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Definitions

• 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 .
• ARSA deficiency leads to damage to the myelin sheath covering most of the nerve fibers of the central (CNS) and peripheral nervous system (PNS).
• the claimed technical solution ensures the delivery of ARSA to the CNS and PNS by minimally invasive procedures.
• AAV5 adeno-associated viral vector serotype 5
• 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.].
• AAV9 encoding ARSA and the green fluorescent protein reporter gene
• 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.
• CSF cerebrospinal fluid
• 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.
• 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).
• 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.
• nucleic acid encoding an AAV capsid protein
• 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.
• 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.
• 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.
• BDNF brain-derived neurotrophic factor
• 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
• 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.
• 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.
• 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.
• AAV9-coARSA 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.
• 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.
• 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.
• composition contains lentiviral vector and recombinant lentivirus.
• the product contains a lentiviral vector, recombinant lentivirus, recombinant cell or pharmaceutical.
• the composition further includes a pharmaceutically acceptable excipient.
• 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.
• 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.
• 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).
• MRS proton magnetic resonance spectroscopy
• 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 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 comprises an amino acid sequence containing no more than one mismatch with SEQ ID NO: 1.
• 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).
• AAV9-coARSA 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.
• 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.
• 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 (CL
• 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.
• AAV adeno-associated virus
• 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.
• 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.
• 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.
• 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.
• GC genome copies
• GC/g genome copies
• 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.
• 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.
• 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.
• 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).
• AAV9-coARSA codon-optimized ARSA gene
• MSCs mesenchymal human stem cells
• 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.
• 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.
• bone marrow transplantation including stem cells .
• 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.
• 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.
• MLD oligodendrocytes
• microglia some CNS neurons
• Schwann cells 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.
• 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.
• 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 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.
• gene and gene-cell preparations and methods for the treatment of MLD 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.
• 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 1 ⁇ 2 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 1 ⁇ 2 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:
• Pig plasma sample Na 1 - IT designation AAV9-coARSA Pig plasma sample Na 1 - IT designation AAV9-coARSA.
• Pig plasma sample Na 2 - 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 3 - IT designation AAV9-coARSA.
• Pig plasma sample 1 ⁇ 2 9 - designation IV AAV9-coARSA Pig plasma sample 1 ⁇ 2 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)).
• a - cerebellar cortex A - cerebellar cortex
• B - cerebral cortex A - cerebral cortex
• C - lumbar spinal cord A - spinal ganglia.
• 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.
• RNA samples of the cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord are indicated
• 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.
• FIG. 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).
• ELISA enzyme immunoassay
• 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.
• BBB blood-brain barrier
• cDNA complementary deoxyribonucleic acid
• MLD metachromatic leukodystrophy
• mRNA - matrix ribonucleic acid
• AAV9-coARSA serotype 9 adeno-associated virus containing a unique codon- optimized ARSA gene sequence
• ARSA is the gene encoding arylsulfatase A.
• 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.
• 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.
• 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.
• 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.
• 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
• the p AAV- ARSA genetic construct 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.
• ARSA enzyme activity is determined in HEK293T lysate 24 hours after transfection.
• the concentration of total protein in the samples was determined using the PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, USA). Samples are normalized to total protein concentration.
• Sulfatase dilutions (#S9626, Sigma) are used as standards.
• Optical density is measured at a wavelength of 515 nm.
• 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.
• a preparation based on the recombinant AAV9-coARSA virus is obtained.
• AAV Helper free system is used to obtain recombinant AAV9 viruses.
• 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.
• mice Na 4-6 which are intravenously injected with MSCs genetically modified with AAV-coARSA in the amount of 2.7 million cells/kg.
• 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.
• 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.
• Steps 1-3 The results described in Steps 1-3 are shown in FIG. 1 - FIG.6.
• 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.
• ARSA expression was lower and most often localized only on the periphery of the cytoplasm of the cell body.
• Purkinje neurons overexpressing ARSA were most often localized singly within the boundaries of one gyrus.
• 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.
• statistical processing of the average ARSA luminescence intensity in this area did not reveal any significant difference between the groups.
• 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.
• 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.
• ARSA 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.
• ARSA expression in the spinal ganglia 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.
• 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.
• 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 “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.
• 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.

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