized ARSA gene sequence presented by SEQ ID NO: 1.

- treatment of metachromatic leukodystrophy using the claimed product for gene therapy, namely a single intrathecal injection of a drug that includes a recombinant adeno- associated virus serotype 9 containing a codon-optimized ARSA gene sequence presented by SEQ ID NO:l.

- treatment of metachromatic leukodystrophy using the claimed product for genecell therapy, namely a single intravenous injection of a drug consisting of mesenchymal stem cells genetically modified with recombinant adeno-associated virus serotype 9 containing a codon-optimized ARSA gene sequence presented in SEQ ID NO:l .

At the same time, the applicant makes a logical conclusion that, since the use of each claimed method separately allows achieving the claimed technical result, it (the claimed technical result) will undoubtedly also be achieved with any combination of the claimed methods. In this case, the combination of the claimed methods can be used, for example, in more severe forms of the disease.

Based on the obviousness of the conclusion, the applicant did not provide examples of the claimed methods in any combination thereof, while the combination of methods is included in the claims as dependent clauses 6, 7, 8, since they are special cases of the methods according to clauses 3, 4, 5 , namely:

- a method consisting in a combination of methods according to claim 3 and claim 4;

- a method consisting in a combination of methods according to claim 3 and claim 5;

- a method consisting in a combination of methods according to claim 4 and claim 5.

From the above, we can make a general conclusion that the claimed technical solution has achieved all the goals and implemented all the claimed technical results, namely:

1. Gene and gene-cell preparations for the treatment of MLD have been developed. 2. A method for gene and gene cell therapy of MLD using recombinant AAV9- coARSA and MSCs genetically modified with recombinant AAV9-coARSA has been developed.

3. Achieved a decrease in invasiveness in the treatment of MLD compared with the prototype.

4. Increased efficiency in the treatment of MLD compared with the prototype.

5. A uniform distribution of the therapeutic enzyme was achieved.

At the same time, in specific examples, it has been experimentally proven that by using the assembled genetic preparation and the developed method, an increase in the amount and activity of the ARSA enzyme, which is deficient in patients with MLD, in blood plasma, in CSF and in homogenate of pig organs is achieved.

In the claimed technical solution, the applicant has developed a gene and gene-cell preparation in its entirety, characterized in that it provides the possibility of treating patients suffering from MLD. At the same time, according to the applicant, taking into account the fact that from the studied prior art, the technology of overcoming BBB serotype 9 by mesenchymal stem cells and adeno-associated virus 9 of the BBB serotype and delivering the missing enzyme to the CNS in lysosomal storage diseases is known as such. Given this fact, it can be argued that the claimed technical solution allows you to restore the activity of the enzyme in the CNS and, consequently, the metabolism of sulfatides, thus stopping neurodegeneration and curing patients.

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.

The claimed technical solution satisfies the “inventive step” patentability condition for inventions, since the applicant has not identified technical solutions from the studied state of the art, characterized by the creation of a gene and gene-cell preparation and the use of a method for treating MLD, which involves intrathecal and intravenous administration of recombinant AAV9-coARSA and intravenous administration of genetically modified stem cells overexpressing the ARSA gene to restore enzyme activity in the CNS and stop neurodegeneration. 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.

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.

Table 1. List of diseases